Biochem. J. (2012) 441, 39–59 (Printed in Great Britain)
39
doi:10.1042/BJ20111226
REVIEW ARTICLE
Insights into the PX (phox-homology) domain and SNX (sorting nexin)
protein families: structures, functions and roles in disease
Rohan D. TEASDALE and Brett M. COLLINS1
Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland 4072, Australia
The mammalian genome encodes 49 proteins that possess a PX
(phox-homology) domain, responsible for membrane attachment
to organelles of the secretory and endocytic system via binding
of phosphoinositide lipids. The PX domain proteins, most of
which are classified as SNXs (sorting nexins), constitute an
extremely diverse family of molecules that play varied roles
in membrane trafficking, cell signalling, membrane remodelling
and organelle motility. In the present review, we present an
overview of the family, incorporating recent functional and
structural insights, and propose an updated classification of
the proteins into distinct subfamilies on the basis of these
insights. Almost all PX domain proteins bind PtdIns3P and
are recruited to early endosomal membranes. Although other
specificities and localizations have been reported for a select
few family members, the molecular basis for binding to other
lipids is still not clear. The PX domain is also emerging as an
important protein–protein interaction domain, binding endocytic
and exocytic machinery, transmembrane proteins and many other
molecules. A comprehensive survey of the molecular interactions
governed by PX proteins highlights the functional diversity of the
family as trafficking cargo adaptors and membrane-associated
scaffolds regulating cell signalling. Finally, we examine the
mounting evidence linking PX proteins to different disorders, in
particular focusing on their emerging importance in both pathogen
invasion and amyloid production in Alzheimer’s disease.
INTRODUCTION
binds to PtdInsP lipids (phosphoinositides) on the cytoplasmic
leaflets of different organelles, similar in function to other
lipid-binding domains such as the PH (pleckstrin homology),
FYVE (Fab1p/YOTB/Vac1p/EEA1) and ENTH (epsin N-terminal
homology) domains. This association defines the cellular
localization of the different PX proteins and hence is critical
to their function. The present review summarizes recent progress
in defining the structural and functional properties of the PX
domain-containing proteins, and presents an update of some
previously ambiguous structural classifications of the proteins.
We provide a survey of the diverse biomolecular interactions
that are regulated by these molecules, demonstrating their central
functions as both cargo adaptors in cell trafficking and scaffolds
regulating signal transduction, and discuss some of the recent
work on previously poorly characterized PX domain-containing
protein families. Finally, we highlight the emerging roles of
PX proteins in disease.
Eukaryotic cells contain numerous membrane-bound organelles
involved in secretion and endocytosis. Transmembrane proteins,
lipids and other critical cargo molecules must be selectively sorted
and transported between these compartments via membrane
trafficking processes, involving either vesicular or tubulovesicular
carriers or compartmental maturation and fusion events.
Furthermore, it is now apparent that endocytic compartments
are also critical platforms for the regulation of cell signalling,
well beyond the traditional function of controlling receptor
degradation [1,2]. The mammalian Phox-homology (PX) domaincontaining molecules constitute a large family of at least
49 proteins. They are predominantly found in the endosomal
system, but also localize to other cellular compartments and
regulate diverse trafficking and signalling processes in the
cell. The PX domain is a membrane recruitment module that
Key words: Alzheimer’s disease, endosome, phosphoinositide,
phox-homology domain (PX domain), retromer, sorting nexin
(SNX).
Abbreviations used: AD, Alzheimer’s disease; ADAM, a disintegrin and metalloproteinase; AP2, adaptor protein complex 2; ApoE, apolipoprotein E; APP,
amyloid precursor protein; BACE, β-secretase; BAR, Bin/amphiphysin/Rvs; CASP, cytohesin-associated scaffolding protein; CGD, chronic granulomatous
disease; CI-MPR, cation-independent mannose-6-phosphate receptor; DGK, diacylglycerolkinase; EGF, epidermal growth factor; EGFR, epidermal growth
factor receptor; ERK, extracellular-signal-regulated kinase; FERM, band4.1/ezrin/radixin/moesin; FHA, forkhead association; FISH, five SH3 domains; FYVE,
Fab1p/YOTB/Vac1p/EEA1; GAP, GTPase-activating protein; GC-GAP, GAB/cdc42 GAP; GPCR, G-protein-coupled receptor; Hrs, hepatocyte growth
factor-regulated tyrosine kinase substrate; HS1BP3, HS1-binding protein 3; 5-HT4 R, 5-hydroxytryptamine type 4 receptor; IRAS, imidazoline receptor
antisera selected; IRS, insulin receptor substrate; KIF, kinesin-family protein; Krit1, Krev-interaction trapped 1; LDLR, low-density lipoprotein receptor;
LRP1, LDLR-related protein 1; LRR, leucine-rich repeat; MIT domain, microtubule-interacting and trafficking molecule domain; MMEP, microcephaly,
micropthalmia, ectrodactyly and prognathism; MVB, multivesicular body; NCF, neutrophil cytosolic factor; NMDA, N -methyl-D-aspartate; NOXO1, NADPH
oxidase organizer 1; NR, NMDA receptor; PA, phosphatidic acid; PDZ, postsynaptic density 95/discs large/zonula occludens; PDZbm, PDZ-binding
motif; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PLCγ1, phospholipase Cγ1; PLD, phospholipase D; PS1, presenilin 1; PSGL, P-selectin
glycoprotein ligand; PTB, phosphotyrosine binding; PX, phox-homology; PXA domain, PX-associated domain A; PXC, PX-associated domain C; RA,
Ras-association; RGS, regulator of G-protein signalling; RPS6K, ribosomal protein S6 kinase; SCV, Salmonella -containing vacuole; SGK, serum- and
glucocorticoid-induced protein kinase; SH, Src homology; SH3PXD2A, SH3 and PX domain protein 2A; siRNA, small interfering RNA; SLIC-1, selectin
ligand interactor cytoplasmic-1; SNARE, soluble N -ethylmaleimide-sensitive factor-attachment protein receptor; snazarus, sorting nexin lazarus; SNX,
sorting nexin; S/T kinase, serine/threonine kinase; STx, Shiga toxin; TC-GAP, TC10/cdc42 GAP; TGF-β, transforming growth factor β; TGN, trans -Golgi
network; TPR, tetratricopeptide repeat; VPS, vacuolar protein sorting-associated protein; VSV, vesicular stomatitis virus; WASP, Wiskott–Aldrich syndrome
protein; WASH, WASP and SCAR (suppressor of cAMP receptor) homologue; N-WASP, neuronal WASP.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
40
R. D. Teasdale and B. M. Collins
THE PX DOMAIN
Structure of the PX domain and binding to the endosomal lipid
marker PtdIns3P
The PX domain is a globular fold approximately 110 residues
in length, composed of three β-strands followed by three
α-helices (Figure 1). Specific features of this protein fold are
a long proline-rich loop between helices α1 and α2, and an
electropositive basic pocket that is involved in direct binding of
negatively charged phosphate groups of phosphoinositides [3].
In addition several studies have highlighted the role of adjacent
hydrophobic residues in membrane penetration in at least some
PX domains, and an additional positively charged surface patch,
which is able to associate weakly with other anionic lipids, such
as phosphatidylserine [4–8].
A survey of the known structures of mammalian and yeast PX
proteins, their localization and lipid binding in vitro reveals a number of insights into the PX domain phosphoinositide specificity
(Figure 1, and Supplementary Movie S1, Table S1 and Figure S1 at
http://www.BiochemJ.org/bj/441/bj4410039add.htm). A global
assessment of Saccharomyces cerevisiae PX domain molecules
demonstrated an almost absolute PtdIns3P specificity [9], and a
review of the literature presents an overwhelming consensus that
mammalian PX domains also show a preference for PtdIns3P (see
Supplementary Table S1). PtdIns3P is primarily found on early
endosomal limiting membranes and therefore most commonly
directs PX protein recruitment to PtdIns3P-enriched endosomal
organelles. It should be noted, however, that distinct pools of
PtdIns3P can be found in other compartments, such as the plasma
membrane, being often generated during specific signalling
processes [10]. Currently there are 27 structures of mammalian
PX domains available in the PDB (from 14 different proteins),
but only two have been solved in complex with PtdIns3P: p40phox
(PDB code 1H6H) and SNX9 (sorting nexin 9; PDB code
2RAK). There are five known structures of yeast PX domains,
with one structure of Grd19p/Snx3p in complex with PtdIns3P
(PDB code 1OCU). In addition, the binding of PtdIns3P to the
SNX17 and SNX22 PX domains has been analysed using NMR
and restrained computational docking approaches [11,12]. These
studies reveal the key determinants for PtdIns3P binding (Figure 1
and Supplementary Table S1) [3]. Each moiety of the PtdIns3P
headgroup interacts with a specific and strictly conserved side
chain of the PX domain. The defining 3-phosphate forms
electrostatic bonds with a conserved arginine side chain. The
inositol group forms a key stacking interaction with a conserved
tyrosine residue (or in some cases phenylalanine) immediately
one residue downstream of the conserved arginine. The 4- and
5-hydroxy groups of the inositol form hydrogen bonds with a
conserved arginine side chain, which helps orient the PtdIns3P in
the binding pocket and also sterically precludes binding of 4- or
5-phosphorylated lipids. In most cases, the polyproline loop also
creates a steric boundary against incoming 5-phosphate groups,
although this loop is inherently flexible and may be able to alter
its conformation. Finally, the 1-phosphate is co-ordinated by a
conserved lysine side chain, which is immediately downstream
of the polyproline loop. The necessary consensus sequence in PX
domains for PtdIns3P binding is thus R[Y/F]x23–30 Kx13–23 R.
Apart from lipid binding by the PX domain, a very
important additional factor is the contribution of associated
domains and other molecular interactions that drive and enhance
membrane attachment through ‘coincidence detection’ [13].
Thus membrane attachment specificity may be controlled not
only by the PX domain, but also by combination with domains
that associate independently with membrane lipids [e.g. BAR
(Bin/amphiphysin/Rvs), FERM (band4.1/ezrin/radixin/moesin)
c The Authors Journal compilation c 2012 Biochemical Society
Figure 1
Structure of the PX domain
(A) Ribbon and surface structures of a representative PX domain from p40phox in complex with
PtdIns3P (PDB code 1H6H). Key determinants of the interaction are the arginine side chain
electrostatic association with the 3-phosphate (ArgP3 ), stacking of the inositol ring with the
tyrosine (or phenylalanine) side chain immediately downstream from the conserved arginine
residue (Tyrinositol ), contact of a lysine side chain with the 1-phosphate (LysP1 ), and hydrogen
bonds of the 4- and 5-hydroxy groups to a second arginine side chain (Arg4,5-hydroxyl ). The
polyproline loop also forms a steric block of potential 5-phosphate groups in the binding
pocket. The PtdIns3P headgroup is located within a deep positively charged pocket. The
consensus PX domain-binding sequence is shown below. See also Supplementary Movie S1
at http://www.BiochemJ.org/bj/441/bj4410039add.htm. (B) Structure of the human SNX5 PX
domain [22] overlaid with the human PX domain of SNX9 within the SNX9 PX-BAR assembly
[18]. The SNX9 protein is coloured in grey, whereas SNX5 is coloured dark blue to light blue (Nto C-terminal). Bound PtdIns3P molecules are shown as spheres, with carbon atoms coloured
green. A schematic membrane is indicated with a curvature that follows the curvature of the
SNX9 PX-BAR assembly. The SNX5 helical insertion, indicated by the broken red circle, will
clearly form steric interactions with the membrane environment in this orientation. SNX5 is only
shown aligned with one subunit of the PX-BAR dimer for clarity.
and PH domains] or bind to other membrane-associated proteins
such as Rabs or cargo molecules.
Do PX domains bind other phosphoinositides?
PtdIns3P is the most common phosphoinositide bound by PX
domains; however, many other phosphoinositide interactions have
The PX protein family
been reported, suggesting a diverse role in membrane trafficking
or signalling at different cellular compartments (Supplementary
Table S1). Typical reported affinities (K d values) in vitro are of the
order of 0.1–10 μM, but vary wildly from nanomolar to millimolar
values depending on the PX domain studied and sometimes even
for the same PX domain. Assessing the phosphoinositide binding
of PX domain proteins is complicated by the range of different
methods used and the relative reliability of these methods [14,15].
Despite the wide variety of other phosphoinositide specificities
reported, there is still no conclusive structural indication of
how these interactions occur. Indeed, a naı̈ve analysis of the
available structural data predicts that other phosphoinositides
apart from PtdIns3P will not be able to associate with PX domains,
unless their binding mode is significantly different from that
demonstrated for PtdIns3P (Supplementary Table S1 and Figure
S1). In most cases there are steric considerations that preclude the
association of 4- and 5-phosphate groups, and where this is not
the case there are no obvious determinants for specific coordination of these phosphates as might be expected.
This lack of a clear mechanistic basis for binding of alternative
phosphoinositides to PtdIns3P suggests that cases where other
specificities are reported could merit further scrutiny, and we have
selected two cases to illustrate this point. SNX9 has been reported
to have a functional preference for PtdIns(4,5)P2 in liposomebinding assays and in vitro assays for stimulation of dynamin and
N-WASP [neuronal WASP (Wiskott–Aldrich syndrome protein)]
activities, but it can associate with all other phosphoinositide
lipids, including PtdIns3P [16–21]. This liposome association,
however, requires co-operation of the SNX9 C-terminal BAR
domain, thus clouding the PX domain specificity [18,20]. The
structure of SNX9 was determined by crystallography in
the presence of PtdIns3P (PDB code 2RAK), but notably,
despite soaking with other phosphoinositide lipids, none were
observed to bind the PX domain [18]. Furthermore, additional
biochemical data shows that mutation of the PX domain abrogates
binding of the PX-BAR unit of SNX9 to PtdIns3P- but not
PtdIns(4,5)P2 -containing liposomes [18]. This suggests that the
SNX9 PX domain is specific for PtdIns3P, whereas the binding of
PtdIns(4,5)P2 is mediated primarily by the SNX9 BAR domain.
SNX5, and its related homologues SNX6 and SNX32 have an
unusual PX domain with a large conserved insertion following
the polyproline loop [3]. The structure of the SNX5 PX domain
shows that this insertion forms an extended helix–turn–helix
structure [22] (PDB code 3HPC). The authors used NMR to
map the binding of different phosphoinositides and suggested
that PtdIns(4,5)P2 binds to a site overlapping the approximate
location where PtdIns3P binds other PX domains. However, their
data shows that the affinity is extremely weak (K d > 400 μM). It
also contradicts previous studies from their own group [23] and
perhaps, more importantly, does not explain cell localization data
showing that SNX5 is only membrane-associated in the presence
of SNX1 [24]. As the structure of SNX5 reveals that it lacks both
conserved residues required for phosphoinositide binding and an
identifiable binding pocket for PtdIns(4,5)P2 , the specificity of
SNX5 certainly merits closer scrutiny. In conclusion, although
mammalian PX domains have been reported to have specificities
for phosphoinositides other than PtdIns3P, a detailed structural
explanation is required to understand precisely how they are
associated.
The PX domain as a protein-interaction module
Studies of the PX domain have almost exclusively focused on
its ability to bind phosphoinositides, but there is accumulating
41
evidence that the PX domain can also act as a protein-scaffolding
device. We have surveyed all reported protein–protein interactions
of PX domain-containing molecules, and a number of these occur
directly via the PX domain itself (Supplementary Table S2 at
http://www.BiochemJ.org/bj/441/bj4410039add.htm). Compared
with the detailed molecular understanding we now have of
PtdIns3P binding, how PX domains control protein–protein
interactions remains very poorly understood. One common
feature of PX domains is the presence of a polyproline loop
between helices α1 and α2, which may potentially be involved
in interactions with SH3 (Src homology 3) domains. Indeed
both intramolecular and intermolecular SH3 domain binding has
been observed. An example of the former is the binding of
C-terminal SH3 domains of p47phox to the p47phox PX domain
[25], an interaction that regulates its activation [26]. An example
of an intermolecular association is the binding of the PLCγ 1
(phospholipase Cγ 1) SH3 domain to the polyproline loop of the
PLD1 (phospholipase D1) and PLD2 PX domains, important
in EGF (epidermal growth factor) signalling [27]. Given the
importance of the dynamic remodelling of the polyproline loop on
PtdIns3P binding, this also suggests a possible role for protein–
protein interactions in regulating membrane association [28].
A number of other direct protein–protein interactions involving
PX domains have also been reported. The PX domains of PLD1
and PLD2 have been found to bind to the GTPase domain
of dynamin and are able to act as GAPs (GTPase-activating
proteins) affecting dynamin-mediated endocytosis [29]. PLD1
and PLD2 PX domains are also able to associate with the exocytic
SM (Sec/Munc) protein Munc18-1, and this association potently
inhibits the phospholipase activity of the proteins [30]. The PX
domains of a number of PX proteins, including SNX1, SNX2,
SNX4 and SNX6, can associate with the cytoplasmic tails of
members of the TGF-β (transforming growth factor β) receptor
family [31]. The functional significance of these interactions have
not been well characterized, but overexpression of SNX6 was
found to downregulate TGF-β signalling. The SNX2 PX domain
was also found to bind to the DEAD-box helicase Abstrakt [32],
although again the functional importance of this interaction is
not clear. The vesicle coat protein clathrin can bind to several
PX domains through a novel inverted clathrin box sequence,
including SNX1, SNX2, SNX3 and SNX4 [33]. The SNX6
PX domain can drive association with the oncogene Pim-1,
resulting in SNX6 phosphorylation and nuclear translocation [34],
although once more the role of this nuclear localization remains
a mystery. Intriguingly, the SNX9 PX domain is able to bind to
and stimulate phosphoinositide kinases, implying that a positive
feedback mechanism between phosphoinositide generation and
effector binding may be involved in SNX9-mediated endocytosis
and signalling [16,19]. A final example of PX domain protein
interactions involves the binding of SNX20 to the cytoplasmic
tail of the transmembrane cell-surface protein PSGL-1 (Pselectin glycoprotein ligand 1), discussed below in more detail
[35].
Taken together, the evidence is substantial that PX domains
act not only as lipid recognition modules, but play a key role
in protein–protein interactions, and it will be of major interest
to determine the molecular details of how these interactions
occur. There is an obvious parallel here between the PX domain
and the PTB (phosphotyrosine binding)/PH domains [36]. Like
the PX domain, these are small modules of approximately
120 amino acids that bind to different phosphoinositides
and are found in a number of different endocytic adaptors and
signalling molecules. In addition, they are known to associate
directly with other proteins, typically via short peptide motifs. A
similar understanding of the protein-binding partners of the PX
c The Authors Journal compilation c 2012 Biochemical Society
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Table 1
R. D. Teasdale and B. M. Collins
Subfamilies of mammalian PX proteins on the basis of domain architecture
Subfamily
Member(s)
PX-BAR
SNX1, SNX2, SNX4, SNX5, SNX6, SNX7, SNX8,
SNX30, SNX32
SNX9 (SH3PX1), SNX18, SNX33
SH3-PX-BAR
PX-SH3
PX-SH3-GAP
PX-FERM
PXA-RGS-PX-PXC
PX-serine/threonine kinase
PI3K-PX
PX-PH-PLD
PX-PXB
PX-only
Kinesin-PX
PX-MIT
PX-LRR-IRAS
PX-SNX16
SNX29-PX
PX-SNX34
Notes
Each protein has a C-terminal BAR domain involved in membrane tubulation. Several also have N-terminal
extended sequences, thought to be involved in protein–protein interactions (e.g. SNX1 and SNX2)
These molecules are very similar in architecture to the PX-BAR proteins, except that they have an N-terminal
SH3 domain that binds to polyproline sequences and an intervening low-complexity region containing AP2and clathrin-binding motifs
SH3PXD2A (SH3MD1, FISH), SH3PXD2B, SNX28 SNX28, p47phox and p40phox are components of the NADPH oxidase complex. The PX-SH3 proteins have
various numbers of SH3 domains C-terminal to the PX domain (from 1 to 5)
(NOXO1), p47phox (NCF1), p40phox (NCF4)
SNX26 (TC-GAP), PX-RICS (GC-GAP, p250GAP) The PX-SH3-GAP proteins have very long extended C-terminal domains with no identified structural motifs
SNX17, SNX27, SNX31
The FERM domain has all three modules of a typical FERM domain (F1, F2 and F3), except that the F2 module
is significantly smaller. SNX27 contains an additional N-terminal PDZ domain
SNX13, SNX14, SNX19, SNX25
Each PXA-RGS-PX-PXC protein possesses two N-terminal hydrophobic stretches predicted to form
transmembrane helices. Each PXA-RGS-PX-PXC protein possesses an RGS domain, except for SNX19
PXK, RPK118 (RPS6KC1), SGK3 (CISK)
PXK and RPK118 are predicted to have non-catalytic serine/threonine-kinase domains. RPK118 may also
possess an MIT domain
PI3K-C2α, PI3K-C2β, PI3K-C2γ
These are type II PI3K enzymes with multiple domains, including Ras-binding domains, C2 domains, a PI3K
domain and a PI3KC2 homology domain of unknown structure and function
PLD1, PLD2
These proteins have two separate membrane-recruitment domains (PX and PH) coupled to a PLD domain
SNX20 (SLIC-1), SNX21
SNX20 and SNX21 are highly homologous and share a conserved helical domain (PXB) of ∼ 140 residues
downstream of the PX domain. The PXB domain may be composed of up to three TPR helical repeats
SNX3, SNX10, SNX11, SNX12, SNX22, SNX24,
These proteins have been annotated as having no identified domains outside of the PX domain [3,37]. In all
SNX29, HS1BP3
cases, the proteins possess short extra sequences without predicted secondary structure. The C-terminal tail
of HS1BP3 is a proline-rich region
SNX23 (KIF16B)
SNX23 is a member of the kinesin family of microtubule motors. It includes an N-terminal kinesin motor
domain, a putative forkhead association (FHA) domain and a central coiled-coil dimerization domain
SNX15
SNX15 includes a C-terminal MIT domain
IRAS (nischarin)
IRAS is a large protein ∼ 1504 residues in length. Apart from the N-terminal PX domain, IRAS has a predicted
leucine-rich repeat region from approximately residues 220 to 397, and secondary structure predictions
indicate that the C-terminal region from approximately residue 570 onwards is composed of one or more
structural domains with mixed α/β topologies.
SNX16
SNX16 possesses significant predicted helical structure from between approximately residues 220 and 310
downstream of the PX domain
SNX29
SNX29 possesses significant predicted helical structure between approximately residues 80 and 250 upstream
of the PX domain
SNX34 (C6ORF145)
SNX34 has not been annotated previously and was identified in the present study via bioinformatics searches.
It potentially contains a small C-terminal β-sheet domain between approximately residues 160 and 230
domain will allow us to discern common features governing their
association, will lead to the identification of other interacting
molecules, and will drive the design of specific mutants aimed
at dissecting the functional importance of PX domain protein
recruitment.
THE MAMMALIAN PX PROTEIN FAMILY – AN UPDATE
There are currently 49 known proteins that contain a PX domain
encoded in the human genome. Most of these are modular
proteins containing one or more additional domains with diverse
functions, including membrane remodelling, phosphoinositide
kinase activity, phospholipase activity, protein–protein interaction
and numerous other functions both known and unknown. We have
revisited recent catalogues of the PX domain proteins [3,37–40],
and in Table 1 and Figure 2 we propose a modified classification
based not only on sequence homology or known domains, but
also on the presence of conserved structural elements defined by
careful analysis of secondary-structure predictions. This reveals
that in some cases proteins have been misclassified and in other
cases there is strong evidence for the presence of conserved
domains not previously characterized. The classification of PX
proteins into related structural subfamilies provides an important
basis for considering both the common and distinct functions of
these diverse proteins. Some of these subfamilies contain many
members, such as the PX-BAR and PX-only families, whereas
c The Authors Journal compilation c 2012 Biochemical Society
others contain only a single member. The majority of PX domain
proteins have been named SNXs [3,37]. This terminology was
originally used by Kurten et al. [41], after identification of SNX1,
and later this nomenclature was refined to refer to proteins that
have greater than 50 % sequence similarity to SNX1 in the PX
domain [42,43]. A difficulty with this nomenclature arises from
the fact that the classification of PX proteins as SNXs remains
somewhat arbitrary. As just one example, it is not obvious why
TC-GAP (TC10/cdc42 GAP) should also have the name SNX26,
whereas GC-GAP (GAB/cdc42 GAP) does not have an SNX
moniker. Therefore we propose that current common names for
these proteins remain in place, but that a new SNX name is
assigned for any future molecules identified for the PX family
(e.g. C6ORF145, which we have named SNX34; see below).
We have also produced a curated list of proteins that
are found to associate with PX domain proteins and, where
possible, have defined interacting domains, further highlighting
the functional relationships between members of the same
subfamilies (Supplementary Table S2). In constructing this Table,
we restricted interactions to those that appeared to be direct
as shown by immunoprecipitations or other assays, including
yeast two-hybrid, pull-downs with purified proteins or cocrystal structures. Importantly, these analyses also reveal that
ligands for PX proteins can be assigned to several major
functional groups: transmembrane cargo molecules, regulators
of intracellular membrane trafficking, cellular signalling and
cytoskeletal reorganization/attachment.
The PX protein family
Figure 2
43
Classification and domain organization of human PX proteins
Structural classification of PX proteins was based on known domains, and novel conserved domains were identified by secondary-structure prediction and sequence comparison. Note that the
diagrams are not to scale. Domains with broken outlines indicate a domain that is only found in some members of the subfamily (PDZ domain in SNX27; MIT domain in RPK118; RGS domain
missing in SNX19; TM domains missing in SNX25), or found in variable numbers (SH3 domains of PX-SH3 proteins).
PX-BAR and SH3-PX-BAR subfamilies
The subfamily of PX proteins possessing a C-terminal BAR
domain are perhaps the best characterized of all PX subfamilies.
An excellent review by van Weering et al. [39] highlights
their importance in endocytic and endosomal membrane sorting
processes. Sorting of proteins by PX-BAR molecules is dependent
on the membrane remodelling and sensing properties of the BAR
domain, which drives the formation of membrane tubules and
preferentially associates with curved membrane domains [44,45].
Examples include the formation of endosomal transport tubules
by SNX1 [46] and membrane deformation during clathrin-coated
vesicle formation by SNX9 [18,47]. Therefore the overarching
question about the PX-BAR proteins is this: why are there
so many? Does each protein control the sorting of cargo
molecules within specific membrane domains, are they regulated
by different signals or do they function in different intracellular
compartments? Attempts to answer these questions have borne
significant fruit in the last few years; however, dissecting the
individual roles of PX-BAR proteins is severely complicated by
the requirement that they form homo- or hetero-dimers (a feature
of the BAR domain) and higher-order oligomeric complexes
during membrane tubulation.
It is plain that in some cases different PX-BAR proteins
can have overlapping functions. For example, one of the bestcharacterized roles of the PX-BAR proteins is to co-operate with
the retromer protein complex in endosome-to-TGN (trans-Golgi
network) recycling of a variety of transmembrane cargo proteins.
Retromer is a heterotrimeric assembly composed of the proteins
VPS35 (vacuolar protein sorting-associated protein 35), VPS29
and VPS26 thought to participate in cargo loading into membrane
tubules coated by PX-BAR proteins, including SNX1, SNX2
c The Authors Journal compilation c 2012 Biochemical Society
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R. D. Teasdale and B. M. Collins
SNX5 and SNX6 [40,48,49]. The highly homologous SNX1
and SNX2 have been shown to participate somewhat interchangeably in retrograde endosomal protein trafficking of both the
bacterial toxin StxB (Shiga-like toxin β subunit), where siRNA
(small interfering RNA) knockdown of both proteins results in a
greater defect in protein trafficking than that of either one alone
[50], and the CI-MPR (cation-independent mannose-6-phosphate
receptor) whereby knockdown of both proteins together is
required to observe a defect in retromer membrane tethering
and CI-MPR endosome-to-Golgi retrieval [51]. Each protein has
been shown to both self-associate and form heterodimers with
each other, suggesting that they have very similar biochemical
properties [51–56]. These data are somewhat clouded, however,
by separate reports that SNX2 does not participate in either
trafficking process [46,57,58], whereas other evidence suggests
that SNX1 and SNX2 can also have independent functions, as
shown by their differential roles in down-regulation of activated
EGFR (EGF receptor) or PAR1 (protease-activated receptor 1)
[52,59]. Perhaps the most compelling evidence for functional
overlap is that deletion of both SNX1 and SNX2 in mice results
in a lethal phenotype, strongly implying that the two proteins can
functionally compensate for each other to a certain extent [60].
Other examples of PX-BAR proteins with overlapping
functions include the highly similar SNX5 and SNX6 proteins,
as well as the endocytic SH3 domain-containing proteins SNX9,
SNX18 and SNX33. In the former case, both SNX5 and SNX6
were found to play similar roles in the endosome-to-Golgi
retrieval of the CI-MPR, and both form heteromeric complexes
with the retromer-associated SNX protein SNX1 [24,56,61].
Interestingly, SNX5 has been shown to have a significantly
different membrane recruitment response to EGFR-mediated
signalling, with SNX5 showing both endosomal and plasma
membrane localization in stimulated cells, perhaps regulated by
an affinity for PtdIns(4,5)P2 , whereas SNX1 remains endosomally
bound [22,62]. This is strong evidence that PX-BAR proteins may
have differing functions depending on whether they are partnered
with one particular PX-BAR protein or another.
SNX9 plays a well-established role in clathrin-mediated
endocytosis, in part controlling the membrane remodelling of
the narrow neck region of the forming vesicle, and acting as
a molecular scaffold for assembly and recruitment of essential
vesicle components, such as dynamin, AP2 (adaptor protein
complex 2), clathrin and components of the actin cytoskeleton
(reviewed in [47]). Studies of SNX18 and SNX33 indicate that
they have overlapping, but also distinct, intracellular localizations
with SNX9 [63–65]. However, all bind to dynamin and the
effector WASP, and SNX9 and SNX33 have a similar ability
to affect endocytosis of APP (amyloid precursor protein) [66].
The data are conflicting as to whether these proteins can form
heteromeric assemblies with each other [63–65], but these initial
studies suggest that the three proteins probably have both
overlapping and distinct roles in dynamin-dependent endocytosis
and protein trafficking. The functional interplay between the
different members of the PX-BAR subfamilies is therefore highly
complex, and the task now will be to dissect both the common and
unique properties of each PX-BAR protein and protein assembly.
Ongoing goals will be to determine the repertoires of functional
PX-BAR dimeric complexes, examine which of these possess
inherent membrane associating and tubulating properties, and
how these complexes correlate to the in vivo assemblies based
on criteria such as overlapping tissue distribution and cellular
colocalization.
Studies of the SNX9 PX and BAR domains provide important
details on how dimeric SH3-PX-BAR complexes are assembled
and how their structures contribute to membrane attachment and
c The Authors Journal compilation c 2012 Biochemical Society
remodelling (Figure 1B) [18,67]. An interesting finding was the
presence of a so-called ‘yoke’ domain, which is critical for holding
the PX domain in a fixed orientation with respect to the BAR
domain. Although it is assumed that related PX-BAR proteins
will adopt overall similar tertiary assemblies, the recent structure
of the SNX5 PX domain may have important implications
for the assembly of SNX5 into functional complexes [22].
SNX5 possesses a novel helix–turn–helix insertion following the
polyproline loop that forms an extended structure protruding from
the PX domain. When the SNX5 PX domain is aligned with
SNX9, the helical insertion protrudes in such a way that it will
penetrate significantly into the predicted plane of the membrane
(Figure 1B). This suggests that either the PX domain is oriented
differently in SNX5 compared with SNX9, or that SNX5 may
have different membrane-modulating properties compared with
related SH3-PX-BAR proteins. High-resolution studies of homoand hetero-dimeric PX-BAR complexes will be required for a firm
understanding of their assembly and membrane interaction.
To generate or sense tubular membrane structures, BAR domain
proteins must organize into polymeric arrays, involving lateral
interactions between membrane-associated dimers. Multiscale
structural characterization, such as performed in landmark studies
of F-BAR proteins (where F is the FCH domain) [68], will
hopefully provide a more detailed understanding of the membrane
association and higher-order oligomerization properties of PXBAR proteins. An important question, as outlined previously
[37,39], is how different SNX proteins may control formation
or sorting into alternative membrane transport tubules in vivo. For
example, how exclusive are PX-BAR membrane tubules? Can
mixtures of different PX-BAR proteins exist on the same tubules,
and how do cargo receptors and accessory proteins (for example
the retromer complex) integrate into these membrane domains?
All of this information will ultimately help us to understand how
different homo- and hetero-dimeric combinations of PX-BAR
proteins control protein sorting within the endocytic system.
Other questions of immediate interest include determining the
function of the uncharacterized PX-BAR proteins. For example,
does SNX32 participate in endosomal retrograde transport
similarly to the homologous proteins SNX5 and SNX6? Given the
highly dynamic nature of PX-BAR- and SH3-PX-BAR-derived
membrane structures, another question of prime interest will be
to determine how these proteins associate with other regulatory
molecules and with cargo receptors themselves (Supplementary
Table S2). For example SNX1 was originally identified by its
ability to associate with EGFR and other signalling receptors
[41,54], and with the protein Hrs (hepatocyte growth factorregulated tyrosine kinase substrate) [69]. Subsequent studies
have also suggested direct interactions between SNX1 and
GPCRs (G-protein-coupled receptors) [59,70]. Perhaps the bestcharacterized interaction of SNX1 is with the retromer complex
[51–53,71], but even in this case the mechanism of association is
very poorly understood. One of the most exciting discoveries has
been the identification of interactions between endosomal PXBAR proteins and components of the cytoskeleton [39,56,72].
These findings demonstrate the importance of dynein-mediated
microtubule translocation for PX-BAR trafficking processes
and have opened an important area of investigation for these
molecules.
PX-FERM subfamily
The PX-FERM molecules serve as a prime example of the
important endosomal scaffolding and trafficking functions of
many PX proteins. Recent work from our laboratory has shown
The PX protein family
Figure 3 A network of molecular interactions regulated by the PX-FERM
proteins in endosomal membrane transport and signalling
The PX domain binds to PtdIns3P with high specificity, and the similarity of the F3 module
of the FERM domain suggests that it will be responsible for binding cargo receptors via NPxY
sequences. The proteins can bind Ras-GTP, and this is proposed to be via the F1 module similar
to structurally related Ras-association domains. SNX17 has been reported to use its polyproline
sequence within the PX domain to interact with a variety of SH3 domain proteins, including
Src-related kinases and PLCγ 1. SNX27 possesses an additional N-terminal PDZ domain that
binds cytosolic and transmembrane proteins via C-terminal type I PDZbms, ExE[S/T]x[F/V].
β2-AR, β 2 adrenergic receptor; CNKRS2, connector enhancer of kinase suppressor of Ras2;
FEEL1, fasciclin, EGF-like, laminin-type EGF-like and link domain-containing scavenger
receptor 1 (also called stabilin-1); Grb2, growth-factor-receptor-bound protein 2; MAP1A,
microtubule-associated protein 1A; Nck1, non-catalytic region of tyrosine kinase adaptor protein
1; PTCH1, protein patched homologue 1; PTPN2, protein tyrosine phosphatase non-receptor
type 2; RhoGDI, Rho GDP-dissociation inhibitor; VLDLR, very-low-density lipoprotein receptor;
vl-GPCR, very large GPCR.
that three proteins, SNX17, SNX31 and SNX27, constitute a
conserved subfamily containing a C-terminal FERM domain [11].
In addition SNX27 possesses an N-terminal PDZ (postsynaptic
density 95/discs large/zonula occludens) domain. FERM domains
are approximately 300 amino acids in length and are found in
numerous signalling and scaffolding proteins, where they serve
a role in membrane tethering and/or binding to the cytosolic
domains of transmembrane proteins [73]. The FERM domain
is composed of three subdomains or modules named F1, F2 and
F3. The F1 subdomain possesses a ubiquitin-like fold and the
C-terminal F3 subdomain possesses a similar structure to the PH
and PTB domains. The F1 and F3 modules are oriented with
relation to each other by the central F2 subdomain composed
of four α-helices. The sequence homology of the PX-associated
FERM domains with the canonical FERM domain is low, and the
central F2 subdomain is significantly smaller than normal [11].
The PX domains are essential for endosomal localization via
PtdIns3P binding [11,74–80], and the FERM domains of these
proteins regulate endosomal cargo interactions and, possibly,
scaffolding of signalling complexes [11] (Figure 3).
45
SNX17 and SNX31 are the most closely related within this
subfamily. There are currently no published data on the function
of SNX31, but several groups have determined that SNX17 is
endosome-associated and controls intracellular trafficking of a
number of transmembrane cargo proteins. Proteins whose cellular
localization is regulated by SNX17 include LRP1 [LDLR (lowdensity lipoprotein receptor)-related protein 1] [76,81–83], LDLR
[83,84], P-selectin [75,85,86] and APP [87]. Overexpression of
SNX17 enhances LDLR endocytosis [83], whereas expression
of a truncated SNX17 molecule causes LDLR to be mislocalized
[84]. It was subsequently found that SNX17 regulates the
recycling of LDLR from endosomes to the cell surface in nonpolarized cells [76] and from basolateral sorting endosomes to
the basolateral plasma membrane in polarized MDCK (Madin–
Darby canine kidney) cells [82]. The role of SNX17 in trafficking
of LDLR and related lipoprotein receptors suggests a potentially
central function in lipid metabolism, and it will be important
to determine whether this is indeed the case. SNX17 was also
found to promote cellular uptake of the endothelial receptor
P-selectin and, additionally, it colocalizes with P-selectin on
endosomal organelles where it is able to inhibit its transport to
late endosomes/lysosomes for degradation [75]. Finally, SNX17
is expressed in neurons, associates with APP on endosomes
and inhibits its proteolytic processing to the toxic amyloid Aβ
peptide [87]. The role of PX proteins in inflammation and AD
(Alzheimer’s disease) is discussed further below.
In addition to transmembrane cargo proteins, SNX17 has
been found to associate with the cytosolic protein Krit1 (Krevinteraction trapped 1), which is able to enhance recruitment of
SNX17 to endosomes [74]. SNX17 binds receptors and effectors
via short peptide motifs with the consensus Asn-Pro-Xaa-Tyr
(NPxY) via its C-terminal FERM domain. In addition to these
NPxY-motif interactions, a proteomics study has also identified
SNX17 as a ligand for various SH3 domain-containing proteins,
including a number of Src-related tyrosine kinases and PLCγ 1
[88]. In these cases, the interaction involves the SH3 domain
binding directly to the SNX17 PX domain polyproline loop.
The determination that SNX27 shares structural similarity with
SNX17 and SNX31 led to the demonstration that it also possesses
NPxY-motif binding affinity, although it has yet to be confirmed
that SNX27 is able to direct endosomal sorting of NPxY cargo
proteins [11]. SNX27 was annotated as possessing a C-terminal
RA (Ras-association) or B41 domain [3,37,43]. However, it is
now clear that it possesses a similar FERM C-terminal domain
to SNX17 and SNX31. This confusion is due to the fact that
both RA and B41 domains share a ubiquitin-like fold, as does the
F1 module of FERM proteins. Nonetheless, as mentioned above,
the F1 module of FERM domains shares structural similarity
with the RA domain, and this prompted an investigation of the
Ras-binding properties of the PX-FERM proteins [11]. These
preliminary experiments show that PX-FERM proteins are able
to bind the activated H-Ras GTPase, and suggest a potential
link between endosomal Ras-mediated signalling and membrane
trafficking that requires further investigation [89]. A hint that the
PX-FERM proteins can indeed affect Ras-mediated signalling
comes from recent studies showing that SNX27 knockdown by
siRNA results in enhanced ERK (extracellular-signal-regulated
kinase) phosphorylation [79]. However, the actual Ras isoform(s)
targeted by the PX-FERM proteins in vivo remains unknown.
SNX27 is unique amongst the PX-FERM proteins in that it
also contains a PDZ domain upstream of the PX domain. Like
SNX17, SNX27 is highly expressed in brain tissue, and was in
fact first identified as a molecule up-regulated upon stimulation of
dopamine receptors with methamphetamines [90–92]. The PDZ
domain of SNX27 has been implicated in binding to a number
c The Authors Journal compilation c 2012 Biochemical Society
46
R. D. Teasdale and B. M. Collins
of proteins via a C-terminal type I PDZbm (PDZ-binding motif)
(E[S/T]x[V/F]), and structural studies of the SNX27 PDZ domain
show that the preferred motif is enhanced by the presence of
an upstream glutamic acid side chain (ExE[S/T]xV/F]) [90].
The first identified binding partner for SNX27 was 5-HT4 R
(5-hydroxytryptamine type 4 receptor), a GPCR involved in feeding and respiratory control [77]. SNX27 was found to be essential
for transport of 5-HT4 R to EEA1-positive early endosomes.
DGKζ (diacylglycerolkinase ζ ) was identified as a SNX27 binding partner by proteomics and can regulate SNX27 association
with early and recycling endosomes [80]. Other proteins found
to associate with the SNX27 PDZ domain are CASP (cytohesinassociated scaffolding protein), a cellular adaptor protein found
in cells of haemopoietic origin [93,94], Kir3, G-protein-gated inwardly rectifying potassium channels that control a slow
inhibitory postsynaptic response in neurons [78,90,95], and most
recently the NR2C [NMDA (N-methyl-D-aspartate) receptor 2C]
ligand-gated ion channel [96]. SNX27 and Kir3 channels show
colocalization in endosomes, and overexpression of SNX27
results in reduced Kir3.3 expression at the cell surface, suggesting
that it either promotes Kir3.3 endocytosis or inhibits its recycling.
Finally, a recent paper by Lauffer et al. [97] demonstrated
that SNX27 can bind to β2AR (β 2 -adrenergic receptor) via its
C-terminal PDZbm and regulate its endosomal recycling to
the cell surface, perhaps analogously to the recycling role
demonstrated for SNX17.
The exact function of the PX-FERM molecules in protein
trafficking remains unclear and their mechanisms of action are
unknown. Do they form a protein coat regulating membrane
transport, do they act as adaptors for other membrane scaffolding
molecules or do they play a facilitating role by concentrating transport cargoes into membrane domains destined for recycling to
the cell surface? The first clue to the function of these proteins
emerged with the identification of SNX27 as a binding partner
for the WASH [WASP and SCAR (suppressor of cAMP receptor)
homologue] complex [98]. The WASH complex is an endosomeassociated assembly that induces actin polymerization in an
Arp2/3 complex (actin-related protein 2/3 complex)-dependent
manner, and has an important function in endosomal trafficking
and membrane dynamics (reviewed in [99]). Interestingly, WASH
is believed to co-ordinate endosomal sorting in association with
the retromer complex [100,101]. Knockdown of retromer perturbs
cell-surface recycling of the β 1 - and β 2 -adrenergic receptors
similarly to SNX27, and SNX27 was found to be associated with
retromer after cross-linking [98]. The authors propose that SNX27
can act as a cargo-specific adaptor for endosomal sorting of
PDZbm-containing receptors within retromer-coated membrane
tubules, and also propose a role for retromer and SNX27 in
rapid cell-surface recycling via a Rab4-dependent pathway, which
contrasts significantly with the canonical role of retromer in
endosome–TGN retrograde transport. This specific process is
unique to SNX27 as it only operates on PDZbm-containing
proteins, so it remains to be seen whether PX-FERM molecules
also co-ordinate endosomal trafficking of NPxY cargo, such as
APP and LDLR family members, with WASH and retromer.
SNX14 and SNX25. Two other domains (each approximately
150 amino acids) have previously been annotated as the PXA
domain (PX-associated domain A) lying N-terminal to the RGS
and canonical PX domains, and the C-terminal PX-associated
domain lying downstream of the PX domain [3,37]. For clarity,
we refer to the N- and C-terminal domains as the PXA and PXC
(PX-associated domain C) domains respectively, to denote that
they are as yet uncharacterized PX-associated domains. Finally,
each member of the family possesses two putative N-terminal
transmembrane helices resulting in a predicted topology of a
short cytoplasmic leader, two closely spaced transmembrane
domains, and a long C-terminal structure containing the three
(SNX19; PXA-PX-PXC) or four (SNX13, SNX14 and SNX25;
PXA-RGS-PX-PXC) modular domains (Figure 4B). Currently,
SNX25 is predominantly annotated as lacking transmembrane
sequences, but our analyses of the rat, mouse and human genomes
identified a conserved N-terminal coding sequence that is clearly
transcribed and encodes two potential transmembrane helices.
There are only a small number of reports that address the
function of the PXA-RGS-PX-PXC proteins. These describe
the role of SNX13 (also called RGS-PX1) as a GAP and
regulator of the trimeric G-protein subunit Gα s via its RGS
domain [102–104], and of SNX25 as a modulator of TGF-β
signalling [105]. The former studies confirmed SNX13 as an
important member of the family of RGS proteins, a family
with diverse roles in modulating GPCR-mediated signalling
[106,107]. Initial work identified SNX13 after database searches
for the putative GAP for Gα s , which at the time had not been
identified. SNX13 was subsequently cloned and shown to both
bind to and promote GTPase activity of Gα s with high specificity
and attenuate Gα s -mediated signalling [103]. Endosomes are
increasingly being recognized as critical sites of signal regulation
[1,2,108], and the affinity of SNX13 for PtdIns3P and localization to endosomes suggests a potential role in GPCR signal
attenuation in this compartment. SNX13 was subsequently shown
to form a heteromeric complex with both Gα s and Hrs on
endosomes and co-operate in the lysosomal targeting of the
EGFR, suggesting a role both in signalling and trafficking [102].
A model for SNX13 function in endosomal signal attenuation
is presented in Figure 4(B). SNX13-null mice show embryonic
lethality, and yolk-sac endoderm cells of these embryos display
highly disrupted endosome morphology, demonstrating that it
plays a role in endosomal function that is critical for normal
development and that other PXA-RGS-PX-PXC proteins are
unable to compensate for its loss [104]. SNX25 was identified
through bioinformatics searches for novel PX domain proteins and
is proposed to modulate TGF-β signalling via endosomal sorting
of TGF-β receptors for lysosomal degradation [105]. Another
study has implicated SNX19 in chondrogenic regeneration in
cartilage, although the mechanism for this function is not proposed
[109]. A final interesting finding is that the Drosophila protein
snazarus (sorting nexin lazarus), for which SNX25 is the closest
homologue, plays a role in regulating longevity, as snazarus
mutants display significantly increased lifespans [110].
PX-only and PX proteins with putative novel domains
PXA-RGS-PX-PXC subfamily
Four proteins, SNX13, SNX14, SNX19 and SNX25, display a
unique architecture consisting of a central PX domain flanked
by several conserved domains (Figure 4A). The first domain
identified was the RGS (regulator of G-protein signalling)
domain, found in a number of molecules that attenuate GPCR and
related G-protein signalling. This domain is seen only in SNX13,
c The Authors Journal compilation c 2012 Biochemical Society
The subfamily of proteins that have been annotated as PX-only
proteins is a relatively poorly characterized group of molecules
[3,37]. As the name suggests, bioinformatics analyses do not
detect any conserved domains outside the defining PX domain.
Structurally, however, these proteins are of various lengths and
typically contain long extended sequences with no predicted
secondary structure. In several cases, such sequences have
The PX protein family
Figure 4
47
Architecture and interactions of PXA-RGS-PX-PXC proteins
(A) Schematic diagrams of the human PXA-RGS-PX-PXC proteins SNX13, SNX14, SNX19 and SNX25. Transmembrane (TM), N-terminal PXA, RGS, PX and C-terminal PXC domains are indicated
with shaded boxes. Proteins and domains are drawn to scale. (B) Model for SNX13 function in GPCR signalling [103]. SNX13 is predicted to possess two transmembrane domains. After activation
of a GPCR by ligand binding, GDP is displaced and exchanged for GTP in the Gα s subunit of the trimeric G-protein, releasing the Gβ and Gγ subunits and resulting in signal transduction.
The RGS domain of SNX13 binds to Gα s and SNX13 acts as a GAP to stimulate GTPase activity. The G-protein complex can then reform and signal transduction is ablated. Because SNX13
has a PtdIns3P -binding PX domain and is localized to endosomes, it probably functions in signal attenuation in this intracellular compartment and also interacts with Hrs, a component of the
ubiquitin-mediated protein sorting and degradation pathway in late endosomes.
been found to be critical for function [111,112]. Of the PXonly proteins, SNX3 is perhaps the best characterized. Like
the majority of PX proteins, SNX3 binds to PtdIns3P via
its PX domain and this interaction drives its association with
the limiting membrane of early endosomes [113] and MVBs
(multivesicular bodies) [114]. Overexpression of SNX3 was
found to alter endosome morphology [113], and the protein is
essential for the formation of the intralumenal vesicles of MVBs
[114]. Ubiquitylation is a critical feature of protein sorting into
MVBs, and SNX3 itself has been found to be ubiquitylated,
with its stability being regulated by the de-ubiquitylating enzyme
Usp10 [115]. SNX3 has also been linked to neurite outgrowth,
and its expression is induced by lithium treatment [116]. The
yeast homologue Grd19p/Snx3p appears to co-operate with the
retromer coat complex in endosome-to-Golgi retrieval [117–119],
and elegant studies have recently shown that it also co-operates
with retromer in endosome-to-Golgi recycling of the Wnt receptor
Wntless in Caenorhabditis elegans, Drosophila melanogaster and
mammalian cells, indicating an evolutionarily conserved function
in retromer-mediated membrane trafficking [120].
HS1BP3 (HS1-binding protein 3) is a unique PX domain
protein identified by proteomic screening as a binder of the
HS1 protein, which in turn is a substrate of the Lck tyrosine kinase
involved in T-cell differentiation [121]. HS1BP3 has an extended
C-terminal region rich in proline residues that is bound by the
HS1 SH3 domain. Expression of a truncated HS1BP3 construct
resulted in reduced IL-2 (interleukin-2) production, suggesting
a role in T-cell-receptor-mediated signalling, and genetic studies
also indicate that a variant (A265G) is associated with familial
essential tremor [122–124]. The only other PX-only protein that
has been characterized to any extent is SNX10, which induces
the formation of giant vacuoles when overexpressed [111]. This
vacuolization is sensitive to disruption of the Golgi, suggesting
that SNX10 may in some way enhance the fusion of Golgi-derived
vesicles with endosomes.
In addition to the PX-only proteins, several PX molecules
possess regions of predicted secondary structure that almost
certainly form folded domains of unknown function. SNX20
and SNX21 are a prime example. These proteins are highly
homologous and share a conserved domain of ∼ 140 residues containing six α-helices downstream of the PX domain on the basis
of secondary-structure predictions (Supplementary Figure S2
at http://www.BiochemJ.org/bj/441/bj4410039add.htm). We propose that this be named the PXB domain (PX-associated domain
B), continuing the nomenclature adopted for the PXA-RGSPX-PXC proteins. Results from the TPRpred server suggest
that this region may contain up to three TPRs (tetratricopeptide
repeats), α–α-hairpin structures that form helical solenoids [125].
SNX16 possesses a predicted helical structure between residues
∼ 220 and 310 C-terminal to the PX domain. SNX29 has
significant predicted helical structure between residues ∼ 80 and
250 upstream of the PX domain. Therefore technically, SNX16,
SNX29, SNX20 and SNX21 proteins should not be classified
as ‘PX-only’ molecules. During bioinformatic searches we also
identified the novel open reading frame C6ORF145, which we
have designated SNX34. SNX34 is 231 residues in length and
secondary-structure predictions indicate the presence of a small
domain composed of β-strands at the C-terminus (residues 160–
225).
SNX16 associates with endosomes via PtdIns3P binding to
its PX domain, and the helical C-terminal domain appears to be
important for self-association, possibly by forming a coiled-coil
[126,127]. This domain regulates SNX16 distribution between
early and late endosomes and is important for processing of
c The Authors Journal compilation c 2012 Biochemical Society
48
R. D. Teasdale and B. M. Collins
cargo molecules, such as EGFR, into the late compartments.
Interestingly, SNX16 was recently found to be a PtdIns3P
effector that is able to inhibit the export of viral RNA into
the cytoplasm from late endosomes, although the mechanism of
function is unknown [128]. A recent study of D. melanogaster
SNX16 identified an interaction with the WASP-activating F-BAR
molecule Nwk on recycling endosomes, and highlighted a role for
this interaction in down-regulating BMP (bone morphogenetic
protein) and Wg (Wingless) synaptic growth signalling pathways
in nerve terminals [129].
One final PX protein of interest with novel structural domains
is IRAS (imidazoline receptor antisera-selected), also called
nischarin in mice. IRAS/nischarin is a large protein of 1504 amino
acids. Apart from the N-terminal PX domain, IRAS/nischarin also
has a predicted leucine-rich repeat region (residues ∼ 220–397).
Leucine-rich repeats display a characteristic three-dimensional
structure composed of repeating β-strands and α-helices, and
often participate in protein–protein interactions [130]. In addition,
secondary-structure predictions indicate that the C-terminal
region of IRAS/nischarin (from residue ∼ 570 onwards) is
composed of one or more structural domains with mixed
α/β topologies. IRAS/nischarin was originally cloned as a
putative imidazoline receptor, although it is probably not a bona
fide receptor [131], and is localized to endosomes in HEK
(human embryonic kidney)-293 cells via its PX domain [132].
It was subsequently found to bind to IRSs (insulin receptor
substrates) and enhanced IRS-dependent insulin stimulation of
ERK [133]. A number of other papers have centred on the
ability of IRAS/nischarin to bind to α5 integrins [132,134–138].
IRAS/nischarin binds to a short peptide in the cytoplasmic tail of
α5 integrin via a C-terminal region (residues ∼ 710–810) [132].
It also regulates Rac-induced cell migration and Rac/PAK (p21activated kinase) signalling [134,135,137,138], although these
data must be viewed with some degree of caution as all of
these studies employed a mouse version of IRAS/nischarin that
lacks the N-terminal PX domain [132,135], apparently due to
a truncation in the original IRAS/nischarin clone. The current
mouse IRAS/nischarin entry in the NCBI database (accession
number NM_022656) has the PX domain and is homologous
with the full-length human gene. It is possible, therefore, that
the previous studies of IRAS/nischarin function in Rac signalling
employed a dominant-negative version of the protein.
Other unique PX proteins
SNX15, which includes a C-terminal MIT domain (microtubuleinteracting and trafficking molecule domain), was first identified
via database searches for SNX1 homologues, and despite the
lack of a C-terminal BAR domain for dimerization was found
to associate with SNX1, SNX2 and SNX4 [139]. It was
also found to associate with the receptor for PDGF (plateletderived growth factor), and interestingly all of these identified
interactions were dependent on the PX domain itself. SNX15
is localized to endosomes and overexpression of the protein
has numerous phenotypic consequences, including disrupted
endosomal morphology, reduced processing of several growth
factor receptors and inhibited trafficking of several proteins such
as furin, TGN38 and transferrin [139,140]. Although its exact
function is not clear, it appears that SNX15 plays an important
role in endosomal transport processes. MIT domains are found
in a number of endosomal proteins, including Vps4 {an ATPase
that controls the disassembly of the ESCRT (endosomal sorting
complex required for transport) complex [141]}, spastin and
another PX protein, the serine/threonine-pseudokinase RPK118
c The Authors Journal compilation c 2012 Biochemical Society
[142]. The structure of the MIT domain reveals a small three-helix
bundle, and its function appears to be to act as a protein–protein
interaction module, specifically binding small MIT-interaction
motifs [143,144]. The exact role of the SNX15 MIT domain,
however, is unknown.
KIF16B (kinesin-family protein 16B, also known as SNX23)
is a member of the kinesin superfamily of proteins [145]. It
contains kinesin motor, FHA (forkhead association) and coiledcoil dimerization domains. KIFs are microtubule-binding motors
with numerous roles in the transport of organelles, protein
complexes and RNA [146,147]. KIF16B is a plus-end-directed
microtubule motor that associates with PtdIns3P-containing
endosomes via its C-terminal PX domain and regulates the
localization of endosomal organelles in the cell as well as receptor
recycling within the endosomal system [145,148].
EVOLUTIONARY INSIGHTS FROM THE YEAST PX PROTEOME
The conservation of PX proteins in yeast and other simple
eukaryotes demonstrates a deep evolutionary heritage in eukaryotes, in line with their critical function in membrane trafficking,
organization and cell signalling. Several interesting observations
are made when the human PX proteome is compared with that
of yeast. First, it is clear that higher eukaryotes have evolved an
arsenal of PX domain proteins more diverse than those of their
single-celled cousins. Secondly, it is seen that not all subfamilies
of PX proteins are found in all eukaryotes. There are 15 known
PX domain proteins in yeast, divided into several subfamilies that
generally reflect those found in humans (Supplementary Table
S3 at http://www.BiochemJ.org/bj/441/bj4410039add.htm). The
most populous subfamily consists of the PX-BAR molecules
(seven in yeast compared with nine in humans). Homologues
are also seen for the PX-PH-PLD (Pld1p), PXA-RGS-PXPXC (Mdm1p) and PX-only subfamilies (Snx3p and Ypt35p).
However, in yeast each of these families has only one or two
members. Yeast does have a PX domain-containing GAP, Bem3p;
however, this molecule has a different architecture to the PX-SH3GAP proteins found in humans, replacing the central SH3 domain
with a membrane-binding PH domain.
Many PX subfamilies found in humans are not identified in
yeast, including the PX-FERM, PX-S/T-kinase (serine/threonine
kinase) and SH3-PX-BAR proteins; conversely, yeast has three
PX proteins not identified in humans. The SNARE (soluble
N-ethylmaleimide-sensitive factor-attachment protein receptor)
protein Vam7p has, in addition to the canonical PX domain,
C-terminal t-SNARE (target SNARE) motifs for forming SNARE
complexes with Vam3p and Vti1p during vacuolar membrane
fusion. The SH3-PX-CAD (caspase-activated DNase) protein
Bem1p has two SH3 domains N-terminal to the PX domain and
a C-terminal CAD/PB1 domain, which belongs to the ubiquitinfold superfamily. The poorly characterized Ypr097Wp is a large
protein and appears to contain a highly divergent PX domain on
the basis of sequence and secondary-structure predictions (see
[9]). Outside the PX domain, it is predicted to be primarily
α-helical in structure, although no other known domains can
be identified. Overall, it appears that eukaryotic organisms have
employed both overlapping and distinct PX protein families
throughout evolution.
PX PROTEINS AND DISEASE
There are now many reported links between PX proteins and
various disease processes (Table 2). Currently, there is only one
known case where a specific PX protein mutation is known to be
The PX protein family
Table 2
49
PX proteins implicated in disease
Family
PX protein
Disease
References
PX-BAR
SNX1
STx uptake
Salmonella uptake
Down-regulated in ovarian cancer
Gefitinib-sensitive non-small lung cancer
Down-regulated in colon cancer
Aβ production (AD)
STx uptake
Salmonella uptake
Epilepsy
–
Uptake of Ebola virus via macropinocytosis
Aβ production (AD)
–
STx uptake
–
–
EPEC (enteropathic Escherichia coli ) infection
Aβ production (AD)
–
Prion disease mediated by PrPc (normal cellular prion protein)
Aβ production (AD)
Aβ production (AD)
Tumour metastasis
–
–
–
Chronic granulomatous disease
–
–
Aβ production (AD)
–
–
–
–
Candidate gene involved in Jacobsen syndrome
May be a chondrogenic factor in osteoarthritis
Genetic risk factor in myocardial infarction
Transcriptionally up-regulated in thyroid oncocytic tumours
Potential oncogene in myeloid leukaemia
Homozygous deletion of 3 exons found in B-cell non-Hodgkin’s lymphoma cell lines
Systemic lupus erythematosus
Akt-independent cancer cell survival
Up-regulated and promotes cell survival in oestrogen-receptor-positive breast cancer
–
Prostate cancer risk
Up-regulated in some glioblastoma brain tumours
–
Aβ production (AD)
Up-regulated in endometrial cancer cells
Invasion by metastatic breast cancer cells
Colon cancer
Aβ production (AD)
Invasion by metastatic breast cancer cells
Metastatic mammary adenocarcinoma
Lymphoma cell proliferation
Colon cancer
–
–
Salmonella uptake
An effector of lithium treatment for bipolar disorder, and a regulator of neurite outgrowth
Gene disrupted in MMEP
–
–
–
–
Oestrogen-regulated expression in breast cancer cell lines
Familial essential tremor
–
[50,57,58,221]
[212]
[224]
[225]
[166,167]
[186]
[50]
[213]
[156]
SNX2
SH3-PX-BAR
SNX4
SNX5
SNX6
SNX7
SNX8
SNX30
SNX32
SNX9
SNX18
SNX33
PX-SH3
PX-SH3-GAP
PX-FERM
PXA-RGS-PX-PXC
PX-S/T kinase
PI3K-PX
PX-PH-PLD
SH3PXD2A (SH3MD1, FISH, Tks5)
SH3PXD2B (Tks4)
SNX28 (NOXO1, p41NOX, SH3PXD5)
p40phox (NCF4, SH3PXD4)
p47phox (NCF1, SH3PXD1A)
SNX26 (TC-GAP)
PX-RICS (GC-GAP, p250GAP, p200GAP, Grit)
SNX17
SNX27
SNX31
SNX13 (RGSPX1)
SNX14
SNX19
SNX25
PXK (MONaKA)
RPS6KC1 (RPK118, humS6PKh1)
SGK3 (CISK, SGKL)
PIK3C2A (PI3K-C2α)
PIK3C2B (PI3K-C2β)
PIK3C2G (PI3K-C2γ )
PLD1
PLD2
PX-PXB
PX-only
SNX20
SNX21
SNX3
Kinesin-PX
SNX10
SNX11
SNX12
SNX22
SNX24
HS1BP3
KIF16B (SNX23)
[223]
[190]
[222]
[226,227]
[66]
[66,228]
[66]
[199,200]
[163,164]
[149–151]
[87]
[229]
[109]
[230]
[231]
[232]
[233]
[155]
[168]
[169]
[165]
[234,235]
[202–206]
[236]
[237–240]
[241]
[207]
[240,242]
[162]
[243]
[244,245]
[213]
[112,116]
[153,154]
[246]
[122–124]
c The Authors Journal compilation c 2012 Biochemical Society
50
Table 2
R. D. Teasdale and B. M. Collins
Continued
Family
PX protein
Disease
References
PX-MIT
SNX15
[162]
[213]
PX-LRR-IRAS
PX-SNX16
IRAS (nischarin)
SNX16
SNX29-PX
PX-SNX34
SNX29
SNX34 (C6ORF145)
Metastatic mammary adenocarcinoma
Salmonella uptake
–
VSV infection
Differential expression used as a biomarker in bladder cancer
Undergoes alternative splicing in certain melanoma cell lines
–
–
the causative agent of a disorder: mutation of the p47phox subunit
of the NADPH oxidase in CGD (chronic granulomatous disease)
[149–151]. Patients with CGD have a primary failure in neutrophil
function and do not mount the normal NADPH oxidase-mediated
respiratory burst required for phagocytosis. In particular, it is
known that the R42Q mutation is implicated in CGD and that this
mutation disrupts the phosphoinositide-binding site and membrane coupling of the protein [152]. Another PX protein, SNX3,
was found to be disrupted in a patient with MMEP (microcephaly,
microphthalmia, ectrodactyly and prognathism) [153]; however,
subsequent studies were unable to detect coding mutations in
SNX3 in MMEP patients [154], so it remains to be determined
whether SNX3 is directly involved in this family of diseases.
Other PX molecules have been implicated in disease on the
basis of differential transcription or protein expression. PXK (PX
domain-containing serine/threonine-kinase) has been linked to
SLE (systemic lupus erythematosus) by genome-wide association
studies [155]. SNX19 was found to be up-regulated in cartilage
cells during progression of osteoarthritis, and studies in a model
chondrogenic cell line implicated it as a protective factor against
cartilage degradation [109]. SNX2 was also isolated in a screen
for genes with potential involvement in epilepsy, but it remains to
be confirmed that SNX2 is important for the disease [156].
Several reviews highlight the critical role of dysregulation of
phosphoinositide homoeostasis in many different disorders [157–
160]. Enzymes that regulate phosphoinositide generation and
breakdown (kinases and phosphatases) are found to be mutated in
a number of diseases. Of particular relevance to the PX proteins
are disorders relating to enzymes regulating PtdIns3P synthesis
at the early endosome, PtdIns(3,5)P2 generation from PtdIns3P at
late endosomes and their respective levels during endosomal
maturation (reviewed in [160]). These include disorders such
as the family of Charcot–Marie–Tooth neuropathies caused by
mutations in the 5-phosphatases (Sac3/Fig4 and myotubularins)
regulating conversion of PtdIns(3,5)P2 into PtdIns3P, and corneal
fleck dystrophy caused by mutations in the PIKfyve kinase that
converts PtdIns3P into PtdIns(3,5)P2 . Further evidence for the
importance of PtdIns3P levels comes from studies highlighting
the role of PIKfyve in Salmonella uptake into macropinosomes
and progression of the pathogen through the endosomal system
[161]. Although the underlying mechanisms that lead to these
different pathologies are still not clear, the PX domain proteins
are extremely strong candidates for molecules whose function
will be perturbed by defects in PtdIns3P metabolism.
PX proteins in cancer
Many PX proteins have been linked to cancers on the basis
of differential expression and other criteria (Table 2). Both
the unique PX protein SNX15 and the PX-PH-PLD family
c The Authors Journal compilation c 2012 Biochemical Society
[128]
[247]
[248]
member PLD2 are up-regulated in metastatic mammary tumour
cells [162]. SH3PXD2A (SH3 and PX domain protein 2A) is
essential for the formation of podosomes, actin-rich structures
required for invasion of many types of cancers. It has been shown
that SH3PXD2A is required for podosome formation and invasion
of several cancer cell lines in a PX domain-dependent manner
[163], and that inhibiting its expression correlates with reduced
tumour growth in mouse transplantation models [164]. It is
hypothesized that SH3PXD2A may act to regulate the recruitment
of essential factors, such as N-WASP, to podosomes via its SH3
domains. Single-nucleotide polymorphisms in the gene encoding
PI3K (phosphoinositide 3-kinase)-C2β (PI3KC2β) have been
associated with increased risk of prostate cancer, suggested to
be due to either enhanced cell migration or increased insulin
signalling [165]. There is strong evidence for a role for SNX1
as a tumour suppressor. SNX1 expression assessed by both
immunohistochemistry and microarray data indicates that SNX1
is reduced in colon carcinoma cells, and, furthermore, inhibition
of SNX1 expression by shRNA (small hairpin RNA) leads
to increased cell proliferation and decreased apoptosis [166].
The authors provide evidence that SNX1 depletion leads to
increased EGFR phosphorylation and subsequent ERK signalling
from endosomes, and that this may be important for tumour
progression. Further support for a role for SNX1 depletion
in colorectal tumour progression comes from a study showing
that a microRNA up-regulated in colon carcinomas, miR-95,
specifically targets SNX1 [167]. This model fits with the original
identification of the SNX1 protein as a molecule that can
enhance the degradation of the EGFR [41], but it does not fit
with subsequent knockdown data indicating that depletion of
SNX1 by siRNA has little effect on EGFR degradation [46]
and therefore will need further investigation before it can be
confirmed.
Two other families of PX proteins have been implicated
in cancer due to their function as cell signalling modulators:
the serine/threonine kinase SGK3 (serum- and glucocorticoidinduced protein kinase 3) and the phospholipases PLD1 and
PLD2. Vasudevan et al. [168] established that oncogenic
mutations in the PI3K subunit PIK3CA can result in cancers
that are either dependent or independent of up-regulation in
Akt signalling. In Akt-independent cancer cells, the key factor
promoting cell survival is increased activation of SGK3 by
the concerted signalling of PI3K and PDK1 (phosphoinositidedependent kinase 1). Furthermore, studies have found that
SGK3 is up-regulated in ER (oestrogen receptor)-positive breast
tumours, is required for oestrogen-mediated cell survival and
can protect cells against apoptosis induced by chemotherapeutics
[169]. Few substrates for SGK3 have been identified; however,
SGK3 can attenuate the endosomal activity of ubiquitin ligases
and thus impair lysosomal degradation of signalling molecules,
The PX protein family
such as the chemokine receptor CXCR4 involved in breast cancer
[170]. Enhanced signalling due to inhibited receptor degradation
is one possible mechanism by which SGK3 could promote cell
survival. Perhaps the best-characterized PX proteins in terms of
oncogenic potential are PLD1 and PLD2. There is not scope in
the present paper to review all of the evidence for the role of PLD
isoforms in cancer, so instead we refer to several previous reviews
[171–173]. Very briefly, it appears that their importance in cancer
is due to their role in the synthesis of PA (phosphatidic acid) from
PC (phosphatidylcholine) and the effect of PA on downstream
signalling, anti-apoptotic and membrane-remodelling processes.
Importantly, PLD isoforms are now validated targets for the
development of small-molecule inhibitors as chemotherapeutics
[173–175].
PX proteins in inflammation
An interesting example of how endosomal trafficking of receptors
by PX proteins can affect disease-related processes is how the
PX proteins SNX17 and SNX20 play complementary roles
in haemopoietic cells during the inflammatory response. The
importance of platelets in inflammation lies in their ability to
co-operate with endothelial cells to co-ordinate the adherence
and extravasation of leucocytes during the inflammatory response
[176]. The release of chemokines and cytokines by macrophages
within injured tissue initiates a sequence of events whereby
leucocytes are attached to the endothelium, first by low-affinity
interaction of adhesion molecules called selectins on endothelial
and platelet cells with glycoproteins on the leucocytes (‘leucocyte
rolling’), and secondly by high-affinity attachment via cellsurface integrins that are able to resist the fluid shear forces
within the blood vessel. Similar mechanisms are also known to
contribute to coagulation and haematogenous tumour metastasis
[177]. The reciprocal arrangement of adhesion molecules
on leucocytes, platelets and endothelial cells is carefully
controlled by regulating their subcellular localization. PX domain
proteins have been linked to the trafficking and cell-surface
presentation of two key molecules in this process: P-selectin on
endothelial and platelet cells by SNX17 [75,85,86], and PSGL-1
on leucocytes by SNX20 [35] (Supplementary Figure S3 at
http://www.BiochemJ.org/bj/441/bj4410039add.htm). P-selectin
is stored within specific intracellular compartments from which
it is released to the cell surface upon stimulation: α-granules in
platelets and Weibel-Palade bodies in endothelial cells. It also
undergoes endocytosis, after which it is either recycled through
the endocytic system or degraded within lysosomes [177,178].
SNX17 was identified as a P-selectin-interacting protein [75,85],
and we have also observed binding of SNX27, suggesting that this
may be a common function of the PX-FERM subfamily (R. Ghai
and B.M. Collins, unpublished work). Overexpression of SNX17
was found to enhance the internalization of P-selectin from the cell
surface, and to retard the degradation of P-selectin in lysosomes by
restricting its transport into late endosomes/multivesicular bodies
and causing an accumulation in SNX17-positive endosomes
[75,86]. Within the opposing leucocyte cells, SNX20 was
identified as a binder of the cytoplasmic tail of PSGL-1 [35].
Overexpression of SNX20 caused a significant redistribution of
PSGL-1 from the cell surface into endosomes; however, mice
deficient in SNX20 appeared to be normal and healthy and their
neutrophils displayed no significant defects in PSGL-1-mediated
cell adhesion or signalling, possibly due to compensation by the
highly homologous SNX21. Further studies will be required to
determine if this is true and whether the SNX20/SNX21 proteins
control trafficking of other transmembrane cargoes.
51
PX proteins in Alzheimer’s disease
AD is caused, at least in part, by the accumulation of the toxic
amyloid peptide Aβ [179]. Aβ is derived from the sequential
cleavage of the APP by transmembrane enzymes called secretases,
first by β-secretase to release a soluble ectodomain fragment, and
subsequently by γ -secretase within the transmembrane domain to
form the 40–42-residue Aβ peptide. This degradation is thought
to occur primarily within intracellular organelles, whereas a
non-amyloidogenic pathway involving initial cleavage by the
α-secretase is thought to occur primarily at the cell surface
[180]. For detailed reviews of APP trafficking and processing see
[181–185]. The production of Aβ is therefore dependent on a
delicate balance between the trafficking of APP, the trafficking
of secretase enzymes and the degree to which the molecules
encounter each other within the organelles of the neuronal cell. It
is increasingly apparent that many PX proteins and their partners
such as the retromer complex play important roles in APP and
secretase trafficking, regulating the subsequent accumulation of
toxic Aβ peptide (Figure 5). This can occur in a number of ways,
as described below.
(i) Trafficking of β-secretase and SorLA, which is a co-receptor
involved in the retrograde trafficking of APP from endosomes to
the TGN, is co-ordinated by the retromer retrograde trafficking
complex [186–189]. Removal of APP from the endosomal system
is expected to result in less Aβ production, due to avoidance of
β- and γ -secretases, which function predominantly within these
organelles. SNX1, SNX2, SNX5 and SNX6 are key regulators
of retromer-mediated trafficking, although a direct role for these
PX-BAR proteins in APP trafficking and degradation is yet to be
demonstrated.
(ii) A recent study has shown an important role for SNX6
in regulating BACE1 (β-secretase 1; also called β-site APPcleaving enzyme 1) trafficking and APP degradation [190]. It
was found that SNX6 forms a complex with β-secretase, and that
SNX6 suppression leads to modulation of β-secretase retrograde
trafficking, increased levels of β-secretase and subsequent
increased production of Aβ peptide. In contrast with the normal
function of the retromer complex and SNX1, this study suggests
that SNX6 inhibits the retrograde traffic of BACE1 from
endosomes to the Golgi. It is proposed that SNX6 may either
antagonize SNX1 function in BACE1 transport or form retromer
complexes with differential cargo selectivity. Indirect support for
this latter hypothesis is supplied by data showing that alternative
retromer-associated PX-BAR proteins can mediate trafficking of
different cargo proteins [58].
(iii) SNX17 is highly expressed in neurons [83,87] and is
implicated directly in the recycling of APP from endosomes
to the cell surface, as it binds to the APP NPxY sorting motif
and inhibition by siRNA results in an increased level of APP
breakdown and Aβ peptide production within the endosomal
compartment [11,87]. In addition, one of the most intriguing
aspects of SNX17-receptor trafficking is the relationship between
many of the identified SNX17 cargos. Many studies over the last
few years have highlighted the complexity of APP processing, and
in particular have shown a critical role for ApoE (apolipoprotein
E) and its receptors from the LDLR family in APP processing and
Aβ clearance in AD (reviewed in [181–183]). SNX17 regulates
the endosome-to-cell surface recycling of many receptors of the
LDLR family, including LRP1, which facilitates the transport
of the APP protein through an interaction mediated by FE65
[81–85], and others implicated directly in both the clearance
and generation of Aβ plaques via their role as receptors for the
Aβ chaperone ApoE [181–183]. Therefore SNX17 appears to be
functioning at a nexus of trafficking pathways critical for APP
c The Authors Journal compilation c 2012 Biochemical Society
52
Figure 5
R. D. Teasdale and B. M. Collins
Cartoon outlining potential roles of PX proteins in the trafficking and processing of APP to neurotoxic Aβ implicated in AD
For reviews of APP trafficking and processing, see [181–185]. See the text for a detailed discussion.
processing, not only controlling APP directly, but also affecting
other receptors with important roles in the process of Aβ peptide
production and clearance. It is possible that trafficking of APP
and LDLR family proteins may be influenced by the other PXFERM molecules SNX27 and SNX31, but this remains to be
confirmed.
(iv) SNX33 and SNX9 have been shown to affect the uptake of
APP from the cell surface, although the mechanism is poorly
understood [66]. Increased expression of SNX33 or SNX9
was shown to significantly increase the level of soluble APP
released into the extracellular space by α-secretase (sAPPα).
A similar effect was seen for dominant-negative constructs of
the dynamin GTPase required for scission of endocytic vesicles.
SNX33 was shown to bind dynamin via its SH3 domain and
its expression was able to inhibit the endocytosis of APP.
Therefore the SH3-PX-BAR proteins in this system are able
to affect the balance of α- and β-secretase cleavage and the
level of Aβ production. The authors suggest that SNX33 and
SNX9 may be working to inhibit dynamin during endocytosis.
Given the role of SNX9 in promoting clathrin-dependent and
-independent endocytosis, this may at first appear counterintuitive
[17,21,47,191,192]. However, other studies have also shown that
either overexpression or knockdown of SNX9 can effectively
perturb clathrin-mediated endocytosis, indicating that altered
SNX9 protein levels may impair the homoeostasis of proteininteraction networks required for endocytic vesicle formation
[191–193]. One possible explanation is that overexpression of
SNX33 or SNX9 results in competition for APP-specific adaptors
such as Dab2 [87,194] for binding to the AP2 complex, clathrin
c The Authors Journal compilation c 2012 Biochemical Society
and dynamin during endocytic vesicle formation, and therefore
acts to slow the dynamics of APP endocytosis. More work will
be required to dissect the mechanisms by which SH3-PX-BAR
proteins affect APP uptake.
(v) SH3PXD2A was shown to play a role in mediating Aβinduced neurotoxicity through its interaction with members of the
ADAM (a disintegrin and metalloproteinase) family of enzymes
(for reviews of ADAMs, see [195,196]). SH3PXD2A can bind to
(at least) ADAM12, ADAM15 and ADAM19 through association
of its fifth SH3 domain with polyproline sequences in the
cytosplasmic tails of the transmembrane proteases [197,198].
Exposure of neurons to cytotoxic Aβ peptide results in tyrosine
phosphorylation of SH3PXD2A and relocalization of the protein,
and SH3PXD2A is then able to stimulate the metalloproteinase
activity of ADAM12 [199]. Treating cells with toxic Aβ peptide
results in increased ADAM12 proteolytic activity, whereas
expression of protease-deficient ADAM12 or an ADAM12binding mutant of SH3PXD2A blocks Aβ-induced cell death. In
addition, SNPs (single nucleotide polymorphisms) in ADAM12
and SH3PXD2A may confer susceptibility to late-onset AD
[200]. Although the mechanism is very poorly understood, the
hypothesis is that neuronal cell death induced by toxic Aβ
peptides is at least partly mediated by ADAM12 and SH3PXD2A.
These proteins therefore may be potential targets for therapeutic
applications.
(vi) Another family of PX proteins implicated as negative
regulators of Aβ production in AD are the PX-PH-PLD proteins
[201]. PLD1 is significantly up-regulated in brains of AD patients,
in particular within mitochondrial fractions, and is able to interact
The PX protein family
with the cytoplasmic tail of APP via its PH domain [202,203].
There is now strong evidence that PLD1 can influence the
trafficking of both APP and the PS1 (presenilin 1) subunit of
γ -secretase (and, through PS1, presumably other γ -secretase
subunits), in particular promoting the formation of TGN-derived
transport vesicles destined for the cell surface [204–206]. It
is thought that PLD1 enhances egress of APP from the TGN
and internal endosomes where Aβ production predominantly
occurs, thereby reducing the level of toxic Aβ production. In
addition to a role in APP trafficking, PLD1 was also found to
inhibit Aβ production directly, by interfering with the assembly
of γ -secretase via association with PS1 [204]. Adding to the
complexity of PLD function in AD, other studies indicate that
PLD activity is increased in neurons exposed to Aβ, that PLD2 is
critical for mediating Aβ neuronal toxicity and that PLD2 ablation
in amyloidogenic transgenic mouse models improves memory
deficits and synaptic function, suggesting that PLD activity may
be a valid target for therapeutic action [207].
It is becoming increasingly apparent that intracellular
trafficking is central to the process of APP homoeostasis and
Aβ manufacture [180,208]. Taken together, the evidence is overwhelming that PX proteins regulate various APP and secretase
trafficking pathways and modulate production and toxicity of the
Aβ peptide, so determining the precise roles of the PX proteins
and the molecular mechanisms that underpin their function in Aβ
regulation represents an important area of investigation.
PX proteins in pathogen invasion
Another disease process that has gained recent attention is
that of pathogen invasion via corruption of the endocytic
system (reviewed in [209]). The invasion of Salmonella enterica
via coercion of normal cellular macropinocytic processes
[161,210,211] has been found to be critically dependent on
the action of PX proteins. Bujny et al. [212] found that SNX1
undergoes rapid translocation to sites of bacterial entry and SCVs
(Salmonella-containing vacuoles), resulting in the formation of
extensive long-range membrane tubules that are thought to
mediate membrane contraction during SCV maturation. Critically,
suppression of SNX1 was found to halt the progression of SCVs
into the cell. Recently, Braun et al. [213], demonstrated that
the PX-only protein SNX3 is also important for the process.
Similarly to SNX1, SNX3 is recruited to SCVs and to the
SCV membrane tubules, and its depletion leads to impaired SCV
maturation. However it was found that SNX3 recruitment occurs
after SNX1, and in fact SNX3 depends on SNX1 (and SNX2)
for localization to these tubules. From a mechanistic standpoint,
this correlates well with the known role of SNX1 and SNX2 as
proteins regulating membrane tubulation. The generation of an
SCV is dependent on bacterially secreted enzymes that promote
the generation of PtdIns3P on its limiting membrane during the
early stages of formation. Given the central role of PtdIns3P
in early stages of Salmonella invasion, it is very likely that other
PX proteins will also be important for the process. Indeed, SNX15
was recently found to be recruited to early SCVs (but not to
tubules) [213], and given the role of PX-BAR proteins other than
SNX1 in macropinocytosis [24,214], these are also likely to be
involved in pathogen invasion. SNX1, SNX6, SNX9 and SNX33
have all been identified as being involved in the internalization
of apoptotic cells via phagocytosis and the subsequent transport
into a degradative organelle [215–217]. However, their role
in phagocytosis of pathogens remains uncharacterized. Other
PX family members have central roles in host defence against
pathogens, with p47phox , p40phox and NOXO1 (NADPH oxidase
53
organizer 1) associated with the NADPH oxidase complex,
which is responsible for the formation of superoxide anions
[218,219]. Following phagocytosis of pathogens, the NADPH
oxidase complex is activated, resulting in an oxidative burst that
can kill the pathogen directly, or indirectly by activating other
components of the innate immune system.
In addition to a role in bacterial invasion, endocytosis and
trafficking through the endosomal system is also critical for
the uptake of bacterial toxins and the processing and assembly
of viruses [209]. STx (Shiga toxin) is secreted by Shigella
dysenteriae and is endocytosed by target cells by clathrinmediated uptake, bypasses the degradative pathways and is
eventually translocated into the cytosol from the endoplasmic
reticulum [220]. SNX1 has been found to be important for the
efficient passage of STx [50,57,58,221]. SNX2 has also been
implicated [50], although other studies suggest that SNX2 does
not play an important role in the process [57,58]. In contrast,
depletion of another PX-BAR protein SNX8 increases the rate
of endosome–Golgi transport of STx, suggesting an antagonistic
role in STx passage through the endosomal compartment
[222]. Finally, SNX5 has been implicated in the uptake via
macropinocytosis of Ebola virus [223], and the protein SNX16
has been found to be important for infection by VSV (vesicular
stomatitis virus) [128]. For infection, VSV and other enveloped
viruses must be endocytosed and transported to late endosomes
where the acidic pH of the lumen triggers fusion of the viral
envelope with the endosomal membrane and release of the
nucleocapsid into the cytosol for viral replication. SNX16 was
found to localize to these late endosomes and its overexpression
significantly inhibited the export of the nucleocapsid. It remains to
be determined, however, how SNX16 acts to regulate this process.
CONCLUDING REMARKS
In the last few years there has been major progress in
understanding the functions of PX proteins. Of course, the
diversity of these proteins makes it impossible to present a
single unified picture of how they work. It is clear, however,
that the coupling of the membrane-localizing PX domain with
different functional modules is a mechanism by which eukaryotic
cells have constructed tools to control a multitude of protein
binding, membrane remodelling, signalling, motor and enzymatic
functions localized to specific regions of the secretory and
endocytic system.
Many questions remain regarding the functional, structural
and pathological roles of the PX proteins. From a cellular
perspective, it will be important to develop quantitative tools
for analysing the localization, interactions and, most importantly,
the spatiotemporal regulation of PX proteins by different cellular
signals. Structurally, although we now have a firm grasp of how
the PX domain associates with PtdIns3P, there is a severe paucity
of information regarding how the PX domain regulates protein–
protein interactions. There is also a significant lack of knowledge
regarding the structures of many of the PX-associated protein
domains and the overall tertiary structures of the full-length
molecules. Most importantly, it will be essential to combine these
cellular and structural insights with broader systems approaches
to develop an understanding of how the vast networks of
interactions controlled by the PX proteins regulate cellular fate.
Finally, it is imperative to determine how PX proteins contribute
to different disease processes, such as pathogen invasion and
amyloid production, both in order to determine whether they
might be suitable therapeutic targets and also to gain insights
into how such interventions might be achieved.
c The Authors Journal compilation c 2012 Biochemical Society
54
R. D. Teasdale and B. M. Collins
FUNDING
Research in the laboratories of B.M.C. and R.D.T. is supported by funds from the Australian
Research Council (ARC) and the National Health and Medical Research Council (NHMRC).
B.M.C. is an ARC Future Fellow [grant number FT100100027]. R.D.T. is an NHMRC Senior
Research Fellow [grant number 511042].
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Received 11 July 2011/26 July 2011; accepted 27 July 2011
Published on the Internet 14 December 2011, doi:10.1042/BJ20111226
c The Authors Journal compilation c 2012 Biochemical Society
Biochem. J. (2012) 441, 39–59 (Printed in Great Britain)
doi:10.1042/BJ20111226
SUPPLEMENTARY ONLINE DATA
Insights into the PX (phox-homology) domain and SNX (sorting nexin)
protein families: structures, functions and roles in disease
Rohan D. TEASDALE and Brett M. COLLINS1
Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland 4072, Australia
See the following pages for Supplementary Figures S1–S3 and
Supplementary Tables S1–S3.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
R. D. Teasdale and B. M. Collins
Figure S1
Known structures of mammalian PX domains, including yeast Grd19p/Snx3p
There are three known structures of PX domains in complex with PtdIns3P : SNX9, p40phox and yeast Grd19p/Snx3p. These structures show identical binding modes for PtdIns3P . In many other
structures, SO4 ions are found in the putative binding site, usually mimicking the location of the 3-phosphate group (associated with the conserved arginine side chain). In this Figure, all 15 known
mammalian PX domain structures are shown in surface representation (PDB codes are given below each structure). Only one representative structure is shown for each protein, and for proteins
c The Authors Journal compilation c 2012 Biochemical Society
The PX protein family
Figure S2
Sequence alignment of human SNX20 and SNX21
The PX domain and the C-terminal domain of unknown structure and function are indicated. We refer to this domain as the PXB domain (PX-associated domain B) to fit with terminology adopted for
the PXA-RGS-PX-PXC proteins. Secondary-structure predictions calculated with JPRED [1] (http://www.compbio.dundee.ac.uk/www-jpred/) are indicated for SNX20 (above) and SNX21 (below).
The alignment was generated using ESPript 2.2 [2] (http://espript.ibcp.fr/ESPript/ESPript/).
where more than one structure is available, only PtdIns3P or SO4 -bound structures are shown. Surfaces of the PX domains are coloured according to electrostatic potential red to blue from − 0.5
to + 0.5 V. An Ins(1,3,4,5)P 4 molecule has been positioned in the putative binding site of each structure, based on the known binding orientation of PtdIns3P , to provide an indication of whether
3-, 4- and 5-phosphates of phosphoinositides are likely to fit within the binding pocket. In all cases, steric clashes are observed with at least one of the modelled 4- or 5-phosphate groups,
and in some instances the residues required for 3-phosphate co-ordination are missing. Phosphate positions are labelled (3, 4 and 5), and whether the phosphate groups are able to associate is
indicated. Reported lipid-binding specificities and key side chains of the binding site are given in Supplementary Table S2. There are no clear cases where structures support the contention that
a 4- or 5-phosphorylated phosphoinositide would be able to actively engage the PX domain, without binding in a significantly different orientation to PtdIns3P . In the cases of SNX5, PI3K-C2α
and PI3K-C2γ , there appears to be little possibility that any phosphoinositide (including PtdIns3P ) will bind in the preferred conformation. Note 1: structures marked with asterisks (*) are NMR
structures, and in these examples the polyproline loop is highly dynamic in conformation and often obscures the binding pocket. This highlights the flexible nature of the polyproline loop (and to a
lesser extent the loop between the β1 and β2 strands) and the importance of its conformation for phosphoinositide binding. Note 2: in the IRAS crystal structure, both the flexible polyproline loop
and the loop between strands β1 and β2 are in conformations that obscure the binding pocket, but the binding determinants for the PtdIns3P are present.
c The Authors Journal compilation c 2012 Biochemical Society
R. D. Teasdale and B. M. Collins
Figure S3
Cartoon outlining a potential role for PX proteins in inflammation
(A) Inflammatory signals trigger the attachment of leucocytes to blood-vessel endothelial cells mediated by interactions of the leucocyte PSGL-1 glycoprotein with endothelial and platelet P-selectin
adhesion proteins. The P-selectin protein is stored within α-granules in platelets and Weibel-Palade bodies (WPBs) in endothelial cells. (B) Secretion of P-selectin from Weibel-Palade bodies, along
with von Willebrand factor, is promoted by secretagogue signals. In endothelial cells, SNX17 interacts with P-selectin on early endosomes (EE). Overexpression of SNX17 promotes recruitment of
P-selectin to endosomes, and retards its degradation in lysosomes. Whether SNX17 is able to promote cell-surface recycling as for members of the LDLR family is unknown. (C) In leucocytes, the
glycoprotein PSGL-1 interacts with P-selectin via its glycans containing sialyl Lewis X. Its localization to endosomes is strongly enhanced by SNX20, but it still remains unknown exactly what role
SNX20–PSGL-1 interaction plays in PSGL-1 trafficking and signalling. It is also unknown whether the close homologue SNX21 is able to perform similar functions to SNX20 in this process. LE,
late endosome; RE, recycling endosome.
c The Authors Journal compilation c 2012 Biochemical Society
Table S1
Critical phosphoinositide binding determinants in human PX proteins, and structurally predicted phosphoinositide specificities
n.d., not determined.
P3
Tyrinositol (aromatic
stacking with
inositol ring†)
LysP1 (binds
to 1phosphate‡)
Arg4,5-hydroxy (binds to
4- and 5-hydroxy
groups of inositol
ring§)
Polyproline loop
(potentially clashes
with 5-phosphate
groups)
Reported
phosphoinositide
preferences
PI3P
PI3P > PI(3,5)P 2
None
PI(4,5)P 2
None
n.d.
PI3P
PI3P
PI3P
PI3P
None
n.d.
n.d.
n.d.
PI3P
PI(3,4)P 2 ≈
PI(4,5)P 2 ≈
PI(3,5)P 2 ≈
PI(3,4,5)P 3 >
PI3P ≈ PI4P
PI(4,5)P 2 > PI(3,4)P 2 >
PI(3,5)P 2
n.d.
Subfamily
Protein
PDB code (method, ligand)
Arg (binds to
3-phosphate*)
PX-BAR
SNX1
2I4K (NMR, apo)
R186
F187
K214
R239
Polyproline loop
occludes
5-phosphate
SNX2
SNX4
SNX5
R183
R106
3HPC (X-ray, apo), 3HPB
(X-ray, apo)
F184
Y107
Q68
K211
K132
H69
R236
R155
K96
?
?
T139
PI3P
PI3P
Polyproline loop
occludes
5-phosphate
SNX6
Q68
H69
R96
T139
?
SNX7
3IQ2 (X-ray, SO4 )
R73
Y74
K99
R117
R109
R132
Q63
Y110
Y133
H64
K135
K158
R91
R150
R177
T134
The structure of
homologue
SNX5 suggests
that the normal
PtdIns-binding
pocket is simply
not present
Polyproline loop is
open
?
?
?
R286
Y287
K313
R327
Polyproline loop
occludes
5-phosphate
SNX18
R312
Y313
K338
R353
?
PI3P
SNX33
R266
Y267
K292
R308
?
PI3P
SNX8
SNX30
SNX32
SH3-PX-BAR
SNX9
2RAJ, (X-ray, SO4 ), 2RAK
(X-ray, PI3P ), 2RAI
(X-ray, apo), 3DYU
(X-ray, apo), 3DYT
(X-ray, apo)
Comments
In the reported NMR structure
there are significant local
conformational differences
that occlude the binding site.
Nevertheless specific side
chains are present in
approximately the correct
positions compared with
known co-crystal structures
of other PX domains
PI3P > PI(3,5)P 2
n.d.
The structure of SNX5 suggests
that the normal
PtdIns-binding pocket is
simply not present
K99 will need to reorient to bind
the 1-phosphate
The structure of homologue
SNX5 suggests that the
normal PtdInsP -binding
pocket is simply not present
Note: although the reported
specificity is PI(4,5)P 2 for
liposomes, PI3P is also
bound and only PI3P [not
PI(4,5)P 2 ] is able to bind in
soaked crystals
The PX protein family
c The Authors Journal compilation c 2012 Biochemical Society
Structurally
predicted
phosphoinositide
specificity¶
Continued
PDB code (method,
ligand)
ArgP3 (binds
to
3-phosphate*)
Tyrinositol (aromatic
stacking with
inositol ring†)
LysP1 (binds
to 1phosphate‡)
Arg4,5-hydroxy
(binds to 4- and
5-hydroxy groups
of inositol ring§)
Polyproline loop
(potentially
clashes with
5-phosphate
groups)
Structurally
predicted
phosphoinositide
specificity¶
Subfamily
Protein
PX-SH3
SH3PXD2A
R43
Y44
K78
R93
?
PI3P
SH3PXD2B
SNX28 (NOXO1)
R44
S41
Y45
W42
K78
K71
R94
R90
?
?
PI3P
None
PI3P
p40phox
2DYB (X-ray, apo) 1H6H
(X-ray, PI3P )
R58
Y59
K92 (plus
R60)
R105
p47phox
1KQ6 (X-ray, SO4 ), 1O7K
(X-ray, SO4 ), 1GD5
(NMR, apo)
3LUI (X-ray, SO4 ), 3FOG
(X-ray, apo)
R43
F44
K73
R90
R36
Y37
K62
R75
PX-FERM
SNX17
PXA-RGS-PX-PXC
SNX27
SNX31
SNX13
R196
R37
R601
Y197
Y38
Y602
K222
K62
K628
R236
R76
R642
Polyproline loop
occludes
5-phosphate
Polyproline loop
occludes
5-phosphate
Polyproline loop
occludes
5-phosphate
?
?
?
SNX14
SNX19
SNX25
R616
R582
R552
Y617
Y583
L553
K642
K611
K580
R657
R629
K595
?
?
?
SNX3
SNX10
SNX11
SNX12
SNX22
SNX24
HS1BP3
R70
R53
R59
R71
R43
R38
K70
Y71
Y54
Y60
Y72
Y44
Y39
Y71
K95
K79
K85
K96
K66
K61
K96
R118
R95
R100
R119
R79
R75
R109
?
?
?
?
?
?
?
PX-only
2CSK (NMR, apo)
2ETT (NMR, apo)
Comments
The SNX25 PX domain has
conservative alterations
in two of the four
canonical binding
residues (Tyr/Phe to Leu
at inositol-binding site,
and Arg to Lys at
4-phosphate-binding
site). It remains to be
seen if these
substitutions can
support PtdIns binding
HS1BP3 has a conservative
substitution (Arg to Lys
in P3-binding site). It
remains to be seen if
such a substitution can
support binding to
PtdIns3P
Reported
phosphoinositide
preferences
PI3P ≈ PI(3,4)P 2 >
PI(4,5)P 2
PI3P ≈ PI(3,4)P2
PI4P ≈ PI5P ≈
PI(3,5)P 2 > PI(3,4)P 2
PI3P
PI3P
PI(3,4)P 2 > PI3
P > PI(3,5)P 2
PI3P
PI3P
PI3P
PI3P
PI3P
PI3P
PI3P
PI3P ?
PI3P
n.d.
PI3P ≈ PI5P >
PI(3,5)P 2
n.d.
n.d.
n.d.
PI3P
PI3P
PI3P
PI3P
PI3P
PI3P
PI3P ?
PI3P
n.d.
n.d.
n.d.
PI3P
n.d.
n.d.
R. D. Teasdale and B. M. Collins
c The Authors Journal compilation c 2012 Biochemical Society
Table S1
Table S1
Continued
Tyrinositol (aromatic
stacking with
inositol ring†)
LysP1 (binds
to 1phosphate‡)
R55
R53
R44
Y56
Y54
Y45
K79
K83
K69
R92
R99
R84
S99
Y100
–
–
S172
T1462
Y173
F1463
R200
R1488
–
R1503
?
Polyproline loop
is open
T1405
S1237
F1406
F1238
R1431
W1263
R1446
R1275
?
Polyproline loop
occludes
5-phosphate
PLD1
K119
F120
R165
R180
?
PLD2
K103
Y104
R149
K164
?
Protein
PX-S/T-kinase
PXK
RPK118
SGK3 (CISK)
PX-SH3-GAP
SNX26 (TCGAP)
PX-PI3K
PX-RICS (GCGAP)
PI3K-C2α
PI3K-C2β
PI3K-C2γ
PDB code (method,
ligand)
1XTN (X-ray, SO4 ), 1XTE
(X-ray, apo)
2IWL (X-ray, SO4 ), 2AR5
(X-ray, apo), 2REA
(X-ray, apo), 2RED
(X-ray, apo)
2WWE (X-ray, apo)
Comments
Structurally
predicted
phosphoinositide
specificity¶
PI3P
PI3P
PI3P
?
?
Polyproline loop
occludes
5-phosphate
?
c The Authors Journal compilation c 2012 Biochemical Society
None
None
PI3P
PI3P
PI3P > PI(3,5)P 2 ≈
PI(4,5)P 2 ≈
PI(3,4,5)P 3
PI(4,5)P 2 ≈ PI(3,4)P 2 >
PI3P ≈ PI4P
PI3P ≈ PI4P ≈ PI5P
PI(4,5)P 2
None
None
n.d.
n.d.
PI3P ?
PI(3,4,5)P 3 PI3P >
PI5P >
PI(4,5)P 2 >
PI4P > PI(3,5)P 2 >
PI(3,4)P 2
PI3P ?
n.d.
None
D1464 occludes the normal
1-phosphate-binding
site. No density is
observed in the structure
for R1488 or the
polyproline loop. T1462
in the ArgP3 position will
preclude 3-phosphate
interaction
E1239 occludes the normal
1-phosphate-binding
site. W1263 will
preclude electrostatic
contact with the
1-phosphate. S1237 in
the ArgP3 position will
preclude 3-phosphate
interaction
The PLD1 PX domain has
conservative alterations
in two of the four
canonical binding
residues (Arg to Lys at
3-phosphate-binding
site, and Lys to Arg at
1-phosphate-binding
site)
The PLD2 PX domain has
conservative alterations
in three of the four
canonical binding
residues (Arg to Lys at
3-phosphate-binding
site, Lys to Arg at
1-phosphate-binding
site and Arg to Lys at the
4-hydroxy-binding site)
Reported
phosphoinositide
preferences
The PX protein family
ArgP3 (binds
to
3-phosphate*)
Subfamily
PX-PH-PLD
Polyproline loop
(potentially
clashes with
5-phosphate
groups)
Arg4,5-hydroxy
(binds to 4- and
5-hydroxy groups
of inositol ring§)
Continued
Subfamily
Protein
PX-PXB
SNX20
Kinesin-PX
SNX21
Kif16B
PX-MIT
SNX15
PX-LRR-IRAS
IRAS
PX-SNX16
SNX29-PX
PX-SNX34
Yeast PX protein
SNX16
SNX29
SNX34
Snx3p
Polyproline loop
(potentially
clashes with
5-phosphate
groups)
ArgP3 (binds
to
3-phosphate*)
Tyrinositol (aromatic
stacking with
inositol ring†)
LysP1 (binds to
1-phosphate‡)
Arg4,5-hydroxy
(binds to 4- and
5-hydroxy groups
of inositol ring§)
R116
Y117
K143
R158
?
R171
R1220
Y172
Y1221
K198
K1246
R212
R1260
R51
Y52
R81
R97
?
Polyproline loop
occludes
5-phosphate
?
3P0C (X-ray, apo)
R49
Y50
K75
R88
1OCU (X-ray, PI3P)
R144
R309
R55
R81
Y145
Y310
S56
Y82
K168
K336
R84
K112
R184
R350
R99
R127
PDB code (method,
ligand)
2V14 (X-ray, apo)
Polyproline loop
and loop
between
strands β1 and
β2 occlude the
binding site
?
?
?
Polyproline loop
occludes
5-phosphate
Comments
K1246 will need to reorient
to bind the 1-phosphate
SNX15 has a conservative
substitution (Lys to Arg
in 1-phosphate-binding
site)
The occluding loops will
need to alter their
conformations to
accommodate binding
Structurally predicted
phosphoinositide
specificity¶
Reported
phosphoinositide
preferences
PI3P
PI3P ?
PI4P ≈ PI5P ≈
PI(3,5)P 2 ≈
PI(3,4)P 2
n.d.
PI3P > PI(3,4)P 2 >
PI(3,4,5)P 3 >
PI(3,5)P 2
n.d.
PI3P
PI3P
PI3P
PI3P
PI3P ?
PI3P
PI3P
n.d.
n.d.
PI3P
PI3P
PI3P
*The Arg side chain makes specific electrostatic contact with the 3-phosphate group and is therefore required for binding of 3-phosphorylated PtdIns.
†The aromatic groups of Tyr or Phe make stacking interations with the inositol ring and are therefore required for binding of PtdIns.
‡The lysine side chain at the end of the polyproline loop is involved in electrostatic contact with the 1-phosphate group and is thus important for PtdIns orientation and binding.
§The Arg side chain makes specific H-bonds to the 4- and 5-hydroxy groups of PtdIns3P and therefore precludes binding of 4- and 5-phosphorylated PtdIns.
The polyproline loop generally orients in such a way as to sterically preclude binding of 5-phosphates. However this loop is relatively flexible and therefore might accommodate binding via conformational changes. For all proteins without a known structure
we have used ‘?’ to indicate that their orientation is unknown.
¶The phosphoinositide preferences are based on a critical evaluation of binding experiments reported in the literature.
R. D. Teasdale and B. M. Collins
c The Authors Journal compilation c 2012 Biochemical Society
Table S1
The PX protein family
Table S2
Identified interacting molecules for the mammalian PX protein subfamilies
Interactions between PX proteins are in bold. Ligands have been classified into functional families: PX protein, transmembrane cargo, trafficking, cytoplasmic signalling protein, cytoskeleton and
other. Transmembrane signalling receptors (e.g. EGFR) have been designated as transmembrane cargo for the purposes of this Table.
PX sub-family
PX protein
Ligand
Ligand class
Domain from PX protein
Domain from ligand protein
Reference(s)
PX-BAR
SNX1
SNX1
SNX2
SNX5
SNX6
Tyrosine kinase receptors (EGFR, IR, PDGFR)
Type I and II TGF-β receptors
Transferrin receptor
Dopamine receptors (D1, D5)
GPCRs (M1, NK1, NK2, DOP, US28, GLP)
P-selectin
Hrs
Retromer
Clathrin heavy chain (CHC17)
PX protein
PX protein
PX protein
PX protein
Cargo
Cargo
Cargo
Cargo
Cargo
Cargo
Trafficking
Trafficking
Trafficking
–
–
BAR domain
–
Cytoplasmic domain
Intracellular domain
–
C-terminal tails
C-terminal tails
–
C-terminus excluding the VHS domain
VPS35 and VPS29 subunits
–
[3–5]
[6–8]
[5,8–10]
[5,8,11,12]
[6,13]
[11]
[6]
[14]
[15]
[16]
[17,18]
[3,5,7,19,20]
[21]
Enterophilin
Rab6IP1
RME-8 J-domain protein
Trafficking
Trafficking
Trafficking
–
C-terminus
IWN3 and ARM-like domains
[22]
[5]
[18,23]
Arf6-interacting protein 1
Ypt Interacting protein 1
Ras-like protein (RLP)
Trafficking
Trafficking
Signalling
–
–
GTPase domain
[5]
[5]
[24]
Kalirin-7 (RhoGEF)
WASH complex
Rho GTPase (inactive form)
SNX2
SNX1
SNX5
SNX6
Tyrosine kinase receptors (EGFR, IR, PDGFR)
Type I and II TGF-β receptors
TPR/MET tyrosine kinase
Retromer
Clathrin heavy chain (CHC17)
Cytoskeleton
Cytoskeleton
Cytoskeleton
PX protein
PX protein
PX protein
PX protein
Cargo
Cargo
Cargo
Trafficking
Trafficking
–
Strumpellin subunit of WASH complex
–
–
–
–
–
Cytoplasmic domain
Intracellular domain
–
VPS35 and VPS29 subunits
–
[25]
[26,27]
[25]
[3,6,7]
[6–8]
[5,8]
[5,8,11]
[6]
[11]
[28]
[3,5,7]
[21]
Enterophilin
Archain
Kalirin7 (RhoGEF)
WASH complex
Rho GTPase (inactive form)
Formin-binding protein 17 (FBP17)
Zinc finger CDGSH
Abstrakt (DEAD-box helicase)
SNX4
SNX2
SNX6
Tyrosine kinase receptors (EGFR, PDGFR)
Type I and II TGF-β receptors
CFTR
Clathrin heavy chain (CHC17)
Trafficking
Trafficking
Cytoskeleton
Cytoskeleton
Cytoskeleton
Cytoskeleton
Other
Other
PX protein
PX protein
PX protein
Cargo
Cargo
Cargo
Trafficking
–
–
–
–
–
–
Residues 1–162
–
–
–
Cytoplasmic domains
Intracellular domain
–
–
[22]
[5]
[25]
[26]
[25]
[29]
[5]
[30]
[31]
[6]
[11]
[6]
[11]
[32]
[21]
Amphiphysin 2
KIBRA (Dynein interactor)
Dynein
Trafficking
Cytoskeleton
Cytoskeleton
Residues 1–304 (BAR domain)
C-terminal domain
–
[33]
[31]
[21,31]
SNX1
SNX2
SNX6
Retromer
Clathrin heavy chain (CHC22)
Dynactin
DOCK180
Stathmin-2
CREB bZIP transcription factor
CSE1
Fanconi anemia proteins (FANCs)
Mind bomb 1 (Mib1)
PX protein
PX protein
PX protein
Trafficking
Trafficking
Cytoskeleton
Cytoskeleton
Cytoskeleton
Other
Other
Other
Other
–
–
BAR domain
–
BAR domain
PX domain
–
–
–
–
BAR domain
N-terminus
Inverted clathrin box within
PX domain (DFLGL)
BAR domain
–
C-terminal segment of BAR
domain
–
–
Linker between PX and BAR
domain
–
–
BAR domain
–
–
–
–
–
PX domain
PX + BAR domains
–
Inverted clathrin box within
PX domain (DFLGL)
BAR domain
–
–
–
BAR domain
–
–
PX domain
–
–
–
–
PX domain
–
Inverted clathrin box within
PX domain (EFELL)
Helix 3 of BAR domain
BAR domain
Inverted clathrin box within
PX domain (EFELL)
BAR domain
–
–
–
Helix 2 of BAR domain
–
Helix 1 of BAR domain
–
–
–
BAR domain
–
BAR domain
–
–
–
Hub domain (residues 1074–1640)
C-terminus of p150glued (DCTN1)
DHR1 domain
–
–
–
–
–
[8–10]
[5,8]
[5,8]
[5,20]
[34]
[5]
[35]
[5]
[5]
[5]
[36,37]
[38]
SNX2
SNX4
SNX5
c The Authors Journal compilation c 2012 Biochemical Society
R. D. Teasdale and B. M. Collins
Table S2
Continued
PX sub-family
PX protein
Ligand
Ligand class
Domain from PX protein
Domain from ligand protein
Reference(s)
SNX6
SNX6
SNX1
SNX2
SNX4
SNX5
Tyrosine kinase receptors (EGFR, IR, PDGFR,
LR)
Type I and II TGF-β receptors
BACE-1
Retromer
Arf6 Interacting protein 5
GIT1
Dynactin
PX protein
PX protein
PX protein
PX protein
PX protein
Cargo
–
–
–
–
–
–
–
–
–
–
–
–
[11]
[8,11,12]
[5,8,11]
[11]
[5,8]
[11]
Cargo
Cargo
Trafficking
Trafficking
Signalling
Cytoskeleton
PX domain
PX domain
–
–
BAR domain
Residues 71–140
[11,39]
[40]
[5]
[5]
[41]
[5,8]
NudE-like 1
MLLT10
CREB bZIP transcription factor
Pim1 kinase
Translationally controlled tumour protein
(TCTP)
p27Kip1
BRMS-1
Cytoskeleton
Other
Other
Other
Other
–
–
–
PX domain
–
Intracellular domains
–
–
–
Coiled-coil domain 2
C-terminus of p150glued
(DCTN1); residues
1061–1282
–
–
–
–
–
Other
Other
Residues 300–406
[43]
[44]
USP16
PLCγ 1
Other
Signalling
–
Polyproline loop of PX domain
(residues 90–103;
TLIIPPLPEKFIVK)
Proline-rich domain
Residues 1–88 (coiled-coil
domain)
–
SH3 domain
–
–
–
SNX9
SNX18
SNX33
Metalloprotease disintegrins MDC9 and
MDC15 (ADAM9 and ADAM15)
Insulin receptor
AP2 complex
PX protein
PX protein
PX protein
Cargo
BAR domain
BAR domain
–
SH3 domain
[47–50]
[51]
[52]
[53]
Cargo
Trafficking
Clathrin
Trafficking
Dynamin 1 and dynamin 2
Synaptojanin-1
ITCH ubiquitin ligase
Son of sevenless 1 (Sos1) and Sos2
PI3K p85
PtdIns(4)P -5-kinases Iα, Iβ and Iγ
Cdc-associated kinase 1 (ACK1) and ACK2
WASP and N-WASP
Arp2/3 complex
Trafficking
Trafficking
Trafficking
Signalling
Signalling
Signalling
Signalling
Cytoskeleton
Cytoskeleton
Proline-rich domain
Proline-rich domain
Proline-rich domain
Proline-rich domain
–
–
Proline-rich domain
Proline-rich domain
–
[50,56–59]
[60,61]
[62]
[63]
[64]
[59]
[60,65]
[50,59,64,66]
[59]
Aldolase
Other
Near aldolase active site
[57,67]
USP7
EspF
USP25
PARP11
TL132L
SNX18
SNX9
Dynamin2
AP1 complex
Other
Other
Other
Other
Other
PX protein
PX protein
Trafficking
Trafficking
Trafficking
Trafficking
Signalling
Cytoskeleton
–
Proline-rich domain
–
–
–
BAR domain
BAR domain
Proline-rich domain
γ 1 subunit appendage
domains
Proline-rich domain
Proline-rich domain
Proline-rich domain
Proline-rich domain
[45]
[68,69]
[45]
[45]
[45]
[51,70]
[51]
[51,71]
[71]
Synaptojanin-1
ITCH ubiquitin ligase
Son of sevenless 1 (Sos1) and Sos2
WASP and N-WASP
–
Low complexity region between SH3
and PX domains
Low complexity region between SH3
and PX domains
SH3 domain
SH3 domain
SH3 domain
SH3 domain
PX domain
PX domain
SH3 domain
SH3 domain
Low complexity region between SH3
and PX domains
Low complexity region between SH3
and PX domains. Minimal
sequence WDEDWDG
–
SH3 domain
–
–
–
BAR domain
BAR domain
SH3 domain
Low complexity region between SH3
and PX domains
SH3 domain
SH3 domain
SH3 domain
SH3 domain
BAR domain
BAR domain
–
PARPAPAPP polyproline
sequence
–
α and β2 subunits
appendage domains
–
SNX7
SH3-PX-BAR
SNX8
SNX30
SNX32
SNX9
SNX18
c The Authors Journal compilation c 2012 Biochemical Society
[5]
[5]
[5]
[42]
[12]
[44]
[46]
[54]
[55,56]
[56]
[51]
[62]
[63]
[51,72]
The PX protein family
Table S2
Continued
PX sub-family
PX-SH3
PX protein
Ligand
Ligand class
Domain from PX protein
Domain from ligand protein
Reference(s)
SNX33
SNX33
SNX9
ADAM15
Dynamin 1 and dynamin 2
WASP
ADAM proteases (ADAM12,
ADAM15, ADAM19)
PX protein
PX protein
Cargo
Trafficking
Cytoskeleton
Cargo
–
–
SH3 domain
SH3 domain
SH3 domain
SH3 domain (number 5)
–
–
Proline-rich domain
Proline-rich domain
Proline-rich domain
C-terminal cytoplasmic tail
polyproline sequences
[52]
[52]
[73]
[71,74]
[52]
[75,76]
Dynamin 2
Grb2
Sos1
Nck1 and Nck2
WIP
N-WASP
Dystroglycan
–
p22phox
Trafficking
Signalling
Signalling
Signalling
Cytoskeleton
Cytoskeleton
Other
SH3 domains (1,2 and 5)
Polyproline sequences
SH3 domains (1–2)
Src-phosphorylated Tyr557
SH3 domains (3 and 5)
SH3 domains (1-5)
SH3 domain (number 3)
Polyproline
N-terminal SH3 domain
Polyproline
SH2 domain
–
–
Polyproline
[77,78]
[77]
[78]
[79]
[77]
[77]
[80]
Other
Tandem SH3 domains
Polyproline sequence
[81–83]
p67phox
NOX1
NOXA1
p47phox
Other
Other
Other
PX protein
Polyproline sequence
–
Polyproline sequence
SH3 domain
SH3 domain
NADPH-binding domain
SH3 domain
Polyproline sequence
[81]
[84]
[83,85,86]
[87–95]
Ku70
Signalling
N- and C-terminal regions
[96]
Coronin
Moesin
p67phox
Thioredoxin
p47phox
Cytoskeleton
Cytoskeleton
Other
Other
PX protein
p40phox
cPLA2
RelA
Akt kinase
Cortactin
Actin
Moesin
p67phox
p22phox
Fyn tyrosine kinase
CrkII
Rho GTPases (Cdc42, TC10β)
TrkA receptor tyrosine kinase
PX protein
Signalling
Signalling
Signalling
Cytoskeleton
Cytoskeleton
Cytoskeleton
Other
Other
Signalling
Signalling
Cytoskeleton
Cargo
C-terminus
PX domain
C-terminal PB1 domain
–
PX domain polyproline
loop
Polyproline sequence
PX domain
SH3 domain
C-terminal regions (last
186 residues)
–
FERM domain
PB1 domain
–
SH3 domain
–
Residues 319–337
PX domain
Polyproline sequence
SH3 domain
Polyproline sequences
Polyproline sequences
GAP domain
C-terminal domain
–
–
FERM domain
C-terminal SH3 domain
Polyproline sequence
SH3 domain
SH3 domain (N-terminal)
GTPase fold
Cytoplasmic domain
NR2B (GluRε2) NMDA receptor
Gab1, Gab2
PSD-95
Crk and CrkL
Nck
p130CAS
p120 Ras GAP
14-3-3 proteins
GABARAP and GABARAPL1
N-Shc
β-Catenin
N-Cadherin
Dynein/dynactin complex
P-selectin
Cargo
Signalling
Signalling
Signalling
Signalling
Signalling
Signalling
Signalling
Signalling
Signalling
Cytoskeleton
Cytoskeleton
Cytoskeleton
Cargo
C-terminal domain
C-terminal domain
–
Polyproline sequences
Polyproline sequences
C-terminal domain
Polyproline sequences
RSKSDP sequence (Ser1796 )
–
C-terminal domain
C-terminal domain
–
–
FERM domain
Cytoplasmic domain
–
–
SH3 domain
SH3 domain
–
SH3 domain
LRP1
LDLR
VLDLR
ApoER2 (LRP8)
APP
FEEL-1/stabilin-1
Cargo
Cargo
Cargo
Cargo
Cargo
Cargo
FERM domain
FERM domain
FERM domain
FERM domain
FERM domain
FERM domain (F3 module)
SH3PXD2A
(SH3MD1, FISH,
Tks5)
SH3PXD2B (Tks4)
SNX28 (NOXO1,
p41NOX,
SH3PXD5)
p40phox (NCF4,
SH3PXD4)
p47phox (NCF1,
SH3PXD1A)
PX-SH3-RhoGAP
SNX26 (TC-GAP)
PX-RICS (GC-GAP,
p250GAP,
p200GAP, Grit)
PX-FERM
SNX17
SH3 domain
C2 domain
Polyproline sequence
–
SH2 domain
–
–
–
FTNAAFDPSP peptide
motif
IGNPTY peptide motif
FDNPVY peptide motif
FDNPVY peptide motif
FDNPVY peptide motif
YENPTY peptide motif
NPVF peptide motif
[97]
[98,99]
[87,88,90,91,94,100–103]
[104]
[105]
[87–95]
[106]
[107]
[108]
[109]
[110]
[98,99]
[89–92,95,111–116]
[92,115,117,118]
[119]
[120]
[120]
[121]
[122–124]
[125]
[122,123]
[121,125]
[125]
[121,125]
[126]
[127]
[128]
[121]
[122,123]
[122,123]
[127]
[16,129,130]
[131–134]
[133,135]
[133]
[133]
[136,137]
[138]
c The Authors Journal compilation c 2012 Biochemical Society
R. D. Teasdale and B. M. Collins
Table S2
Continued
PX sub-family
PX protein
SNX27 (Mrt1)
SNX31
Ligand
Ligand class
Domain from PX protein
Domain from ligand protein
Reference(s)
PTCH1
H-Ras
Krit1
Abl1
Cargo
Signalling
Signalling
Signalling
–
–
–
SH3 domain
[130]
[137]
[139]
[46]
Crk
Signalling
SH3 domain
[46]
Src
Signalling
SH3 domain
[46]
Fyn
Signalling
SH3 domain
[46]
Grb2
Signalling
SH3 domain
[46]
Nck1
Signalling
SH3 domain
[46]
p85A
Signalling
SH3 domain
[46]
PLCγ 1
Signalling
SH3 domain
[46]
KIF1Bβ
Cytoskeleton
FERM domain
FERM domain
FERM domain
Polyproline loop of PX domain
(residues 55–66;
ANVLPAFPPKKLFS)
Polyproline loop of PX domain
(residues 55–66;
ANVLPAFPPKKLFS)
Polyproline loop of PX domain
(residues 55–66;
ANVLPAFPPKKLFS)
Polyproline loop of PX domain
(residues 55–66;
ANVLPAFPPKKLFS)
Polyproline loop of PX domain
(residues 55–66;
ANVLPAFPPKKLFS)
Polyproline loop of PX domain
(residues 55–66;
ANVLPAFPPKKLFS)
Polyproline loop of PX domain
(residues 55–66;
ANVLPAFPPKKLFS)
Polyproline loop of PX domain
(residues 55–66;
ANVLPAFPPKKLFS)
FERM domain (F3 module)
[140]
5-HT4 R
Cargo
PDZ domain
Kir3.3/GIRK3
Cargo
PDZ domain
NMDA receptor 2C (NR2C)
Cargo
PDZ domain
Very large GPCR
Cargo
PDZ domain
β 2 -Adrenergic receptor
Cargo
PDZ domain
APP
CASP
Cargo
Trafficking
FERM domain
PDZ domain
DGKζ
Signalling
PDZ domain
Connector enhancer of kinase
suppressor of Ras 2 (CNKSR2)
Ribosomal protein S6 kinase,
90 kDa, polypeptide 3 (RPS6KA3)
Protein tyrosine phosphatase,
non-receptor type (PTPN2)
H-Ras
Dynactin 1
Trichoplein, keratin filament-binding
protein (TCHP)
Microtubule-associated protein 1A
(MAP1A)
Rho GDP dissociation inhibitor
(GDI) α
WASH complex
Liver mitochondrial glutaminase
(GA)
E2a-Pbx1-associated protein (EB-1)
ATPase, Na + /K + transporting,
β2 polypeptide (ATP1B2)
Component 1, q
subcomponent-binding protein
(C1QBP)
RAN-binding protein 9
G3BP1
H-Ras
Signalling
PDZ domain
Signalling
PDZ domain
Signalling
–
C-terminal residues
813–916
ESCF; C-terminal Type I
PDZ binding motif
ESESKV; C-terminal Type I
PDZ binding motif
ESEV; C-terminal Type I
PDZ binding motif
ETHL; C-terminal Type I
PDZ binding motif
DSLL; C-terminal Type I
PDZ binding motif
YENPTY peptide motif
ESRF; C-terminal Type I
PDZ binding motif
ETAV; C-terminal Type I
PDZ binding motif
ETHV; C-terminal Type I
PDZ binding motif
ETAL; C-terminal Type I
PDZ binding motif
–
Signalling
Cytoskeleton
Cytoskeleton
FERM domain
–
–
–
–
–
[137]
[145]
[145]
Cytoskeleton
–
–
[145]
Cytoskeleton
–
–
[145]
Cytoskeleton
Other
–
PDZ domain
[151]
[145]
Other
Other
–
–
–
ESMV; C-terminal Type I
PDZ binding motif
–
–
Other
–
–
[145]
Other
Other
Signalling
–
–
FERM domain
–
–
–
[145]
[45]
[137]
c The Authors Journal compilation c 2012 Biochemical Society
[141]
[142–144]
[145]
[145]
[146]
[137]
[147,148]
[145,149,150]
[145]
[145]
[145]
[145]
[145]
The PX protein family
Table S2
Continued
PX sub-family
PX protein
Ligand
Ligand class
Domain from PX protein
Domain from ligand protein
Reference(s)
PXA-RGS-PX-PXC
SNX13
Hrs
Gα s
–
IA-2 (islet antigen-2)
Trafficking
Signalling
PX + PXC domains
RGS domain
–
–
[152]
[153]
Other
PX domain (residues 494–676)
[154]
SNX25
PXK (MONaKA)
TGF-β receptor I
Na,K-ATPase subunits β1 and β3
β-Actin
Cargo
Cargo
Cytoskeleton
[39]
[155]
[156]
RPS6KC1 (RPK118,
humS6PKh1)
Sphingosine kinase (SPHK)
Signalling
PXA and PX domains
–
WASP-homology domain
(residues 531–578)
PSK2 domain
Residues 744–979
(C-terminal protein
tyrosine phosphatase
domain)
–
–
–
–
[157]
Peroxiredoxin-3
AIP4 E3 ubiquitin ligase
Other
Trafficking
–
WW domains
[158]
[159]
GSK-3β
Clathrin
Signalling
Trafficking
–
Clathrin terminal domain
[160]
[161,162]
Dynactin subunits (p50 and p150)
EGFR
Cytoskeleton
Cargo
Pseudo-kinase domain
Kinase domain (possible PPFY
motif)
–
N-terminal clathrin-binding
sequence
–
N-terminal domain
–
VENPE(pY)L sequence
[163]
[164]
Clathrin
Trafficking
Clathrin terminal domain
[165]
Shc
Eps8 Rac GEF complex
(Eps8–Abi1–Sos1)
Grb2
Signalling
Signalling
–
Eps8 SH3 domain
[166]
[166]
SH3 domains
[166,167]
–
[168]
SNX14
SNX19
PX-S/T kinase
SGK3 (CISK, SGKL)
PI3K-PX
PIK3C2A
(PI3K-C2α)
PIK3C2B
(PI3K-C2β)
PX-PH-PLD
PIK3C2G
(PI3K-C2γ )
PLD1
PLD2
Tyrosine phosphorylated proteins
(p70 and p110)
–
Other
N-terminal clathrin-binding
sequence
–
N-terminal polyproline
sequences
N-terminal polyproline
sequences
–
PLD1
PLD2
EGFR
APP (Amyloid precursor protein)
Presenilin 1
Arf1
RalA
Secretory carrier membrane protein
(SCAMP)2
Caveolin1 and Caveolin 3
CtBP1/BARS
Munc18-1
Dynamin
Amphyphysin I and II
AP180
PKCα
PLC-γ 1
Type Iα PIP kinase
PEA-15
CKII
c-Src tyrosine kinase
Cdc42
RhoA
Actin
α-Synuclein
PLD1
PLD2
EGFR
Munc18-1
Dynamin
Caveolin 1
Amphyphysin I and II
Type Iα PIP kinase
mTor/Raptor complex
Syk kinase
PLC-γ 1
PX protein
PX protein
Cargo
Cargo
Cargo
Trafficking
Trafficking
Trafficking
–
–
–
PH domain
–
–
–
–
–
–
–
Cytoplasmic domain
Intracellular loop
–
–
–
[169]
[169]
[170]
[171]
[172]
[173]
[174,175]
[176]
Trafficking
Trafficking
Trafficking
Trafficking
Trafficking
Trafficking
Signalling
Signalling
Signalling
Signalling
Signalling
Signalling
Signalling
Cytoskeleton
Cytoskeleton
Other
PX protein
PX protein
Cargo
Trafficking
Trafficking
Trafficking
Trafficking
Signalling
Signalling
Signalling
Signalling
–
–
PX domain
PX domain
–
C-terminal residues 820–1036
C-terminal domain
PX domain
–
C-terminus
–
PH domain
–
C-terminal domain
–
PX + PH domains
–
–
–
PX domain
PX domain
–
–
–
TOS motif in PH domain
PX domain
PX domain
Scaffolding domain
–
–
GTPase domain
N-terminal domain
Residues 312–314 (TSP)
C-terminal peptide
SH3 domain
N-terminal domain
Death-effector domain
–
Kinase domain
–
–
–
N-terminal domain
–
–
–
–
GTPase domain
–
N-terminal domain
N-terminal domain
Raptor WD40 domain
–
SH3 domain
[171,177]
[178]
[179]
[180]
[181]
[182]
[177,183–186]
[187]
[188]
[189]
[190]
[191]
[192]
[189,192–194]
[195]
[196]
[169]
[169]
[170]
[179]
[180,197]
[198]
[181]
[188]
[199]
[200]
[187]
Signalling
c The Authors Journal compilation c 2012 Biochemical Society
R. D. Teasdale and B. M. Collins
Table S2
Continued
PX sub-family
PX-PXB
PX-only
PX protein
SNX20
SNX21
SNX3
SNX10
SNX11
SNX12
SNX22
SNX24
HS1BP3
Kinesin-PX
PX-MIT
SNX23 (KIF16B)
SNX15
PX-LRR-IRAS
IRAS (nischarin)
PX-SNX16
SNX29-PX
PX-SNX34
SNX16
SNX29
SNX34 (C6ORF145)
Ligand
Ligand class
Domain from PX protein
Domain from ligand protein
Reference(s)
PEA-15
c-Src tyrosine kinase
PTP1B
PKC-ζ
Signalling
Signalling
Signalling
Signalling
C-terminus
PH domain
–
PX domain (lysines 101–103)
[189]
[191]
[201]
[202]
Grb2
Signalling
Rac2
Cytoskeleton
Small GTPase domain
[205,206]
β-Actin
RhoA
Cytoskeleton
Cytoskeleton
YLNR and YRNY
phosphotyrosine motifs in
PX domain
PH domain (including two CRIB
sequence motifs)
Residues 613–723
PX domain
Death-effector domain
Kinase domain
–
Kinase domain (residues
348–370 required)
SH2 domain
[207]
[208]
Aldolase
α-Synuclein
PSGL-1
–
ENaC
Clathrin heavy chain (CHC17)
Other
Other
Cargo
PH domain
PX + PH domains
PX domain
–
Small GTPase domain
nucleotide free
–
N-terminal domain
C-terminal cytoplasmic tail
[209]
[196]
[210]
Cargo
Trafficking
–
–
[211]
[21]
Trafficking
Other
–
Inverted clathrin box within PX
domain (DFEWL)
–
–
–
–
[20]
[211]
Signalling
–
SH3 domain
[212]
PX protein
PX protein
PX protein
PX protein
Cargo
PX protein
Cargo
PX domain
PX domain
PX domain
PX domain
PX domain
C-terminal region
Residues 710–810 (human
numbering)
[213]
[213]
[213]
[213]
[213]
[214]
[214–216]
Signalling
C-terminus
PX domain
–
–
–
–
C-terminal region
Cytoplasmic domain
peptide
IYILYKLGFFKRSL
C-terminus
Signalling
Signalling
Cytoskeleton
PX protein
N-terminus
N-terminus
N-terminus
C-terminal coiled-coil
Kinase domain
PDZ and kinase domains
–
C-terminal coiled-coil
[218]
[219]
[220]
[221,222]
Retromer
Usp10
–
–
–
–
–
Haemopoietic cell-specific Lyn
substrate 1 (HS1)
–
SNX15
SNX1
SNX2
SNX4
PDGFR
IRAS
α5 Integrin
Insulin receptor substrates (IRS-4,
IRS-1, IRS-2, IRS-3)
PAK1, PAK4, PAK5 kinases
LIM kinase
Rac1
SNX16
–
–
c The Authors Journal compilation c 2012 Biochemical Society
[201,203–205]
[217]
The PX protein family
Table S3
Yeast PX proteins and their closest mammalian homologues
PX family
Yeast protein
Yeast name
Aliases
Closest human homologue*
PX-BAR
YOR069W
YOR132W
YMR004W
YJL036W
YDR425W
YDL113C
YKR078W
YML104C
YHR105W
YOR357C
YKR031C
YPL115C
GRD2, PEP10, VPT5
PEP21, VPT3
SNX8
CVT13, ATG24
SNX1
None
PX-PH-PLD
PX-PH-GAP
Vps5p
Vps17p
Mvp1p
Snx4p
Snx41p
Snx42p
YKR078Wp
Mdm1p
Ypt35p
Snx3p
Pld1p
Bem3p
PX-SNARE
Vam7p
SH3-PX-CAD
PX-unknown
PXA -RGS-PX-PXA PX-only
GRD19
SPO14
YJL036W
SNX30
SNX4
SNX4
None
SNX13
SNX13, SNX11
SNX3
PLD2
None
YGL212W
VPS43, VPL24
KIF16B
Bem1p
YBR200W
SRO1
SH3PXD2B
YPR097Wp
YPR097W
CVT20, ATG20
None
Notes
Has extended loop between helices H2 and H3 of BAR domain
Has extended loop between helices H2 and H3 of BAR domain
Has a large insert between helices α1 and α2 of the PX domain
Mammalian PX-SH3-GAP proteins have an SH3 domain, not a
PH domain. They have extended regions C-terminal to the PX
and GAP domains, whereas the yeast proteins have extended
regions N-terminal to the PX and GAP domains
The Vam7p SNARE is a clear invention of fungi, on the basis of
detailed phylogenetic analysis [223].
Has two N-terminal SH3 domains upstream of the PX domain
and a CAD/PB1 domain C-terminal to the PX domain. The
CAD/PB1 domain is from the ubiquitin superfamily
The PX domain of YPR097Wp is highly degenerate, with a
predicted insert of ∼130 amino acids between α1 and α2.
YPR097Wp is large (1073 residues) and overall is predicted
to be highly α-helical in structure. However, there are no
other known domains identified outside the PX domain
*The closest human PX protein homologue is listed, although this may not necessarily mean the proteins are orthologues. Where ‘none’ is stated this means that, on default settings, BLAST
searches did not retrieve any known PX domain proteins encoded in the human genome.
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