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Phil. Trans. R. Soc. B (2012) 367, 2556–2573
doi:10.1098/rstb.2012.0107
Review
Evolution of SH2 domains and
phosphotyrosine signalling networks
Bernard A. Liu1 and Piers D. Nash2, *
1
2
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada
Ben May Department for Cancer Research and Committee on Cancer Biology,
The University of Chicago, Chicago, IL 60637, USA
Src homology 2 (SH2) domains mediate selective protein–protein interactions with tyrosine phosphorylated proteins, and in doing so define specificity of phosphotyrosine (pTyr) signalling
networks. SH2 domains and protein-tyrosine phosphatases expand alongside protein-tyrosine kinases
(PTKs) to coordinate cellular and organismal complexity in the evolution of the unikont branch of the
eukaryotes. Examination of conserved families of PTKs and SH2 domain proteins provides fiduciary
marks that trace the evolutionary landscape for the development of complex cellular systems in the
proto-metazoan and metazoan lineages. The evolutionary provenance of conserved SH2 and PTK
families reveals the mechanisms by which diversity is achieved through adaptations in tissue-specific
gene transcription, altered ligand binding, insertions of linear motifs and the gain or loss of domains
following gene duplication. We discuss mechanisms by which pTyr-mediated signalling networks
evolve through the development of novel and expanded families of SH2 domain proteins and the elaboration of connections between pTyr-signalling proteins. These changes underlie the variety of general
and specific signalling networks that give rise to tissue-specific functions and increasingly complex
developmental programmes. Examination of SH2 domains from an evolutionary perspective provides
insight into the process by which evolutionary expansion and modification of molecular protein interaction domain proteins permits the development of novel protein-interaction networks and
accommodates adaptation of signalling networks.
Keywords: SH2; protein-tyrosine kinase; eukaryotes; phosphotyrosine
1. INTRODUCTION
Phosphotyrosine (pTyr)-mediated signalling is a relatively recently evolved cellular communication system
in comparison to more primordial post-translational
modifications (PTMs) such as Ser/Thr phosphorylation or lysine ubiquitination that are used across all
branches of the eukaryota [1]. Some prokaryotes
possess unique tyrosine kinases that are involved in
pTyr-mediated cellular activities but these are distinct
from those found in metazoans (see Grangeasse et al.
[2]). Signalling mediated through the use of pTyr is
essential in metazoans, regulating many key cellular
and developmental processes, including cell growth,
proliferation, differentiation and migration. In
metazoans, pTyr signalling depends upon an essential
triad of signalling molecules: the protein-tyrosine
kinases (PTKs) that add a phosphate onto substrate
tyrosines (conceptually these may be viewed as the
‘writers’); the protein-tyrosine phosphatases (PTPs)
that remove or dephosphorylate substrates (the ‘erasers’); and the modular protein interaction domains
that recognize the phosphorylated ligand (the ‘readers’) and hence recruit the proteins containing these
* Author for correspondence ([email protected]).
One contribution of 13 to a Theme Issue ‘The evolution of protein
phosphorylation’.
domains to specify downstream signalling events [1].
Several modular interaction domains have the capability to bind to tyrosine-phosphorylated ligands.
These include most Src homology 2 (SH2) domains
[3,4], a subset of phosphotyrosine-binding (PTB)
domains [5], at least one C2 domain [6] and the
Hakai pTyr-binding domain [7]. The SH2 domain is
the largest domain family dedicated to pTyr recognition, with 111 proteins containing at least one SH2
domain encoded in the human genome [8]. With the
recent sequencing of the genomes of multiple organisms, the identification of the PTKs, PTPs and SH2
domains has provided insights into the emergent evolution of this PTM signalling system [1,9 – 11]. Recent
examination of the evolutionary history of SH2
domains indicates that PTKs coevolved along with
SH2 domain proteins to bring about organismal and
tissue complexity in metazoans [8]. The diversification
of proteins that contain SH2 domains allows this
singularly remarkable domain to coordinate specificity
in a diverse range of signalling networks.
This review explores the origins of pTyr-mediated
signalling through the expansion and diversification
of SH2-domain-containing proteins. Examination of
the origins of SH2 domains and their respective
protein families reveals that SH2 domains are integrated into a diverse set of protein interaction
modules. In this way, SH2 domains coordinate
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This journal is q 2012 The Royal Society
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Review. Evolution of pTyr signalling
(a)
B. A. Liu and P. D. Nash
54
PTB
90
PTKs
2557
111
SH2
P
P
x–3-x–2-x–1-Y-x+1-x+2-x+3
x–3-x–2-x–1-Y-x+1-x+2-x+3
>10 000 pTyr sites
P
107
PTPs
number of genes
(b)
180
360
450
751–993
540–650
970
990
650
H. sapiens
90
M. musculus
M. domestica
X. tropicalis
D. rerio
C. intestinalis
S. purpuratus
A. aegypti
D. melanogaster
C. elegans
N. vectensis
M. brevicollis
250
761–957
1400
1500
1000 900 800 700 600 500 400 300 200 100
PTK
90
93
90
79
122
49
53
SH2
111
111
115
101
133
54
53
PTP
38 (107)
38
32
28
51
32
36
31
32
90
31
128
33
36
66
29
112
15
15
67
36
38
S. cerevisiae
0
1
3
D. dictyostelium
0
15
4
Ma
Figure 1. The phosphotyrosine signalling circuitry expands in unikonts to become a large and complex signalling system in
mammals. (a) The number of writers (protein-tyrosine kinases, PTKs), erasers (protein-tyrosine phosphatases, PTP) and
readers (Src homology 2, SH2; phosphotyrosine-binding domain, PTB) in humans. PTB (green) and SH2 (light blue)
domain both contain a pTyr binding pocket (yellow circle), yet specificity is determined by a secondary binding site adjacent
to the pTyr binding pocket that recognize sequences surrounding the pTyr. (b) The total number of PTK, classical PTPs (total
number of tyrosine phosphatases) and SH2-encoded genes across eukaryotes. The branched divergence times and lengths in
millions of years (Ma) are indicated.
specificity across a multitude of signalling pathways.
As we trace the evolutionary history of the different
protein families, we discuss the various evolutionary
paths by which these proteins change and diverge. In
detail, we will highlight the key underlying genetic
mechanisms of diversification through gene duplication, transcriptional regulation, domain loss and
gain, and insertion of linear motifs. Lastly, we will
explore both the conserved and divergent nature of
pTyr-mediated signalling networks to provide a
better appreciation for the complexity of mammalian
pTyr networks. This overview will introduce the mechanisms by which adaptive changes within pTyr– SH2
interaction networks act to drive cellular complexity
and participate in the development of tissue specific
signalling networks. Understanding the molecular
changes that drive expansion and complexity of
pTyr signalling networks provides an early glimpse of
some aspects of the process of molecular evolution
that underlies the adaptive changes in organisms as
they adapt and develop more elaborate functions and
increasingly complex tissue types.
Phil. Trans. R. Soc. B (2012)
2. SH2 DOMAINS HAVE A BROAD ROLE
IN PHOSPHOTYROSINE SIGNALLING
AMONG MAMMALS
Much of our knowledge and understanding of pTyr
signalling and SH2 domains comes from studies
in mammals, particularly in mouse and human.
The human genome encodes roughly 90 PTKs,
107 PTPs, 54 PTB domain-containing proteins (only
about one-fifth of PTB domains can bind pTyr) and
111 SH2-domain-containing proteins. This creates a
complex network of regulators in pTyr signalling
(figure 1a) [3,12,13]. Since the discovery of pTyr
nearly three decades ago [14], pTyr signalling has
been implicated in almost every aspect of normal cellular function and found to be a major driver in
human malignancies such as cancer [15,16]. Tyrosine
phosphorylation encompasses a small percentage (up
to 2 per cent) of the total protein phosphorylation
within a mammalian cell, although a list of over
10 000 putative pTyr sites have been identified so far
across various tissues and species [17,18]. The discovery of the SH2 domain [19– 21] led to numerous
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B. A. Liu and P. D. Nash
Review. Evolution of pTyr signalling
studies mapping interactions and SH2 domain selectivity that have led to a growing understanding of
specificity of pTyr signalling (reviewed in earlier
studies [3,4,22]). SH2 and PTB domains determine
selectivity for pTyr ligands by recognizing pTyr and
residues surrounding the pTyr (figure 1a). The preference for þ1 to þ5 residues C-terminus of the pTyr in a
sequence-dependent manner is dictated by the surface
residues on the SH2 domain that lie adjacent to the
pTyr-binding pocket. In contrast, PTB domains recognize sequences N-terminus to the pTyr with a core
consensus of N-P-x-pY, yet only a small fraction of
PTB domains are capable of binding pTyr peptides.
An overwhelming preponderance of what we know
about pTyr signalling is based on studies in human
and mouse systems, with very little known about pTyr
signalling in simpler organisms. Mammalian genomes
and protein interaction maps provide one window
into the role of pTyr signalling in the development of
complex organisms. Indeed, a study of human PTK circuits using prediction networks to identify PTKs, SH2
and PTPs suggests an evolving paradigm of pTyr signalling and evolution [23]. In this review, we describe the
evolutionary provenance and trajectory of aspects of
the cellular pTyr circuitry. The available pTyr machinery in various extant organisms suggests that pTyr use
transitioned from a nuclear and cytoplasmic system in
early unicellular eukaryotes to signalling at the cellular
membrane through use of receptor tyrosine kinases
(RTKs). This evolutionary analysis of pTyr signalling
has implications for the involvement of PTKs and
pTyr signalling in human disease such as cancer [23].
The spatio-temporal regulation of tyrosine phosphorylation is necessarily complex in mammalian
cells. The interplay between the substrate tyrosines
and the array of available PTKs and PTPs governs
the timing, localization and longevity of phosphorylation events. PTKs and PTPs commonly have
somewhat limited substrate specificity for peptide
ligands. They are, however, tightly controlled in both
their activation and spatial localization. The result is
that, as a general paradigm, PTKs and PTPs control
the spatio-temporal localization of phosphorylation
events but have limited deterministic control over
downstream events. Specificity in pTyr signalling is
thus largely determined by recognition modules, such
as SH2 domains, that distinguish specific tyrosine
phosphorylated motifs and act to nucleate complex formation to propagate signalling. Further complexity
arises from tissue- and cell-specific expression profiles
and dynamic sub-cellular localization of reader proteins
that may play a role in the cellular context of protein
phosphorylation. Therefore, dissecting the temporal
and spatial organization of dynamic protein interaction
networks is an essential element in understanding
information flow through the signalling system [24].
Transgenic animal models give a first approximation
of the role of a protein in a signalling network. Over 80
of the 111 SH2 proteins have been genetically disrupted
in mice, revealing a broad range of cellular functions for
SH2 domains in various tissues and cell types [3]. However, in order to extrapolate the multiple biological
functions of these proteins and understand the cellular
context by which these proteins regulate cellular
Phil. Trans. R. Soc. B (2012)
activity, understanding of the PTKs, PTPs and potential interaction networks within specific cell types or
tissues is required. This already challenging problem
is compounded by the copious variety of tissues and
cell states. Further complexity arises when multiple
members of a closely related gene family are present
where some extent of overlapping or redundant functions coexist with specialized roles. For instance, in
the GRB2 family (Grb2, Gads and Grap), Grb2 is ubiquitously expressed and plays an essential role in growth
factor signalling downstream of RTKs. Gads and Grap,
by contrast, are highly restricted in their expression to
a subset of immune cells and appear to function
primarily in signalling downstream of the T-cell and
B-cell receptors. Loss of Grb2 results in embryonic
lethality [25], whereas loss of Gads or Grap leads to
rather specific immune phenotypes [26,27]. While the
three encoded genes are closely related, they possess
distinct roles in their specific cellular context. This is
not always the case for all families, suggesting that
some members have redundant roles while some individual genes within a family have a ubiquitous and
essential role in development [3]. We discuss some of
the evolutionary patterns that drive divergence and
complexity in SH2-domain-containing proteins in §6.
3. IDENTIFICATION AND CLASSIFICATION OF
SH2 DOMAINS
Large numbers of whole genome sequences, combined
with emerging bioinformatics tools, present the opportunity to examine signalling systems across a broad
range of organisms. Modular domain families are
identified from sequence datasets using hidden
Markov models and multiple sequence alignments
tools such as those developed by the protein families
database (Pfam) [28], simple modular architecture
tool (SMART) [29] and conserved domains database
(CDD) [30]. Soon after the availability of their annotated genomes, the human and mouse complements of
all the kinases, including the PTKs, as well as the
PTPs, were reported [12,13,31,32]. This was followed
by the identification of 111 SH2 domain proteins in
the human and mouse genomes [3] and later by the
total complement of PTKs and SH2 encoding genes
across multiple eukaryotic organisms (figure 1b)
[8,10,11]. The 111 SH2-containing proteins contain
a diverse array of additional protein interaction
domains (e.g. SH3, PTB, PH) and catalytic domains
(e.g. TK, phosphatase) that are gained and lost over
time to generate diversity in signalling functions
[3,33]. With over 150 eukaryotic genomes sequenced
till date, bioinformatic approaches are required to
identify protein domains and trace paralogues and
orthologues across these diverse organisms. By examination of SH2 domains across 21 extant species in
the Eukaryota we identified 38 conserved families of
SH2 domains using a hierarchical method combining
sequence homology, domain organization and positions of splice-site patterns [3,8]. Such a hierarchical
method was necessary to avoid improper classification
of a domain family based on sequence homology
alone. Analysis of splice-site position within the open
reading frame provides an easily accessible hallmark
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Review. Evolution of pTyr signalling
B. A. Liu and P. D. Nash
2559
Table 1. SH2 domain families.
family
human gene names
domains (in order)
pseudogenes (yes/no)a
ABL
Abl1, Abl2
no
CBL
Cbl, CblB, CblC
CHN
CRK
CSK
DAPP1
FPS
FRK
GRB2
GRB7
JAK
NCK
PI3KR
PLCg
Chn1, Chn2
Crk, CrkL
Csk, Matk
Dapp1
Fes, Fer
Frk, Brk, Srms
Grb2, Gads, Grap
Grb7, Grb10, Grb14
Tyk2, Jak1, Jak2, Jak3
Nck1, Nck2
Pik3r1, Pik3r2, Pik3r3
Plcg1, Plcg2
PTPN
RASA1
RIN
SH2B
SH2D1
SH2D2
SH2D3
SH2D4
SH2D5
SH3BP2
SHB
SHC
SHIP
SLAP
SLP76
SOCS
Ptpn6, Ptpn11
Rasa1
Rin1, Rin2, Rin3
Aps, Lnk, Sh2b
Sh2d1a, Sh2d1b
Sh2d2a, Hsh2, Sh2d7
Sh2d3a, Sh2d3c, Bcar3
Sh2d4a, Sh2d4b
Sh2d5
Sh3bp2
Shb, Shd, She, Shf
Shc1, Shc2, Shc3, Shc4
Ship1, Ship2
Slap, Slap2
Slnk, Blnk, Slp76, Mist
Socs1, Socs2, Socs3, Socs4,
Socs5, Socs6, Socs7, Cish
Src, Fyn, Lck, Fgr, Yes, Lyn,
Hck, Blk
Bks, Brdg1
Stat1, Stat2, Stat3, Stat4,
Stat5a, Stat5b, Stat6
Supt6h
Zap70, Syk
Bmx, Tec, Btk, Itk, Txk
Tns1, Tns3, Tenc1, Tns4
Vav1, Vav2, Vav3
SH3, SH2, TK , F-actin binding
domain
EF-Hand, SH2, 4-helix bundle,
RING, UBAb
SH2, C1, RhoGAP
SH2, SH3, SH3
SH3, SH2, TK
SH2, PH
F-Bar, SH2, TK
SH3, SH2, TK
SH3, SH2, SH3
RA, PH, SH2
FERM, SH2, TK
SH3, SH3, SH3, SH2
SH3b, RhoGAPb, SH2, SH2
PH, PLCXc, PH, SH2, SH2,
SH3, PLCYc, C2
SH2, SH2, phosphatase
SH2, SH3, SH2, PH, C2, RasGAP
SH2, VPS9, RA
PH, SH2
SH2
SH2
SH2, RasGEF
SH2
PTB, SH2
PH, SH2
SH2
PTB, SH2
SH2, phosphatase
SH3, SH2
SAMb, SH2
SH2, Socs
SRC
STAP
STAT
SPT6
SYK
TEC
TNS
VAV
no
no
no
no
no
no
no
no
no
no
no
no
no
yes
no
no
no
yes
no
no
no
no
no
no
yes
no
no
no
yes
SH3, SH2, TK
yes
PH, SH2
DNA BD, SH2, TA
no
no
CSZ, S1, SH2
SH2, SH2, TK
PH, BTK, SH3, SH2, TK
PTPc, SH2, PTB
CH, RhoGEF, PH, C1, SH3, SH2, SH3
no
no
no
no
no
a
Human pseudogenes identified using the Pseudofam database (http://pseudofam.pseudogene.org/) [35]; bdomains that are not shared
among all family members. For complete domain descriptions visit http://pawsonlab.mshri.on.ca or http://www.sh2domain.org.
of conserved genomic structure that can assist in
accurate classification of a family [8,34]. This provided
a methodology that could trace families as well as
individual proteins across lineages and species.
Domain families assist in defining orthologous and
paralogous proteins across the 21 different eukaryotes.
This in turn provides insights into the evolutionary
mechanisms that drive diversification within these
families that create new proteins with both shared
and novel functions.
The 38 families of SH2 domains in humans encode a
heterogeneous range of proteins that connect pTyr signalling to a diverse array of cellular systems. Many families
have distinctive domain architectures that place the SH2
domain in the context of other modular protein domains
such as SH3, PH, kinase, phosphatase, RING, PTB,
Phil. Trans. R. Soc. B (2012)
RasGAP, RasGEF, FERM and others (table 1) [3,33].
The various mechanisms by which diversity appears to
have arisen within individual families will be examined
in detail in later sections. Examination of these families
across a broad range of eukaryotes suggests points of
origin for each SH2 family at common ancestors representing branch points that separate specific lineages.
The role of each SH2 protein and selective advantage
conferred upon first emergence is rarely clear and presents a major challenge for understanding the core
cellular functions of primordial SH2 domain proteins.
Sequence analysis and functional studies provide a
means to trace the evolutionary history of SH2-mediated
pTyr signalling in metazoans. Examination of the coevolution of PTKs and SH2 domain proteins may provide
insights into their role in the development of
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B. A. Liu and P. D. Nash
Review. Evolution of pTyr signalling
(a)
C-SH2
N-SH2
C-SH2
Dictyostelium
N-SH2
mammals
(b)
Cbl
DIF-1
TK?
JAK
STATc STATc
STATc
PTP-3
STAT1 STAT1
STAT1
TCPTP
DIF-1
CblA
Figure 2. Primordial function of SH2 domains and pTyr signalling. (a) The ribbon structure of the yeast Spt6 tandem SH2
(left panel, PBD: 3PJP). The charged surface of the tandem SH2 (right) indicates the positions of negative (red) and positive
charge (blue) of the tandem SH2 domain. Highlighted in the yellow circle is the phospho-binding pocket for pSer. (b) The
SH2 domain transcriptional activator STAT (red) is present as early as Dictyostelium and found in humans. Analogous to mammalian STAT signalling, STATc is regulated by tyrosine phosphorylation (yellow dot) through PTKs (green) and PTPs (blue).
In Dictyostelium, the SH2/RING domain containing protein CblA (orange) antagonizes the phosphastase, while in mammals
Cbl inhibits the PTK Jak.
multicellularity and subsequent leaps in complexity of tissues and development of complex systems such as the
immune system and the mammalian brain (see §5).
4. SH2 DOMAINS PREDATE DEDICATED
PROTEIN-TYROSINE KINASES
Within Eukaryota, there exist two major divisions: the
bikonts (eukaryotic organisms derived from cells with
two flagella) and unikonts (organisms derived from
cells with one flagella). SH2 domains were previously
described in unikont organisms such as humans,
yeast, amoeba and choanoflagellates [10,11,36].
Recent analysis of SH2 domains across 21 eukaryotic
genomes revealed the presence of SH2 domains in
both divisions [8], including the unicellular organisms
Naegleria gruberi and Phytophthora capsici, and thus
SH2 domains probably predate the emergence of
PTKs [37]. A common SH2 domain protein, Spt6,
is found in both bikonts and unikonts and is universally present in metazoans. A handful of other SH2
domains are observed in certain bikont lineages, but
it remains unclear whether any of these are functional
and capable of binding pTyr [8]. Spt6 was first
described in the budding yeast Saccharomyces cerevisiae
Phil. Trans. R. Soc. B (2012)
[36] as an elongation factor, a histone H3 – H4 chaperone that mediates nucleosome assembly and interacts
with RNA polymerase II (RNAPII) [38,39]. The
SH2 domain of Spt6 is reportedly capable of recognizing the phosphorylated serines (pSer) of RNAPII
C-terminal tail (CTD) [40], with no detectable binding to pTyr peptides. Structural studies indicate
that Spt6 possesses a tandem SH2 fold [41,42]
(figure 2a). Only the first SH2 domain of Spt6 is
recognizable as a canonical SH2 domain by sequence
analysis. The second SH2 fold is highly atypical, lacking the FLVR (Phe-Leu-Val-Arg) motif for phosphobinding, and is not detected using standard SH2
domain models, such as SMART or Pfam. Both
SH2 domains are necessary for this region to recognize
pSer residues on the CTD of RNAPII [40]. The structure of the yeast Spt6 N-SH2 domain possesses many
sequence and structural features that clearly resemble
pTyr-binding SH2 domains in mammals [41,43 – 45].
Binding to pSer/pThr peptides uses the same canonical binding pocket as canonical SH2 domains use to
bind pTyr peptides [45]. Based on sequence and structure, it appears likely that the SH2 domain of Spt6
represents an ancestral pSer/pThr-binding domain
that may have given rise to pTyr-binding SH2 domains
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Review. Evolution of pTyr signalling
somewhere around the divergence between Unikonta
and Bikonta. The Spt6 SH2 remains well conserved
from yeast to humans.
Most branches of Unikonta, including Amoebozoa,
Choanozoa and Animalia, contain diverse sets of SH2
domains, suggesting that pTyr-binding SH2 domains
developed very early in the unikont lineage. In members of the Amoebozoa lineage, such as the
Entamoebae and Mycetozoa, SH2 domains expand
into an independent set of proteins with relatively
few obvious homologues in Metazoa (e.g. Shk, LrrB)
[46,47]. This is despite the absence of dedicated
PTKs that existed within these species. It is not
immediately clear how pTyr signalling networks are
constructed within these organisms. Some Ser/Thr
kinases, such as dual specificity MAPKs [48], as well
as other unique PTKs such as DPYK [49], are
capable of phosphorylating tyrosine residues. Such
non-traditional tyrosine kinases may explain the
expansion and maintenance of SH2 domain proteins
in Amoebozoa such as Dictyostelium discoideum. It is
also in Amoebozoa that we first encounter SH2
domains linked to a dual-specificity kinase, the Ser/
Thr/Tyr kinase Shk [50]. Despite the absence of
PTKs, tyrosine phosphorylation has been reported in
D. discoideum and can be observed in the phosphorylation of the C-terminus of STATc [51,52] (figure 2b).
When STATc is tyrosine phosphorylated, it forms a
homodimer through reciprocal SH2-domain– pTyr
interactions and then accumulates in the nucleus,
functioning in a manner analogous to the mammalian
STAT orthologues. Amoebozoa, Choanozoa and
Metazoa all encode SH2-domain-containing Stat proteins. In addition, D. discoideum contains a Cbl-like
pTyr-binding region, comprising an EF-hand and an
SH2 domain, linked to an E3 ubiquitin-ligase RING
domain. D. discoideum CblA functions as a positive
regulator of STATc by downregulating a tyrosine
phosphatase, whereas metazoan CBL proteins negatively regulate tyrosine kinases [52]. Because D.
discoideum diverged from the lineage that gave rise to
Metazoa before Fungi but after the Bikonta/Unikonta
split, it remains unclear why STATs, CBL and other
SH2 domain proteins were apparently lost in Fungi
[53]. In addition to the SH2 domain proteins, 24
kinase subfamilies shared between D. discoideum and
Metazoa are absent in yeast, suggesting that yeast
developed a specialized biological programme that
does not require many of the kinase signalling networks used in other unikont branches [54]. In fact,
the progenitor to the yeasts may have used pTyr signalling, but, as Gerard Manning’s analysis suggests,
the PTKs and some other kinase families were lost
as the yeasts evolved leaving a few PTP/DSPs (dual
specificity phosphatases). Fungi, which together with
Metazoa are in the opisthokont branch of Unikonta,
lack any apparent pTyr-binding SH2 domains other
than the pSer-binding SPT6. pTyr-binding SH2
domains were lost from Fungi, perhaps as a result of
deleterious outcomes from the occurrence of pTyr.
This is supported by lethal effects of expression of the
PTK v-Src in S. cerevisiae that are associated with
loss of cell cycle control [55,56]. The ability of v-Src
to kill yeast cells is dependent upon its SH2 domain
Phil. Trans. R. Soc. B (2012)
B. A. Liu and P. D. Nash
2561
as an active kinase alone has little effect on yeast
growth [57,58]. This suggests that pTyr-mediated signalling may have been negatively selected out of the
Fungi common ancestor around 1200 Ma [59,60]
and that functional use of pTyr and SH2 domains
may predate dedicated PTKs [47].
Tyrosine phosphorylation and tyrosine kinase signalling is thought to occur primarily in the cytoplasm,
particularly at the plasma membrane, in metazoans.
Upon ligand engagement or cell contact, activated
RTKs phosphorylate their own cytoplasmic tails as
well as membrane-localized substrate proteins. SH2
domain proteins are then recruited to activated
signalling complexes at the plasma membrane or on signalling endosomes to propagate downstream signalling.
In this paradigm, much of the PTK–SH2 signalling is
cytoplasmic in nature with only limited examples of
nuclear involvement (however, more and more nuclear
pTyr proteins are being reported, although admittedly
this does not necessarily tell us where in the cells
these proteins were phosphorylated or whether they
have nuclear SH2-domain-binding partners). In contrast, primordial SH2 domain-containing proteins,
SPT6 and STAT, function in the nuclear compartment
of the cell. There was probably significant nuclear pTyr
signalling in early unicellular eukaryotes that remains a
feature of various lineages. This is supported by several
lines of evidence that tyrosine phosphorylation plays a
role in the nucleus. Tyrosine phosphorylation of nuclear
cell cycle regulators such as Cdc28 and Cdc2 is
observed in mammalian and yeast cells [61–63]. Staining for pTyr in the unicellular organism Giardia lamblia
indicates enrichment in the nuclei that is distinct in
comparison to pSer/Thr staining [64]. This suggests
that in early unicellular eukaryotes, pTyr signalling
may have been more central to the nuclei with concurrent evolution of SH2 domains that may mediate
nuclear functions such as DNA transcription (e.g.
Stat proteins). As cytoplasmic tyrosine kinases
(CTKs) emerged, pTyr signalling may have become
more extensively used for cytoplasmic functions, eventually becoming focused at the plasma membrane
with the emergence of RTKs. Extensive RTK signalling
mediated by SH2 domains is itself a hallmark of multicellularity in Metazoa and represents a key stepping
stone in the emergence of multicellularity in the
animal lineage [23].
Early in the unikont lineage, SH2 domains appear
that have the ability to recognize pTyr-based motifs.
This is a key feature allowing rapid evolution of pTyr
signalling by shuffling SH2 domains into various
proteins [1]. In pre-metazoans, specifically the choanoflagellate lineage, diversification of SH2 domains and
PTKs is tightly linked, suggesting that they coevolved
to create diverse functional signalling systems.
Within the unikont lineages, SH2 domain expansion
may be partially driven by the kinases that generate
the binding partners for SH2 domains through
phosphorylation. True PTKs first appeared in the
unicellular choanoflagellate Monosiga brevicollis [65],
but are absent in amoeba and slime moulds [54]. The
choanoflagellate branch, which includes M. brevicollis,
encodes the core set of PTKs, PTPs and SH2 domains
[10,11] and thus exhibits a functional pTyr signalling
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Review. Evolution of pTyr signalling
phylum
38
+1
+5
+9 –2
–5
–6
+20
Opisthokonta
+1
–2
Unikonta
+3
Homo sapiens
Chordata
+2 –1 37
–3
35 Urochordata
+1 –4
35 Echinodermata
SH2 families
Amoebozoa
–3
genus species
Danio rerio
Ciona intestinalis
Strongylocentrotus purpuratus
27 Arthropoda
25 Nematoda
Drosophila melanogaster
Caenorhabditis elegans
24 Cnidaria
Nematostella vectensis
21 Choanozoa
1
Ascomycota
3 Mycetozoa
Monosiga brevicollis
0
Saccharomyces cerevisiae
Dictyostelium dictyostelium
Entamoebae
Entamoeba histolytica
phylum
genus species
other eukaryotes
29
Homo sapiens
Chordata
tyrosine kinase
families
+1 –2
+3 –1
+5
+12
+6
Opisthokonta
Unikonta
Amoebozoa
–1
–5
29
Danio rerio
27
Ciona intestinalis
Urochordata
25 Echinodermata
22
Strongylocentrotus purpuratus
Arthropoda
18 Nematoda
18 Cnidaria
Drosophila melanogaster
Caenorhabditis elegans
6 Choanozoa
0
Ascomycota
0
Mycetozoa
0
Entamoebae
Monosiga brevicollis
Nematostella vectensis
Saccharomyces cerevisiae
Dictyostelium dictyostelium
Entamoeba histolytica
other eukaryotes
Figure 3. The gain and loss of SH2 and PTK families in Eukaryota. The eukaryotic tree is shown with the phylum and genus species
listed on the right. The number of SH2/PTK families are listed in black text with the gain and loss indicated in grey text. The 38 SH2
and 29 PTK families represent those that are found in humans and are present in other lower eukaryotes [8,10,34].
network reminiscent of metazoans. A total of 19 conserved SH2 families and six conserved PTK families
are found in M. brevicollis, several of which contain
both an SH2 and a TK domain (figure 3). The heterogeneity of the M. brevicollis pTyr system suggests
that SH2 proteins may have cellular roles that diverge
somewhat from their mammalian counterparts. For
instance, the CTK MbCsk is co-expressed with
and can phosphorylate MbSrc1 at tyrosine near
the C-terminus in a manner similar to that of the mammalian Src and Csk pair. However, the negative
regulation of Src through phosphorylation of this residue
is absent from the M. brevicollis pair, suggesting that allosteric regulation of Src developed more recently in the
metazoan lineage [66,67]. The presence of an extensive
and varied collection of pTyr signalling molecules
suggests that M. brevicollis depends upon pTyr to
mediate a multitude of cellular functions, most of
Phil. Trans. R. Soc. B (2012)
which we have yet to understand. It will be fascinating
to see what roles unique SH2 proteins in M. brevicollis
serve in coordinating organism-specific pTyr signalling.
5. EMERGENCE OF PROTEIN-TYROSINE
KINASE AND SH2 DOMAIN FAMILIES FOR
CELLULAR COMPLEXITY
Throughout the evolution of Metazoa, new SH2 and
PTK families appear while others are occasionally
lost (figure 4). The timing of the appearance of new
families may indicate the evolution of specific signalling networks that support an adaptive advantage.
Phenotypic complexity and complex developmental
programmes are hallmarks of metazoan evolution.
Numerous SH2 families emerge in relatively simple
eukaryotes such as Choanozoa and Cnidaria. It
remains unclear what the specific cellular functions
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B. A. Liu and P. D. Nash
Review. Evolution of pTyr signalling
SH2D5
SLAP
SH2D1
AXL
AATYK
TIE
TRK
STAP
JAK
SLP76
MUSK
SHB
ALK
EGFR
MET
SOCS
SH2D3
SH2B
NCK
GRB7
DAPP1
CHN
ACK1
FAK
NOK1
PTK7
ROR
RYK
VEGFR
DDR
INSR
FPS
FRK
SYK
VAV
TNS
SHIP
SHC
SH3BP2
SH2D4
SH2D2
RIN
RASA1
PTPN
PLCG
PIK3R
GRB2
CRK
TEC
SRC
CSK
ABL
EPHR
FGFR
CBL
STAT
SPT6
PTB
SH2
2563
PTB
SH2
SH3
SH2
IG
IG
FN3
T
M
T
M
FN3
SH3
Y kinase
Y kinase
E
G
F
E
G
F
IG
Cys-Rich
PH
T
M
Y kinase
T
M
Y kinase
FN3
IG
Cys-Rich
IG
IG
E
G
F
SH2
FERM
STY kinase
SH2
SAM
Y kinase
SH2
IG
IG
IG
SH2
T
M
kringle
Cys-Rich
Y kinase
SH2
Y kinase
LDLa
FU
FU
FU
SEMA
IG
PSI
IG
IG
T
M
Y kinase
T
M
Y kinase
T
M
Y kinase
SH2 SOCS
phosphatase
RasGEF
Y kinase
SH2
PH
SH3
SH3
FN3
RasGAP
SH2
SH3
RA
RasGEF
SH2
PH
IG
SH2
PH
SH2
C1
SH2
T
M
RhoGAP
CDC42 BD
Y kinase
Y kinase
IG
IG
IG
IG
Y kinase
IG
IG
Cys-Rich
IG
IG
T
M
T
M
Y kinase
T
M
Y kinase
T
M
Y kinase
IG
IG
Kringle
T
M
IG
kringle
FAT
T
M
FERM
Y kinase
IG
IG
Y Kinase
PH
FN3
Cys-Rich
Y kinase
Y kinase
RhoGAP
Y kinase
SH2
SH2
T
M
FN3
SH2
F-Bar
SH3
Cys-Rich
F-Bar
Y kinase
SH2
CH
RhoGEF
C1
PTPc
DSPs
C1
PH
SH2
SH3
SH3
C1
SH2
phosphatase
SH2
PTB
C2
SH2
PH
SH2
SH2
LDLa
SH2
VPS9
SH2
SH2
SH2
SH3
SH2
SH3
C2
PTPc
DSPs
RasGAP
phosphatase
SOCS
PLCXc PH SH2
PH
SH2
SH2
RhoGAP
SH3
RA
PH
SH2
SH3 PH PLCYc
SH2
CDC42 BD
SH3
SH2
SH2
SH3
PH
BTK
SH3
FU
SH3
Y kinase
SH2
SH3
SH2
Y kinase
SH3
SH2
Y kinase
DNA BD
SH3
Y kinase
SH2
IG
IG
4H
IG
4H
FN3
Cys-rich
IG
T
M
FN3
T
M
Y kinase
Y kinase
EF
EF SH2 RING
DNA BD
TEX
SH2
RING
TA
S1
SH2
ns
H. sapie
ulus
M. musc
nstica
M. dome
alis
X. tropic
D. rerio
nalis
C. intesti s
ratu
S. purpu
r
nogaste
D. mela
ti
A. aegyp
ns
C. elega
nsis
N. vecte
ollis
M. brevic
isiae
S. cerev
ostelium
D. dicty um
ure
D. purp
TEX
S1
TA
CH
Figure 4. Emergence and expansion of SH2 and PTK families. The presence and absence of conserved families of SH2 and
PTK proteins is indicated for representative species covering the unikont branch from social amoeba/slime mould Dictyostelium
species to H. sapiens. The families of SH2 and PTK were identified previously [8,10,34]. Text colour indicates SH2 families
that lack a TK domain (black), RTK families (orange), CTKs containing an SH2 domain (blue) and CTKs that lack an SH2
domain (green). The general domain organization of each family is shown on the right.
these proteins serve in primordial cellular signalling.
Certain SH2 families arise at distinct stages coincident
with the development of multicellularity. For example,
Phil. Trans. R. Soc. B (2012)
establishment of cell polarity in cnidarians probably
depended upon the appearance of specific signalling
pathways such as the canonical Wnt and b-catenin
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B. A. Liu and P. D. Nash
Review. Evolution of pTyr signalling
pathway [68,69]. Alongside appeared several SH2
domain families, including NCK and SOCS. Both
Nck and Socs7 play an important role in regulating
cell polarity through Septins in mammals [70], which
suggests that they may have coevolved with the polarity
complexes that emerge in cnidarians [71]. Further
hints of the emerging programmes guiding multicellularity can been seen in the families of PTKs that are
identified in the cnidarian Nematostella vectensis. Only
two families of RTKs are present in M. brevicollis,
whereas nine additional families arise in N. vectensis.
This correlates with studies by Li et al. [23] suggesting
that early pTyr signalling was primarily mediated by
CTK. In cnidarians, additional RTK families (RYK,
ROR, VEGR and others) promote inter-cellular communication for cell – cell contact and cell adhesion
(figure 4). The coincident emergence of FAK, a
PTK crucially involved in regulating focal adhesions
critical for maintaining cell contact [72,73], during
this lineage further supports the development of this
complex cellular process.
In arthropods, complex tissues and organs begin
to emerge, such as the muscle, heart and vascular
system as well as the innate immune system [74]. The
emergence of the muscle-specific tyrosine kinase in
arthropods is coincident with the development of a
muscular system within this lineage (figure 4). In
addition to the emergence of new proteins and protein
families, protein interaction networks can be seen to be
coevolving in parallel. For example, Shc and Grb2 are
both observed in N. vectensis (figure 4). An interaction
between Shc and Grb2 is first seen in arthropods with
the insertion of a pTyr site within the CH1 region of
Shc [8]. Phosphorylation of this site allows for interaction with the SH2 domain of Grb2. In mammals,
this interaction is supported by multiple pTyr sites in
the products of four distinct Shc genes (Shc1, 2, 3, 4)
expressing various isoforms in a wide variety of tissues
[75]. Furthermore, elimination of these pTyr sites in
the Shc1 gene in mice leads to a muscle motor stretch
reflex defect, specifically a decreased number of
muscle spindles of the skeletal muscle sensory organs
that regulate motor behaviour [76]. This highlights
the various processes for emerging novel protein circuits
through evolving not just novel protein families but also
bringing together the pTyr circuit through evolving
pTyr motifs and SH2 domains in a manner that may
have promoted the development of tissue-specialized signalling. Additional analysis regarding evolution of pTyr
signalling networks will be discussed in a later section.
Although many proteins emerge at distinct points in
evolution, mutation, adaptation and selection underlie
a process capable of developing new interactions that
drive the creation of novel signalling networks. Functional interactions in one species do not, therefore,
necessarily represent primordial protein functions.
Studies in mammalian systems suggest that Grb2
associates with Shc and epidermal growth factor
receptor (EGFR), whereas Crk interacts with the
CTK c-Abl and thus plays essential roles in these signalling networks [77– 79]. Yet, these functions may be
later adaptations and signify the role that these proteins served in common ancestor species or the
role that they serve in other lineages. Both Grb2
Phil. Trans. R. Soc. B (2012)
and Shc appear in M. brevicollis and are found across
all metazoans, suggesting that they emerged in a
common pre-metazoan ancestor species (figure 4).
Grb2 associates with Shc through a pTyr-dependent
interaction dependent upon the SH2 domain of
Grb2 and the pY-x-N motifs present in the CH1
region of Shc. However, these pTyr-binding sites do
not appear until arthropods such as Drosophila melanogaster, suggesting that the ancestral role for Grb2 and
Shc were independent of this function, and that this
is a later adaptation. Phenotypic evidence supports
the assertion that the Grb2 – Shc – pTyr-mediated
interaction is indeed functionally important in signalling events in more complex multicellular metazoans
and in particular for signalling networks that underlie
the development of neuromuscular systems. In a similar manner, the Jak-Stat signalling network is
extensively used in adaptive immune signalling in
mammals [80] and gene disruptions in this network
often exhibit specific defects in immunological signalling. Yet, both Jak and Stat proteins predate the
adaptive immune system and appear to have arisen
at different points in evolution. This is consistent
with evolution proceeding by rewiring existing components when adaptive pressure drives the
development of more complex systems. Thus, the
ancestral function of proteins can either change or
become obscured by observed functions in wellstudied mammalian organisms. Shc1, for instance,
serves essential functions in mammals distinct from
the pTyr-mediated interaction with Grb2. Targeted
disruption of the Shc1 gene is embryonic lethal [81],
while disruption of the pTyr sites that allow Grb2
binding has a relatively specific function later in development [76]. The essential function of Shc1 may well
relate to the ancestral function of the protein in singlecelled pre-metazoan species. Evolution in this case has
resulted in the use of existing components to build new
circuits and connections that underlie novel signalling
pathways and add additional levels of complexity.
6. DIVERSIFICATION OF SH2 DOMAIN PROTEINS
THROUGH GENE DUPLICATION
Over 30 years ago, Ohno [82] elucidated the potential
for gene duplication to drive diversification, but it was
not until recently that this concept has become widespread and pivotal in understanding the evolution of
gene families and functional diversification. Approximately 65 per cent of human genes are duplicated,
with 33 of 38 SH2 and 25 of 29 PTK families possessing duplicate copies. Gene duplication provides the
opportunity for the duplicate gene to explore an otherwise prohibited mutational space. Freed from the
pressure to maintain essential function the newly
duplicate gene is free to wander, on condition that
the duplicate genes collectively continue to contribute
their original function. In the case of SH2 domain
proteins, very few new families arise at the split
between arthropods and vertebrates (figure 3).
Instead, gene duplication appears to function by providing an opportunity to build complexity out of
existing components. The expansion and divergence
of SH2 domains and pTyr signalling components
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Review. Evolution of pTyr signalling
(a)
B. A. Liu and P. D. Nash
2565
duplication
duplication
eliminated
fixation
subfunctionalization
(eg. VAV, NCK)
neofunctionalization
(eg. SLAP, SAP, GRB2)
degradation
(pseudogenes eg Grb2-ps1)
(b)
loss or gain
of domains
tissue specialization
transcriptional regulation
evolving domain
specificity
loss or gain of
linear motifs
Figure 5. Fates of duplicate genes. (a) When a new duplicate arises it is eliminated in a majority of cases as fixation in a population is rare event. After fixation, the gene can undergo three general fates that include: neofunctionalization, where a duplicate
copy acquires a novel function; non-functionalization of a gene through mutation of its promoter and/or mutation of its function
creating a pseudogene by inactivation; and subfunctionalization that divides the primary ancestral function between the duplicate
copies possibly across distinct tissue types. (b) Duplicate copies of genes can diverge through means of evolving novel protein
domain specificity for ligands, gain or loss of a protein domain, insertion or deletion of short linear motifs and lastly an alteration
in the promoter of the gene allowing for specialized tissue expression and transcription control.
provides a rich case study for how organisms and
genomes gain complexity by gene duplication followed
by functional divergence [83].
To understand how gene duplicates give rise to
diversification, let us consider the general mechanisms
and consequences. When a gene is duplicated, it faces
a number of hurdles that must be overcome for it to be
fixed into the genome of a species. If a duplicate gene
confers no selective advantage, it is likely to be lost or
mutated. In order for a gene duplicate to be observed
in an extant species, it must be first fixed within the
population, and second it must be preserved over
time [83,84] (figure 5a). Three fates are typically
envisaged for a gene duplicate. One fate, called nonfunctionalization, refers to the loss of all functionality
of a gene. This typically leaves behind a pseudogene
wherein the duplicate gene accumulates mutations
that destroy its function to the point at which it
can no longer be transcribed or translated. The
SH2-homology pseudogene Grb2-ps1 is present
within the mouse genome but is absent in humans,
Phil. Trans. R. Soc. B (2012)
yet no transcript can be identified and the sequence
is recognizably that of a pseudogene. At least 16
human SH2 pseudogenes can be identified using the
Pseudofam database (http://pseudofam.pseudogene.
org/) [35] (table 1). A second fate results when an
advantageous allele arises as a result of one of the
gene copies gaining a new function. This is referred
to as neofunctionalization. For example, the SLAP
family appears to have originated from a duplication
of a SRC family gene. The loss of the TK domain in
SLAP allowed this duplicate to acquire a new function
distinct from its ancestor. Similarly, SAP (SH2D1A),
arose from a duplication of SHIP, and lacks the phosphatase domain. Neofunctionalization is more likely to
occur in genes that are necessarily rapid evolving such
as those involved in immunity and host defence [85].
Lastly, the original functions of the single-copy gene
may be partitioned between the duplicates. This is
known as subfunctionalization. Biological function
suggests that many gene products perform a multiplicity of subtly distinct functions and thus selective
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B. A. Liu and P. D. Nash
Review. Evolution of pTyr signalling
pressures may result in a compromise between optimal
sequences for each role. Partitioning these functions
between the duplicates may increase the fitness of
the organism by removing the conflict between two
or more functions. For example, the families of
NCK and VAV each demonstrate subfunctionalization.
Loss of either Nck1 or Nck2 in mice result in only
subtle phenotypic changes [86 –88], whereas loss of
both alleles results in embryonic lethality. This is
indicative of overlapping and mutually compensatory
functions [89]. In a similar vein, loss of individual
genes within the VAV family causes no obvious phenotypes, whereas loss of all members of the family results
in a severe defect in lymphocyte development [90].
Subfunctionalization can also enable temporal and
spatial partitioning of expression, protein localization
and assembly of signalling complexes.
The driving force during evolution for preserving a
duplicate of an existing gene is to gain a selective advantage. Gene duplicates must confer an immediate
selective advantage (or at least not a selective disadvantage) and this is clearly the case if elevating the level of
protein expression (dosage) is advantageous. The
dosage balance involves changes in protein concentrations relative to those of potential binding partners,
where an imbalance in expression levels may potentially
result in deleterious effects such as improper protein
complex assembly. In order to provide balance in the
system, duplicate genes may undergo various paths for
divergence from its ancestor such as point mutations,
gene fusions, truncations, deletions and gene conversions. Mutation of a duplicate gene can arise through
multiple avenues that allow for divergence in a
manner that includes alterations in gene expression patterns through promoter mutations, changes in ligand
specificity, gain/loss of protein domains and insertion/
deletions of short linear motifs (figure 5b). Many factors
influence the number and divergence patterns for duplicate genes. For example, protein–protein interactions of
the ancestral gene can influence the number of duplicates. The more highly connected and more
centralized proteins found in humans are usually
encoded by duplicated genes, which favour the functional diversification of paralogues [91]. This feature is
unique to vertebrates following whole genome duplication (WGD), as gene dosage, in these cases, is
commonly
controlled
through
tissue-selective
expression and miRNA regulation. High connectivity
may favour the functional diversification of paralogues
through tissue specialization or other mechanisms
[92,93]. A strong correlation (r ¼ 0.93) of the number
of cell types with the number of SH2 domains suggests
that duplication may have driven tissue-specific
expression enabling specialized tissues and developmental programmes to evolve [94]. Dosage balance may act
as an intermediate step to neo- and subfunctionalization,
prolonging the retention of the duplicates before one of
the other mechanisms determines the ultimate fate of
the duplicates. This highlights the important role of
functional divergence of duplicate genes in optimizing
pTyr networks for specialized tissues and creating
novel signalling pathways.
Examination of the 38 conserved SH2 families
reveals 33 families with at least one gene duplicate in
Phil. Trans. R. Soc. B (2012)
the human genome. Within these families, there are
notable examples of some of the mechanisms driving
divergence between duplicate copies. Duplication can
lead to loss or gain of domains, often at the ends
(N-terminus or C-terminus) of the protein [95]. In
addition, duplicate genes can diverge in sequence to
acquire novel binding motifs either within the SH2
domain or elsewhere within the protein (discussed in
detail in §7). Five generalized classes have been
described for the divergence of SH2 families as a
result of duplication events combined with domain
loss or gain [8]. Gene duplication can simply result
in multiple members of a family, though the size of
the family can vary substantially with the SRC family
having eight functional genes in humans. The heterogeneity in family size likely relates to heterogeneity in
both the gain and loss of duplicate genes between
families, though it is not fully understood what evolutionary forces determine the pattern of gene
retention. In some cases, a novel domain may be
inserted or lost such as a SAM domain in SLP76 or
the UBA domain in Cbl-C, respectively. Some families
arise from neofunctionalization, where the gene
family undergoes a change such as a loss of catalytic
domain, as in the case of SH2D1 and SLAP, followed
by an additional level of duplication for subfunctionalization. Lastly, several SH2 families such as RASA1
and SH3BP2 remain single copy, suggesting that
any duplications that arose did not undergo fixation
or that the genes were inactivated through nonfunctionalization. These classes, while general in
definition, encompass the mechanism for divergence
by gene duplication.
7. PHOSPHOTYROSINE NETWORKS
EVOLVE THROUGH SH2-DOMAIN-MEDIATED
INTERACTIONS
The pTyr signalling networks are important contributors to the evolution of organismal complexity in the
unikont branch of Eukaryota, and particularly in
Metazoa. Genome-wide loss of tyrosines in proteins
across metazoans correlates with the expansion and utilization of pTyr as a novel PTM that carries significant
functional importance in signal transduction [59]. Evolutionary forces have driven the assembly of novel
regulatory pathways and networks in pTyr signalling
over the course of metazoan evolution. The pTyr
protein interaction network involves the physical interaction between a pTyr ligand and the recognition by
domains such as SH2 and PTB. Existing interactions
that provide a selective advantage must either coevolve
or remain conserved through evolution. Novel
interactions may evolve rapidly by integrating pTyr
motifs recognized by SH2 domains into scaffolds and
other SH2 domain proteins. The identification of both
conserved and rapidly evolving phosphorylation sites
may have important implications for understanding
human diseases [96]. Understanding of the mechanisms
driving the elaboration of pTyr networks by modification of SH2 domain specificity and the integration
of pTyr signalling into diverse signalling pathways also
have implications for synthetic biology approaches
such as cellular rewiring and creating customized
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Review. Evolution of pTyr signalling
circuits [97]. Within the SH2 domain, an invariant arginine residue within the second b-strand is necessary for
coordinating the negatively charged phosphate of pTyr.
Specificity is then determined through the interactions
with pockets on the surface of the SH2 domain that
recognize the residues primarily C-terminus to the
pTyr, but in various cases extending from the 22 position up to the þ5 position in respect to the pTyr
[98–100]. Loop regions of the SH2 fold play additional
roles in ligand selectivity through their ability to block
certain contact regions and limit ligand access to the
binding surface [98]. Selectivity is not limited to direct
contact of residues that impart binding energy, as
longer range charge repulsion, steric clashes and entropic effects also play a role in selectivity. Interestingly,
SH2 domains explore only a very limited sequence
space in their potential ligands. Two large classes of
SH2 domains exhibit a strong preference for either an
asparagine residue at þ2 (e.g. pY-X-N-X) or a proline
or aliphatic residue at þ3 (e.g. pY-X-X-P/L). Despite
this limited apparent selectivity for permissive residues,
necessary to impart the minimal energies required for
binding, SH2 domains exhibit remarkable selectivity
for physiological peptide ligands. The basis for this is
the extensive use of non-permissive residues that distinguish the binding of SH2 domains that otherwise
have very similar primary motifs for binding [101]. In
this way, the Crk and Brk SH2 domains are able to distinguish ligands using non-permissive residues and
sequence context [101]. In evolutionary terms, this
implies remarkable flexibility in adaptive mechanisms.
A newly arising SH2 domain may preserve the essential
binding motif of its immediate ancestral paralogue, with
adaptation allowing fine-tuning of ligands without major
disruption to the primary binding surface. The use of
non-permissive residues is inherently more flexible
than permissive residues that require direct contacts
and constrained geometry of binding pockets. Non-permissive residues can be accommodated in various ways,
ranging from steric clashes within or adjacent to the
binding groove, longer range charge-repulsion, or even
entropic penalties associated with altering the conformation of the peptide upon binding [101].
The fidelity of pTyr signalling is in large part
determined by the ability of a given SH2 domain to
selectively recognize a specific set of pTyr ligands.
This involves the interplay between the PTK/PTP,
the SH2 domain and the pTyr ligand (figure 6a).
In many cases, this means that over evolutionary distances, SH2 domains display conservation of ligand
specificity, thereby maintaining core-binding partners
whose ligand sequences also do not diverge significantly. Novel binding partners may still arise by
insertion of pTyr motifs or mutations of sequences
surrounding the pTyr to permit or abolish binding
by specific SH2 domains. A complementary mechanism is for specificity to evolve through changes in the
binding surface of an SH2 domain such that it recognizes different ligands than its ancestral relatives,
allowing for novel interactions and re-wiring of the
network. The obvious issue with altering the primary
binding interface is that existing interactions will be
lost, potentially with deleterious consequences for
organismal fitness. Thus, large changes within the
Phil. Trans. R. Soc. B (2012)
B. A. Liu and P. D. Nash
2567
primary binding pocket are uncommon and the peptide binding groove is generally highly conserved
among orthologous SH2 domains across various
species [8]. Duplication events provide the possibility
for more significant alterations, particularly if deleterious dosage effects provide the selection pressure to
adopt alternative binding partners. More subtle
alterations to the binding partner repertoire may be
achieved with changes outside the primary contact
region that impart restrictions on the binding partners, such as prohibiting a specific residue at a
specific position. The refinement of non-permissive
residues that can arise from changes outside the primary pocket can extend to distinguishing physicochemically related amino acid residues. Thus, the
Brk SH2 domain treats a glutamic acid at þ1 as a
permissive residue, whereas an aspartic acid is nonpermissive, the Crk SH2 binds to ligands with a þ3
proline or leucine residues but not to ligands containing the closely related isoleucine or valine residues,
and Grb2 SH2 binds to ligands containing pY-EN-E but not pY-D-N-D, etc. [101] (figure 6b). Such
subtlety in distinguishing binding by related SH2
domains is consistent with the notion that specificity
of SH2 domains coevolves with its pool of available
ligands to fine-tune specificity over time as the size
and complexity of pTyr signalling networks increases.
For SH2 families, such as GRB2 (consisting of Grb2,
Gads and Grap), NCK (Nck1 and Nck2) and CRK
(Crk and CrkL), the specificity pocket appears
highly conserved from fly to humans [8] and these
therefore share a common core set of binding peptides from fly to humans [101] (Nash laboratory
2012, unpublished data). In addition, insertion of
novel binding sites within SH2 domain proteins
is prevalent, particularly between arthropod and
mammal, in a manner that serves to create more
highly connected and potentially robust pTyr interaction networks. The Grb2 family encodes two SH3
domains flanking the central SH2 domain (table 1
and figure 6c). Examination of the conservation of
the specificity pockets of the two SH3 domains
reveals high conservation of the N-terminal SH3
domain pocket but much lower conservation of the
C-terminal SH3 domain ligand binding surface,
suggesting that this domain may have evolved to
recognize novel binding partners that distinguish its
function between arthropod and mammals. Indeed,
the C-SH3 of the GRB2 family recognizes a very
specific and atypical PxxxRxxKxP motif in which
the invariant and essential residues are the RxxK
[102 – 104]. This differs substantially from the canonical PxxP motif recognized by most SH3 domains,
and suggests that mutations in the C-SH3 mutated
to accommodate a novel binding modality that
became fixed owing to selective interactions with a
handful of proteins harbouring the extended RxxK
motif. While these examples demonstrate both conservation and adaptations in specificity, certain SH2
domains appear to have lost the ability to recognize
pTyr ligands at all. For example, individual members
of the RIN (Rin2) and JAK (Tyk2) family harbour a
mutation of the essential bB-Arg to a His residue,
presumably abolishing pTyr ligand binding [3]. In
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2568
B. A. Liu and P. D. Nash
Review. Evolution of pTyr signalling
(a)
ty
ici
cif
ng
d
lvi
ve
vo
ser
e
n
ds
co
an
lig
spe
evolving specificity
conserved ligands
ev
ol
ev ving
ol
s
vi
ng peci
lig fici
t
an
ds y
loss of ligand
binding
(b)
Brk
P
Y-E-x-P-E
+1 +2 +3 +4
(c)
Crk
Grb2
P
Y-D-x-P-R
P
Y-x-x-P-x
+1 +2 +3 +4
P
Y-E-N-E
+1 +2 +3 +4
+1 +2 +3
P
Y-D-N-D
+1 +2 +3
GRB2
C
P-x-x-P
R-x-x-K
pY-x-N
P
C-SH3
N-SH3
SH2
P-x-x-P
N
N-SH3
SH2
C-SH3
0 20 40 60 80 100
percentage conservation (%)
Figure 6. Evolution of protein domain specificity. (a) The specificity of a protein interaction domain, such as an SH2 domain,
can either change or remain conserved over time. Conservation of peptide ligand specificity pocket allows interactions to be
conserved throughout evolution. Insertion of linear motifs and evolving novel binding sites can allow for an SH2 domain to
recognize multiple binding partners. Specificity can also evolve for a domain, yet this can result in acquiring a completely
new set of binding partners while the ancient partners are lost. Lastly, a domain can coevolve over time to maintain ancient
binding partners but also evolve novel binding partners by evolving its peptide-binding pocket. A protein domain can become
inactivated through mutations of its binding pocket such that it no longer can recognize any of its original ligands. (b) SH2
domains use sequence context and non-permissive residues to discriminate pTyr peptide ligands. This underlies a wider channel for information flow between the ligand and the SH2 domain. For example, the SH2 domains of Brk and Crk can each
engage a subset of peptides containing a Pro or Leu at þ3 (three residues C-terminus to pTyr; left panel): Brk and Crk distinguish between peptides containing a þ3 Pro through distinct recognition of permissive and non-permissive residues at þ1
and þ4. Brk SH2-domain binding is favoured with permissive factors for Brk (þ1 Glu and þ4 Glu), which are also non-permissive factors for Crk favour Brk binding (middle panel). A þ1 Asp and þ4 Arg favours Crk binding over Brk (right panel).
This is suggestive of coevolution between SH2 domains and peptide ligands to maximize specific recognition events using
available peptide sequence information. (c) The adaptor Grb2 protein contains two SH3 domains (N-SH3, C-SH3) and a
central SH2 domain. The sequence alignment of the GRB2 family from C. elegans to H. sapiens was mapped onto the structure
of each individual domain. The percentage sequence conservation of the three domains is represented as a heat map on the
surface of the solved ligand-bound structures (see legend for percentage of conservation). Structures used to highlight
conservation are PDB: 1BMB; PDB: 2W0Z; PDB: 1GBR.
Phil. Trans. R. Soc. B (2012)
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Review. Evolution of pTyr signalling
(a)
B. A. Liu and P. D. Nash
2569
(b)
Dm
Sp
Ci
Abl SH3 binding
DE loop
Dr
Xt
(c)
Abl SH3 binding
DE loop
Gg
Mm
Rt
Bt
Hs
Figure 7. Evolution of a short linear motif within the SH2 domain of Crk to accommodate the interaction with the SH3
domain of Abl kinase. (a) Overlay of Crk SH2 domain structures from multiple organisms. The structures are coloured according to the different organisms in (b). The intramolecular pTyr peptide ligand of Crk (Y221) is shown in grey. (b) The SH2
domain structures from D. melanogaster (Dm), S. purpuratas (Sp), C. intestinalis (Ci), D. rerio (Dr), X. tropicalis (Xt),
G. gallus (Gg), M. musculus (Mm), R. norvegicus (Rt), M. domestica (Md), B. taurus (Bt) were modelled using Swiss-model
and UCSF-Chimera of the H. sapiens (Hs) Crk SH2 domain, PDB: 1JU5 (see Liu et al. [8] for detailed methods). (c) Sequence
alignment of the DE loop of Crk and CrkL orthologues is shown. The presence of a splice site within the DE loop (circled in
red) in Crk and absent in CrkL orthologues provides an opportunity for evolving an extended SH3 binding loop.
addition to evolving SH2 domain specificity through
its ligand-binding pocket, domains may evolve secondary binding sites with their ligands to improve
affinity, specificity or alter the biophysical nature of
the interaction (e.g. by reducing off-rate to enhance
downstream signalling). For example, the N-SH2
domain of phospholipase Cg uses secondary contacts
through its BC and DE loops for interaction with the
C-lobe of the fibroblast growth factor receptor
(FGFR) catalytic domain structure, which are important for binding to pY766 in FGFR1 [105]. SH2
domains may also exert allosteric regulation over
other aspects of their host protein. For example,
intramolecular contacts between the SH2 domain
and tyrosine kinase domain directly regulate the enzymatic substrate activity for SH2-domain-containing
kinases such as Fes and Abl [106]. Thus, SH2
domain proteins can evolve in multiple fashions to
achieve selectivity in pTyr-mediated signalling.
Following a WGD event, many duplicate copies
of genes are lost, whereas a fraction of the duplicates survive. Immediately following the WGD event, regulatory
Phil. Trans. R. Soc. B (2012)
networks may be rapidly rewired to integrate the newly
duplicate genes and prevent chaotic loss of control over
cellular processes. The resulting rapid evolution and
functional divergence can occur, in part, at the level of
transcription as mentioned earlier [92]. However, adaption may also occur at the level of post-translational
regulation. Sites of PTMs are typically short motifs
that commonly occur within intrinsically disordered
protein regions that are inherently under less selective
pressure to maintain sequence and thus are capable of
rapid evolution. Changes in PTM sites thus represents
one mechanism of rapidly effecting necessary rewiring.
A study of phosphosites across paralogous yeast genomes
following WGD identified duplicate genes as having on
average more phosphosites than genes that were not
duplicated [107]. This post-translational level of regulation might be a means of rapidly rewiring duplicate
copies, allowing these genes to adapt and to be retained.
SH2 proteins typically contain an assortment of other
modular domains and short linear motifs that prescribe
a range of additional interactions. The product of a gene
duplication may give rise to a new gene that preserves
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B. A. Liu and P. D. Nash
Review. Evolution of pTyr signalling
certain functions of the parent while acquiring specialized
functions to coordinate novel interactions [108]. For
example, the Socs proteins have evolved to fine-tune
their primary binding interface in order to recognize
specific sets of targets. Thus, Socs2 binds specifically to
the growth hormone receptor, whereas Socs4 recognizes
phosphorylated EGFR [109]. Both Crk and Sh2d1a
each evolved as duplications of existing family members
and then acquired new interactions through insertion of
extended loop regions within the SH2 domain that act
to promote the recruitment of SH3 domains via a nonpTyr-dependent, secondary binding surface within the
SH2 domain. The gene family CRK encodes two paralogues, Crk and CrkL, and in mammals the Crk SH2
domain evolved an extended proline rich loop between
the bD and bE strands of the SH2 domain that specifies
an interaction with the SH3 domain of the CTK c-Abl
[78,110] (figure 7). The insertion of a splice site within
the bD–bE loop in vertebrate Crk but absent in CrkL
allowed for evolutionary divergence through extension
of this loop [8]. Splice site slippage is one way that such
sequence fragments may be inserted [111], allowing for
rapid divergence between paralogues [8]. Evolving new
binding sites is both a mechanism for diversifying
duplicate genes as well as enabling the development of
more complex signalling networks within specific
tissues. Collectively, these mechanisms suggest that gene
duplication may have served in part to promote SH2
domain proteins to coevolve and diverge within tissue
types to create specialized cellular signalling networks
such as those involved in signalling in lymphocytes.
8. CONCLUSIONS
The evolution of pTyr signalling involves the complex
interplay of signalling components for control and activation of functional downstream signalling events
through phosphorylation of tyrosine residues by PTKs
and the recognition of specific pTyr sites by SH2 or
other pTyr-binding domains. The site of phosphorylation and the sequence context surrounding the pTyr
is under evolutionary pressure both to optimize kinase/
phosphatase specificity as well as binding specificity
for SH2 or other domains. Therefore, SH2 domains
must coevolve with pTyr sites to optimize the appropriate level of specificity in signalling. Clearly, PTPs also
play a role in the evolution of this system and present a
further opportunity for study. There are still many
unanswered questions, such as what are the primordial
functions of SH2 domain proteins in their earliest
forms? What were the driving forces for diversifying
and expanding these classes of proteins at specific times? And what role did pTyr signalling play in
the emergence and elaboration of multicellularity
in the metazoan lineage. It is likely that important
insights into the role of SH2 domains will come from
future dissection of biological networks and pathways
through genetics in lower organisms. Additional
sequencing of genomes representing early branches of
the unikont lineage and early steps in the development
of metazoan multicellularity as well as biochemical
analysis of pTyr signalling pathways in these organisms
may provide deeper insight into the origins and
evolution of pTyr signalling networks.
Phil. Trans. R. Soc. B (2012)
Addendum: During the publication process, two relevant
articles were published that provide further insight into
phosphotyrosine evolution that we would be remiss to ignore.
Jeffrey G. Williams group identified Pyk2 as the kinase
responsible for phosphorylation and activation of STATc in
Dictyostelium (Proc. Nat. Acad. Sci. PMID: 22699506).
Patrick Cramer and Dirk Eick’s groups showed the tandem
SH2 domains of SPT6 bound well to pTyr1, pTyr1/pSer2,
pTyr1/pSer5 but not to unphosphorylated CTD. This
indicates that Tyr1 phosphorylation stimulates CTD binding
of the bona fide elongation factor SPT6 (Science, PMID:
22745433).
We thank Tony Pawson, Chris Tan and the members of the
Pawson laboratory for helpful discussions. B.A.L is
supported by a Canadian Institutes of Health Postdoctoral
Fellowship. P.D.N. is supported by funds from the
National Science Foundation grant (no. MCB-0819125),
the University of Chicago Cancer Research Center and the
Cancer Research Foundation.
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