Physiol Rev
84: 1315–1339, 2004; 10.1152/physrev.00002.2004.
Paxillin: Adapting to Change
MICHAEL C. BROWN AND CHRISTOPHER E. TURNER
Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York
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I. Introduction
II. Paxillin Superfamily
A. Paxillin
B. Hic-5
C. Leupaxin
III. Paxillin Structure
A. Paxillin LD motifs
B. Paxillin LIM domains
C. Other paxillin binding partners
IV. Paxillin Phosphorylation
A. Tyrosine phosphorylation
B. Serine/threonine phosphorylation
C. Dephosphorylation
V. Paxillin Function
A. Paxillin phosphorylation and migration
B. p21-GTPase regulation and migration
C. Integrin-actin linkages and muscle contraction
D. Gene expression
VI. Development and Disease
A. Paxillin family member expression
B. Potential roles in disease
VII. Future Directions
Brown, Michael C., and Christopher E. Turner. Paxillin: Adapting to Change. Physiol Rev 84: 1315–1339, 2004;
10.1152/physrev.00002.2004.—Molecular scaffold or adaptor proteins facilitate precise spatiotemporal regulation
and integration of multiple signaling pathways to effect the optimal cellular response to changes in the immediate
environment. Paxillin is a multidomain adaptor that recruits both structural and signaling molecules to focal
adhesions, sites of integrin engagement with the extracellular matrix, where it performs a critical role in transducing
adhesion and growth factor signals to elicit changes in cell migration and gene expression.
I. INTRODUCTION
It is well established that an organism’s normal
development and maintenance, as well as its capacity
to recover from injury and infection, is dictated to a
large degree by the ability of individual cells to sense
and respond appropriately to changes in their immediate external environment. Thus a complex, interwoven
array of intracellular signaling pathways is modulated
by cell adhesion to the extracellular matrix, combined
with engagement of soluble growth factors and cytokines with their cognizant receptors to control cell
proliferation and survival as well as changes in cell
shape and motility. Functional defects or imbalance in
these pathways can result in developmental abnormalwww.prv.org
ities, tissue degeneration, hypertrophy, cell transformation, and metastasis.
Cell adhesion signaling from the extracellular matrix
is initiated primarily via engagement of members of the
integrin family of transmembrane receptors (103), with
their appropriate ligands: fibronectin, laminin, vitronectin, etc. Contributions from other matrix receptor types
such as the syndecan family of proteoglycans provide an
additional level of control and complexity (306). In contrast to many growth factor receptors such as receptor
tyrosine kinases, integrins have no inherent enzymatic
activity; instead, intracellular signaling is initiated via
clustering of the receptors in the plane of the plasma
membrane and the concurrent recruitment and activation
of intracellular signaling molecules via association with
0031-9333/04 $15.00 Copyright © 2004 the American Physiological Society
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MICHAEL C. BROWN AND CHRISTOPHER E. TURNER
the cytoplasmic tails of the integrin ␣- and ␤-subunits
(70). Structural proteins are similarly recruited to provide
a physical link to the actin cytoskeleton (153). These
macromolecular adhesion complexes are commonly referred to as focal complexes or focal adhesions (226). The
fidelity of integrin and growth factor receptor signaling
relies on several common features. In particular, multidomain adaptor or scaffold proteins are utilized to recruit
the appropriate complement of signaling intermediates
and effector proteins to discrete subcellular compartments. Not only does this promote efficient activation of
a given pathway but also adaptor proteins provide an
ideal platform for the controlled integration of multiple
pathways, thereby coordinating such diverse cellular responses as changes in gene expression and reorganization
of the cytoskeleton.
First described over a decade ago, paxillin is one of
the prototypical adaptor proteins involved in integrin signaling. In the intervening years, two new paxillin family
Physiol Rev • VOL
members have emerged, and numerous new paxillin binding proteins have been identified. This review presents
our current understanding of the role for the paxillin
family in the integration of integrin and growth factor
signaling (Fig. 1).
II. PAXILLIN SUPERFAMILY
A. Paxillin
Paxillin was initially characterized as a 68-kDa focal
adhesion protein exhibiting a significant increase in tyrosine phosphorylation upon v-src expression (72).
Shortly thereafter, it was purified to homogeneity from
chicken gizzard smooth muscle tissue, an abundant
source of cytoskeletal proteins, and identified as a novel
binding partner for the focal adhesion and actin binding
protein vinculin (279). It was named paxillin, derived from
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FIG. 1. Transmembrane receptors share common adaptor proteins such as paxillin to facilitate signal integration
and transduction. Molecular adapter or scaffold proteins such as paxillin facilitate optimal signal transduction through
their ability to recruit multiple intermediates of specific signaling pathways to specific regions within the cell, in close
proximity to sites of receptor activation. They also provide a platform for signaling pathway integration or cross-talk
through the recruitment of common signaling intermediates. This integration of information permits cells to mount a
multi-faceted response to changes in the external environment, including rapid modulation in cytoskeletal organization
affecting cell shape, adhesion, and motility as well as long-term changes in gene expression. ECM, extracellular matrix;
MAPK, mitogen-activated protein kinase.
PAXILLIN ADAPTING TO CHANGE
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the Latin paxillus, a stake or peg, consistent with its
proposed function in linking actin filaments to integrinrich cell adhesion sites.
Paxillin was cloned by ␭gt11 expression screening in
1994 from an avian cDNA library (281) and shown to
encode for a 559-amino acid protein. This protein is organized into a series of protein binding modules (Fig. 2). The
amino terminus contains five LD motifs and several SH2binding domains while the carboxy terminus consists of
four LIM domains (48, 124, 274). These domains will be
discussed in further detail below. Paxillin encoding
cDNAs have subsequently been isolated from human
(221), mouse (81, 177), frog (193), zebrafish (43), fly (304,
311), slime mold (AAM09351) (73), and yeast (163). Interestingly in nematode, a paxillin that contains both the
amino terminus and carboxy terminus has yet to be identified, although a protein with the four LIM domains well
conserved (including exon splice boundaries) exists
(NM065879 Caenorhabditis elegans, BQ611130 Caenorhabditis briggsae). Further work on nematode genomic
content and organization will reveal whether the fulllength coding sequence remains to be identified or if the
unique physiology of worms mandates the expression of a
paxillin lacking the amino-terminal protein interaction
domains. Regardless, the evolutionary conservation of
paxillin attests to the critical importance of this molecule.
In higher eukaryotes, three paxillin alternative splice
isoforms have been identified. Paxillin ␣ is the principal,
ubiquitously expressed isoform, whereas the ␤- and ␥-isoforms exhibit restricted expression (176). The ␤- and
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␥-isoforms contain a 34- and 48-amino acid insertion,
respectively, between amino acids 277/278 (Fig. 2). Although initial reports suggested the lack of a murine
␥-isoform (177), all three isoforms appear to also be expressed in mouse (308). It is not yet known whether other
splice isoforms exist in lower organisms, although in
zebrafish two proteins that differ by 5 kDa are detectable
by Western immunoblotting (43). A fourth paxillin isoform denoted paxillin ␦ is the product of alternative translation initiation beginning at amino acid 132 (293; unpublished observations). Importantly, the consensus Kozak
for this downstream start methionine is conserved across
species. In total therefore, there is the potential in higher
eukaryotes for the expression of six paxillin isoforms by
a combination of alternative splicing and translation initiation.
Interestingly, a novel splice variant called PDLP is
expressed in Drosophila that encodes a carboxy-terminal
portion of paxillin including LIM domains 1 and 2 as well
as the first zinc finger of LIM3 and a novel second zinc
finger to generate a new LIM3 (304, 311). A Lepidopteran
PDLP ortholog, death-associated LIM-only protein
(DALP), originally proposed to be a Hic-5 ortholog, has
been described (100).
Insofar as genomic organization is concerned, in humans the paxillin gene is comprised of 11 exons located
on chromosome 12, mapped to 12q24.2 (221). In rat, paxillin is located on chromosome 12q16, whereas mouse
paxillin is positioned on chromosome 5. Zebrafish paxillin
is reported at linkage group 5, in Drosophila paxillin is on
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FIG. 2. Domain structure of the paxillin family members. Paxillin ␣ is a 557-amino acid (human), 68-kDa protein that
is comprised of multiple structural domains including 5 leucine-rich LD motifs (consensus LDXLLXXL) (274) and 4
double zinc finger LIM domains (48). These domains are involved in multiple protein-protein interactions. The LIM
domains are also required for targeting paxillin to focal adhesions. Two splice variants of paxillin (␤ and ␥) with a more
restricted distribution have been identified. A fourth isoform, paxillin ␦, is enriched in epithelial cells types and is
believed to be generated from an internal alternative translation start site. Two paxillin paralogs have been identified.
Hic-5 is frequently coexpressed with paxillin and has been shown to bind to many of the same proteins. It appears to
perform both complimentary and antagonistic functions (perhaps due to the lack of key phosphorylation sites, see text
for details). Leupaxin expression is restricted to leukocytes.
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MICHAEL C. BROWN AND CHRISTOPHER E. TURNER
the long arm of chromosome 2 at 37D and chromosome III
in C. elegans.
B. Hic-5
C. Leupaxin
Leupaxin is a 386-amino acid 45-kDa family member
that is predominantly expressed in leukocytes, as is reflected in the naming convention (150). Leupaxin is located on chromosome 11cen-11q12.3 and is encoded by 9
exons. It lacks its paralogs exons 4 and 5 and consequently has a significantly different structural composition and therefore likely unique regulation and function
(Fig. 2). No evidence exists to support leupaxin expression in lower eukaryotes.
III. PAXILLIN STRUCTURE
The multidomain structure of paxillin and the lack of
identifiable enzymatic motifs first suggested it was an
adaptor protein (281). Within the amino terminus are five
leucine-rich regions, termed paxillin LD motifs, that function in protein recognition (23). The carboxy terminus is
comprised of four lin-11, isl-1, mec-3 (LIM) domains that
also mediate protein-protein interactions (48). Dispersed
throughout the molecule are many serine/threonine and
tyrosine phosphorylation sites that will be discussed in
more detail below. In addition, several potential prolinePhysiol Rev • VOL
A. Paxillin LD Motifs
Thus far the most extensively characterized domains
within paxillin are the LD motifs. These protein recognition domains were first identified during a biochemical
microdissection of paxillin initiated to identify the binding sites for the proteins vinculin and FAK (24, 281).
Truncation and deletion analyses delineated two binding
sites for FAK and one for vinculin. Visual sequence gazing
revealed the binding sites to share a leucine-rich motif
that was found to be repeated four times within the amino
terminus of paxillin. Based on this, a “consensus” LDXLLXXL paxillin LD-motif was proposed as an evolutionarily conserved peptide docking site for FAK and vinculin
(23, 24, 274). Tong et al. (269) later proposed a fifth
“degenerate” LD (LD3) that was originally disregarded
due to lack of the conserved “LD” start but has since been
incorporated into the nomenclature (Fig. 2).
The paxillin LD1 motif is conserved across species
and paralogs and is encoded by exons 1 and 2. Notably,
paxillin ␦ and Hic-5 ␤ lack the LD1 motif. The paxillin LD2
motif is encoded by exon 4 and is conserved across
species and in Hic-5 but is absent in leupaxin, which has
evolved a second unique LD motif that is encoded by exon
2. Paxillin LD3 motif is present within the highly divergent
exon 5 and is conserved only among orthologs. The paxillin LD4 motif is encoded by exon 6 and is conserved
across species and paralogs. Interestingly, the coding sequence for this LD motif is the most highly evolutionarily
conserved DNA sequence of the paxillin family outside of
the LIM domains. The paxillin LD5 motif is encoded by
exon 7 and is conserved across species and paralogs.
The individual LD motifs provide specific protein
interaction interfaces (23, 24) (Fig. 3). Within paxillin,
LD1 mediates interactions with actopaxin (183), the integrin-linked kinase (ILK) (184), vinculin (278), and the
papillomavirus protein E6 (268, 288). LD2 binds to vinculin and FAK/PYK2 (24, 278). LD4 binds to actopaxin (183),
FAK/PYK2 (24, 265, 278), the Arf-GAPs p95PKL/GIT2/
GIT1 (278), and perhaps PAK3 (90), clathrin (278), and
PABP1 (307). Thus far no binding partners have been
identified for LD5 or the degenerate LD3 motif. Importantly, the capacity of Hic-5 and leupaxin to interact with
these LD-binding proteins has been confirmed (64, 79,
150, 186, 266, 278).
Upon their identification, the leucine-rich paxillin LD
motifs were modeled and suggested to fold as amphi-
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The paxillin paralog hydrogen peroxide inducible
clone-5 (Hic-5) was first identified in an analysis of mouse
osteoblast transforming growth factor (TGF)-␤- and hydrogen peroxide-inducible cDNAs and encodes a 444amino acid protein (247) (Fig. 2). Human Hic-5 was later
cloned in an androgen receptor two-hybrid screen and
named ARA55 for androgen receptor coactivator 55-kDa
protein (63). A Hic-5 splice isoform that contains an additional 17 amino acids on the amino terminus (containing
an LD motif) was originally identified in a screen for
murine paxillin (266) and later in a differential display
screen of senescent versus nonsenescent human keratinocytes (328). Based on homology with paxillin, we suggest that the “full-length” Hic-5 splice isoform containing
the amino-terminal LD1 “extension” be denoted Hic-5 ␣
and the smaller form Hic-5 ␤. As yet, there is no evidence
that Hic-5 orthologs exist in lower eukaryotes, although
unpublished observations suggest Hic-5 is expressed in
zebrafish (43). Interestingly, Hic-5 shares the same 11exon genomic organization as paxillin, consistent with an
evolutionary duplication event, and is located on chromosome 16p11 in humans (328) and chromosome 7 in mouse
(266).
rich SH3-binding motifs are present within the paxillin
amino terminus, consistent with a described Src SH3paxillin association (302). The paxillin amino terminus is
particularly proline and glycine rich, which in addition to
the existence of a multitude of phosphoisoforms, is reflected by its aberrant electrophoretic mobility of 68 –75
kDa versus a calculated molecular weight of 62 kDa.
PAXILLIN ADAPTING TO CHANGE
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pathic ␣-helices with the leucines providing a hydrophobic interface with its binding partners (23, 228, 274), a
prediction subsequently confirmed experimentally (7, 91,
152). The individual paxillin (and Hic-5) LD motifs are
flanked by proline- and glycine-rich segments (average
composition of 25%), which may also contribute to the
global folding, presentation, regulation, and function of
the individual paxillin protein binding domains. This is
reflected in the ␤- and ␥-paxillin isoforms exhibiting reduced affinity for the LD4 binding proteins FAK and GIT
(175, 176). Also interspersed between the LD motifs are
potential SH3-binding domains and numerous phosphorylation sites, including SH2 binding domains, that may
also regulate the activities and context of the LD binding
partners, thereby imparting an additional means of temporal-spatial regulation of the adaptor functions of paxillin. Hic-5 and leupaxin exhibit significant differences from
paxillin in the length and composition of the LD “spacing”
regions and consequently likely exhibit unique regulatory
and functional capacities. Indeed, Hic-5 binds to PYK2
more efficiently than to FAK (174, 194), and Hic-5 binds to
GIT1 more efficiently than does paxillin to GIT1 (188).
Before our identification of LD motifs as discrete
domains mediating binding to vinculin and FAK, the binding sites for paxillin on the vinculin tail (305) and the FAK
focal adhesion targeting (FAT) domain (95) were identiPhysiol Rev • VOL
fied through truncation and deletion mutagenesis. Subsequently, a FAK point mutagenesis study further localized
the site of paxillin binding and allowed for a comparison
to the known site of paxillin binding to vinculin (305).
This led to the description of paxillin-binding subdomains
(PBS) as an evolutionarily conserved paxillin binding motif (257). Thus a novel protein interaction pair, LD-PBS,
was discovered. These data allowed for the creation of an
algorithm to search and identify the PBS sequences within
the paxillin LD binding partners PKL, ILK, and actopaxin
and other potential candidates (23, 183, 184, 274, 278).
The general utility of LD-PBS associations has emerged
with the discovery of an interaction between gelsolin and
PYK2 (294) as well as between the papillomavirus E6
protein and the ubiquitin ligase E6-AP (14, 47), the RapGAP E6TP1(67), and the Ca2⫹-binding protein ERC-55/
E6BP (38), in addition to paxillin (211).
The crystal structures of the vinculin tail and the FAK
FAT domain, which contain the PBS, have been solved (7,
10, 91, 114). FAK FAT solution structures have also been
reported (66, 152). The vinculin tail and FAK FAT domain
share a parallel up-down-up-down four-helix bundle. This
general structural organization is shared by ␣-catenin,
apolipoprotein E, and the p130Cas family of proteins (7,
10, 91, 114), although only vinculin and FAK bind paxillin.
Interestingly, structural predictions suggest that the PBS-
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FIG. 3. Paxillin binding partners. Paxillin is comprised of multiple protein binding motifs, including the aminoterminal LD motifs, the carboxy-terminal LIM domains, and several phosphotyrosine-SH2 domain docking sites. These
individual motifs have been used in various biochemical GST-pull down, blot overlay assays, and yeast two-hybrid
screens to identify numerous direct binding proteins. These range from structural actin binding proteins including
actopaxin and vinculin to important signaling molecules such as FAK (focal adhesion kinase), ILK (integrin-linked
kinase), and PTP-PEST, a tyrosine phosphatase. Many other potential paxillin binding partners have been identified in
coprecipitation experiments, although in many of these cases it remains to be determined whether the interaction with
paxillin is direct or which domain of paxillin is involved (see Table 1).
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MICHAEL C. BROWN AND CHRISTOPHER E. TURNER
containing PKL carboxy terminus also folds in a similar
manner (7). In contrast, this structural organization is
unlikely in the case of the ILK and actopaxin PBS domains
(7, 183). Such differences are likely to dictate the optimal
binding parameters for each protein and contribute to
selection of the “appropriate” paxillin binding partner(s)
in response to a particular extracellular cue.
B. Paxillin LIM Domains
FIG. 4. Paxillin phosphorylation and function. Paxillin is phosphorylated on multiple tyrosine (PY), serine (PS) and
threonine (PT) residues in response to cell adhesion, and/or exposure to a various soluble growth factors and cytokines
(see text for details). Tyrosine phosphorylation generates docking sites for SH2 domain containing proteins such as Crk
(binds PY31,118) to facilitate downstream signaling. In contrast, phosphorylation of serine and threonine residues is
more likely to influence paxillin conformation and thereby allosterically affect its ability to interact with specific binding
partners.
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LIM domains are double-zinc finger motifs first identified in the lin-11, isl-1, and mec-3 homeodomain proteins.
The four LIM domains of paxillin are present in tandem on
the carboxy terminus, thus designating paxillin a group 3
LIM family protein (48). The structures of several LIM domains have been solved and reveal that LIM domains are
arranged such that each individual zinc finger is comprised
of two antiparallel ␤-sheets that are separated by a tight
turn. In addition, the two zinc fingers pack together due to
hydrophobic interactions, and each LIM domain ends with a
short ␣-helix (48, 203, 289). Interestingly, although it is now
generally accepted that the primary function for LIM domains is in mediating protein-protein interactions (235), the
CRIP and CRP structures are nearly identical to the DNAbinding domains of GATA-1 and the steroid hormone receptor family (203).
The capacity of LIM domains to mediate discrete
subcellular localization to the actin cytoskeleton and focal adhesions was first demonstrated for muscle LIM
protein (MLP) (6) and paxillin (24) and has subsequently
been found to be a common link between many group 3
LIM domain proteins including the zyxin (190), PINCH
(144), and FHL families of proteins (40). Although it is not
believed that tandem LIM domains physically interact,
there is strong evidence for cooperatively between adjacent LIM domains (6, 24, 190).
Functionally, LIM3 (with LIM2 cooperating) mediates
the localization of paxillin (24) and Hic-5 (64) to focal
adhesions with serine/threonine phosphorylation of
LIM2/3 regulating paxillin localization to focal adhesions
and consequently cell adhesion to fibronectin (25) (Figs. 3
and 4). These sites of phosphorylation are largely conserved in paxillin orthologs. The localization of leupaxin
has not been extensively studied, although it colocalizes
with cortical actin in lymphoid cells attached to intracellular adhesion molecule (ICAM)-I (150), podosomes in
osteoclasts (79), and focal adhesions in fibroblastic cells
(D. E. Staunton, personal communication). The insect
paxillin splice isoform (PDLP/DALP) that is comprised
solely of LIM domains localizes to myotendonous junctions and along actin stress fibers (311), whereas the LIM
domains of the yeast ortholog Pxl mediate targeting to the
bud neck (163), thus offering evidence for the ancestral
conservation of LIM function in subcellular compartmentalization. The identity of the paxillin focal adhesion targeting molecule has proven elusive, as it has for these
other LIM domain containing proteins. It will be interesting to determine whether any relationship exists between
the protein(s) that target each of the diverse LIM family
proteins to their respective cytoskeletal compartments.
In addition to the identification of the LIM domains as
mediating paxillin subcellular targeting, tubulin was identified as a LIM2 and LIM3 binding partner (94) and PTPPEST as a binding partner for tandem LIM3 and LIM4 of
paxillin, LIM4 in Hic-5 (41, 186), and leupaxin (79) (Fig. 3).
The ability of Hic-5 and leupaxin to bind tubulin is unknown. Paxillin LIM2 also associates with and precipitates a kinase that can phosphorylate T396/401 (human),
PAXILLIN ADAPTING TO CHANGE
C. Other Paxillin Binding Partners
In addition to the LD and LIM binding partners described above, paxillin binding to many other molecules
has been reported, although in many cases the sites of
interaction are not clearly defined (Table 1). A direct
association of paxillin with ␤1-integrin (231, 259), ␣4- and
␣9-subunit cytoplasmic tails has been reported (153, 155,
156, 322). The ␣4- and ␣9-integrin binding site has been
localized to a region of paxillin amino-terminal and partially overlapping LD4 (154). Interestingly, mutations in
the ␣9-integrin cytoplasmic tail that ablate paxillin and
Hic-5 binding to the ␣-tail only partially disrupt binding to
leupaxin (155). The association between paxillin and the
␣4-tail is inhibited by cAMP-dependent protein kinase
(PKA) phosphorylation of ␣4 S988, thereby triggering ␣4mediated motility (74, 86).
Paxillin can also interact with talin (223), poly(A)binding protein 1 (307), ASAP1/2 (128, 195), the nerve
TABLE
1.
Additional paxillin binding partners
Binding Region
Amino
terminus
ABL
BCL-2
ERK
EF-1␣
HSP72
␤1-Integrin
MEK
Merlin/NF2
Phospholipase D
PP2A
PTP-phi
RNF5
␣-Sarcoglycan
SHP-1/2
Syndesmos
TrkA
⫹
⫹
⫹
⫹
Carboxy
terminus
Unknown
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
Direct?
Unknown
Yes
Yes
Unknown
Unknown
Yes
Unknown
Yes
Unknown
Unknown
Unknown
Yes
Unknown
Unknown
Yes
Unknown
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growth factor (NGF) receptor TrkA (178), the antiapoptosis protein bcl-2 (254, 255), the focal adhesion protein
TES (69), RACK (42), the serine/threonine phosphatase
PP2A (110), the tyrosine phosphatase PTP-␮ (206), Crk
and CrkL (19, 225, 232), the tyrosine kinases Abl (143) and
p210BCR/ABL (225), the protooncogene Cbl (224), and
LIM kinase (62). Paxillin and Hic-5 also bind to the syndecan binding protein syndesmos (52), Hsp27 (118),
Hsp72 (172), and the ring-finger ubiquitination component
Rnf5 (54). Other studies have reported the coprecipitation
of paxillin in complexes that contain ␥-sarcoglycan (320),
the tyrosine phosphatases SHP-1/2 (318), phospholipase
D (113), STAT transcription factors, and translation elongation factor-1␣ (318). Characterization of the molecular
basis of these associations so that discrete perturbation of
paxillin binding can be effected will allow for the determination of the physiological consequences in forming
these interactions.
IV. PAXILLIN PHOSPHORYLATION
A. Tyrosine Phosphorylation
A report characterizing the dramatic changes in paxillin tyrosine phosphorylation that occur during the complex morphogenetic events accompanying embryonic development (a process involving major changes in integrin
and growth factor signaling) provided our first glimpse of
the potential role for paxillin in responding to diverse
extracellular cues (275). Although adhesion-associated integrin ligation is now classically associated with induction of paxillin tyrosine phosphorylation (31) via activation of FAK and Src kinases (15, 232), it is clear that a
wide variety of agents that signal through a range of
transmembrane receptor families trigger paxillin tyrosine
phosphorylation. Paxillin is now considered to be at the
crossroads of cell adhesion and growth factor modulation
of intracellular signal transduction pathways (Fig. 1) (218,
230, 277). A point of commonality between these disparate extracellular stimuli is that they provoke significant
changes in the organization of the cytoskeleton and/or the
state of cellular proliferation.
Among receptor tyrosine kinase ligands, epidermal
growth factor (EGF) (263), growth hormone (219), hepatocyte growth factor (93), insulin-like growth factor I
(IGF-I) (32, 35, 141), MCP-1 (314), NGF (178), plateletderived growth factor (210), SCF (238), steel factor (258),
and vascular endothelial growth factor (1) each stimulates paxillin tyrosine phosphorylation. TGF-␤ and activin
A, which stimulate receptor serine/threonine kinase activity, also indirectly trigger paxillin tyrosine phosphorylation (212). Similarly, agonists of many 7-pass transmembrane serpentine family receptors induce paxillin tyrosine
phosphorylation including acetylcholine (298), epineph-
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and LIM3 binds to a detergent-insoluble kinase that can
phosphorylate LIM3 on S455/479 (25). The identity of
these kinases as well as the capacity of Hic-5 and leupaxin
LIM domains to bind to and be phosphorylated by serine/
threonine kinases remains to be determined. In addition
to these cytoplasmic LIM associations, Hic-5 LIM domains
have been shown to be capable of binding to DNA (187),
and the capacity of paxillin and Hic-5 to interact with the
nuclear matrix (315) perhaps through LIM3 (121) has
been described. Furthermore, both paxillin and Hic-5 can
interact with the androgen and glucocorticoid receptors
through LIM4 (208). The significance of these interactions
and the function of paxillin and Hic-5 in gene expression
is discussed below.
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modulating cellular motility has been an area of active
investigation and is discussed below.
As for tyrosine phosphorylation of other paxillin family members, Hic-5 tyrosine phosphorylation through
PYK2 (174) but not FAK (64) has been described following adhesion, platelet activation (194), hyperosmotic
stress, v-src expression, and stimulation with LPA (174).
Phosphorylation may contribute to Src inactivation via
recruitment of CSK (266) and likely occurs on tyrosine-60
(Y43 Hic-5 ␤), which corresponds to paxillin tyrosine-118
based on exon 3 coding alignment. Leupaxin tyrosine
phosphorylation has been reported (150). This likely occurs on Y20, a site encoded on exon 2, that presumably
corresponds to paxillin Y31.
B. Serine/Threonine Phosphorylation
In addition to tyrosine phosphorylation, changes in
paxillin serine/threonine phosphorylation are well documented (Fig. 4). After cellular activation by integrin ligation, serine residues 188/190 are phosphorylated (16, 53).
Similarly, LIM2 T396/401 and LIM3 S455/479 are phosphorylated after adhesion and stimulation with angiotensin II (26). Growth factors such as activin A (212), EGF
(58, 264), heregulin (286), and interleukin (IL)-3 (221), as
well as the tumor promoter PMA (26, 58), muscle contraction (202), and virus infection (288) each induces serine/
threonine phosphorylation of paxillin. The onset of mitosis also stimulates paxillin serine/threonine phosphorylation (313), which may function to regulate various
paxillin-protein associations leading to focal adhesion
and cytoskeletal disassembly associated with cell rounding before cytokinesis. For the most part, the respective
kinases have not been identified. However, protein kinase
C and mitogen-activated protein (MAP) kinase activities
have been implicated in some instances (53, 264). Of note,
the MAP kinase JNK has been reported to directly phosphorylate paxillin ␤ T178 (102) and the p38 MAP kinase to
target S85 (101) to regulate cell migration and neurite
outgrowth, respectively. Interestingly, a Y88F mutation,
adjacent to the S85 site, attenuated the paxillin electrophoretic mobility that is widely associated with paxillin
serine/threonine phosphorylation (233). Further studies
examining the potential functional interrelationship between sites of paxillin phosphorylation will be necessary.
ERK1/2 (107, 158) and the p21-activated kinase PAK3 (90)
have been reported to directly phosphorylate paxillin as
well. The advent of phosphorylation state-specific antibodies for the serine/threonine paxillin phosphorylation
sites will certainly facilitate further investigation.
C. Dephosphorylation
Cellular responses to extracellular cues are not limited to increases in paxillin phosphorylation. ACTH (290),
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rine (330), angiotensin II, thrombin (283), bombesin, vasopressin, endothelin (326), bradykinin (140), cholecystokinin (68, 324), CSF-1 (318), lysophosphatidic acid (LPA),
and other sphingosine metabolites (218, 240, 241). Binding of ephrins to their cognate receptors also stimulates
paxillin tyrosine phosphorylation and plays a key role in
regulating tissue remodeling and cell migration during
embryonic development (34, 291).
Signaling through immunomodulators such as tumor
necrosis factor-␣ (65) or formyl-methionyl-leucyl-phenylalanine (76), IgE (85), T-cell receptor (55) or complement
receptor ligation (76), Fc-mediated phagocytosis (77), and
reactive oxygen intermediate exposure (75) as well as
viral or bacterial infection (3, 288) and exposure to their
toxins including Pasteurella multocida toxin, cytotoxic
necrotizing factor 1, dermonecrotic toxin, and lipopolysaccharide (136, 137) can also induce paxillin tyrosine
phosphorylation. In each of these scenarios, profound
alterations in the cytoskeleton are effected that are essential to activate and regulate host defenses. Finally,
many different modes of cellular stress such as membrane
depolarization (123, 260), tissue injury (89), hypertonicity
(168), stretch (253, 317), shear stress (83), increased cell
density (13), ␤-amyloid exposures (17), and treatment
with the phorbol ester phorbol 12-myristate 13-acetate
(PMA) (140) have also been reported to induce paxillin
tyrosine phosphorylation.
The second messenger pathways utilized to elicit
these changes in paxillin tyrosine phosphorylation are
beyond the scope of this review; however, as a matter of
course, cell adhesion, an intact dynamic actin cytoskeleton, phosphatidylinositol 3-kinase (PI3K) activity, and
RhoA stimulation are generally common elements. For
the most part the sites of phosphorylation induced by the
stimuli listed above are not known; however, the initial
characterization of adhesion-induced phosphorylation by
FAK and Src revealed tyrosine residues 31 and 118 as the
primary targets (15, 232). The advent of paxillin phosphorylation state-specific antibodies has greatly facilitated the
identification of additional functional paxillin phosphorylation sites Y40, Y88, and Y181 (182, 233), with all but Y181
competent Src substrates (233).
A major consequence of paxillin tyrosine phosphorylation is the generation of functional SH2-binding domains that consequently operate as inducible, regulatable
protein interaction domains (Fig. 4). The most extensively
characterized SH2 association is with the adaptor proteins Crk/CrkL, which binds to pY31 and pY118 (19, 225,
232, 273). Other SH2 interactions that have been described are the association of p85 PI3K with pY31, pY40,
and pY118 and p120RasGAP with pY31 and pY118 (273).
Binding to the Src-inactivating tyrosine kinases Csk via
Y31 and Y118 (220, 273) and Chk (78) have also been
reported. The role of paxillin tyrosine phosphorylation in
PAXILLIN ADAPTING TO CHANGE
V. PAXILLIN FUNCTION
A principal function for paxillin is in the integration
and dissemination of signals from integrins and growth
factor receptors to effect efficient cellular migration. Motility is a complex multistep process that requires the
coordination of membrane trafficking and the reorganization of the actin and tubulin cytoskeleton networks to
realize net cellular movement (reviewed in Refs. 12, 98,
252, 300, 301). The activities of several p21 GTPase families are critical to this process (84, 209), and paxillin is an
important mediator of signal cross-talk between these
families through its phosphorylation and multipotent associations (276, 282, 285) (Figs. 4 and 5).
Physiol Rev • VOL
A. Paxillin Phosphorylation and Migration
The first critical demonstration of a role for paxillin
tyrosine phosphorylation in cell migration was in a study
of NBTII bladder tumor cells in which cell adhesion,
spreading, and motility was associated with induced paxillin tyrosine phosphorylation and consequent association
with CrkII (205). Introduction of a nonphosphorylatable
paxillin tyrosine phosphorylation mutant Y31F /Y118F
blocked migration, which could be reversed by overexpression of wild-type paxillin or CrkII (205). The functional coupling of paxillin to CrkII was also found to be
important in the process of epithelial-mesenchymal transition (138). In this MDCK model system, overexpression
of CrkII induced the localization of paxillin to focal adhesions resulting in the stimulation of lamellipodia and
cell scattering. Expression of a paxillin Y31F/118F mutant
that no longer bound CrkII blocked the effect. Interestingly, expression of a paxillin LD4 mutant molecule no
longer competent to bind PKL, functioned similarly (138).
Furthermore, a recent study of ephrin B1-stimulated cell
migration in a variety of cell types including renal vascular endothelial cells demonstrated a requirement for paxillin tyrosine Y31/118 phosphorylation and the LD4 motif.
In this example, the LD4 motif was required for Nckmediated recruitment to the activated receptor (291). A
model is emerging in which growth factor and integrinmediated phosphorylation of paxillin stimulates the formation of a paxillin-Crk complex at focal adhesions
where Crk-DOCK180 can then locally activate Rac to
produce lamellipodial extension and enhance migration
(Figs. 4 and 5).
Notably, in a study of migration on collagen using
Cos7, NMuMG mammary epithelial, and MM1 hepatoma
cells, no motility defect was identified upon expression of
a non-tyrosine phosphorylatable paxillin mutant (316). In
this study, a role for CrkII binding to p130Cas rather than
paxillin was concluded. Although the specific paxillin
mutants and conditions between the two studies were not
identical, it suggests cell type specificity in signaling cell
motility. In a subsequent study, these investigators determined that tyrosine phosphorylated paxillin associated
with the SH2 domain of p120RasGAP, thereby competing
with and displacing p190RhoGAP from p120RasGAP
(273). The consequence of releasing p190RhoGAP was the
localized suppression of RhoA in focal adhesions that
potentially facilitates Rac-mediated lamellipodial extension, focal adhesion turnover, and thereby stimulates cell
motility.
Roles for the MAP kinase family in the regulation
of cell motility are well established. For instance, in
germline deletion analyses of MEKK1 (325) and JNK
(116), a significant impairment in cellular motility was
observed. A role for paxillin in JNK activation has been
identified (106), and recently JNK phosphorylation of
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EGF (108), ephrin (180), insulin (129), IGF-I (80), and
nitric oxide (122) can initiate tyrosine dephosphorylation
of paxillin. In vivo, myometrial remodeling at the onset of
labor is associated with a drop in paxillin tyrosine phosphorylation (165) as is Heymann nephritis (270) and
ethacrynic acid-induced cell retraction (192). In addition,
deadhesive signals result in rapid paxillin tyrosine dephosphorylation (31), perhaps through activation of PKA
(88, 99). Cell adhesion to matricellular proteins such as
thrombospondin, tenascin, and SPARC can also trigger
paxillin tyrosine dephosphorylation in a PKA- or protein
kinase G-dependent manner. A role for PKA activation
mediating paxillin tyrosine dephosphorylation is intriguing. As RhoA activation is often a critical component of
paxillin phosphorylation, it is interesting to speculate that
PKA activation targets RhoA for phosphorylation and inactivation, which leads to “passive” loss of paxillin phosphorylation by severing the stimulatory signaling pathway
(57, 146).
Along the same vein, with respect to insulin, PTP1D
phosphatase may dephosphorylate and thereby activate Csk
leading to inactivation of Src and consequent “passive” decrease in paxillin tyrosine phosphorylation (196, 267). Conversely, IGF-I induces an association of paxillin with the
phosphatase SHP-2 (170); similarly, the tyrosine phosphatase PTP-PEST is a paxillin binding partner (41, 243). PTPPEST may directly target paxillin for dephosphorylation
(242); alternatively, it may result in a “passive” decrease
through paxillin’s recruitment of PTP-PEST to target Src and
FAK for inactivation (41). Regardless, mutagenesis of the
LIM domains that mediate PTP-PEST binding decreases cell
adhesion and motility on fibronectin, perhaps indicating a
role for paxillin binding to PTP-PEST in focal adhesion
dynamics (27, 41). Finally, paxillin also interacts with the
serine/threonine phosphatase PP2A, and although it is not
known if paxillin is a physiological protein phosphatase 2A
substrate, perturbation of this association and consequent
increase in paxillin LIM3 serine phosphorylation enhances
cell metastasis (110, 323).
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MICHAEL C. BROWN AND CHRISTOPHER E. TURNER
paxillin T178 has been implicated as critical for cell
motility with expression of a paxillin T178A mutant
blocking cell migration (102). Intriguingly, expression
of a c-Jun molecule with alanine substitutions for the
two serines targeted by JNK similarly blocks cell motility (116). Future studies will be required to determine
the precise mechanism by which paxillin influences
JNK signaling and motility. In addition to a role for
paxillin in the JNK MAP kinase signaling pathway, paxillin is essential for adhesion-mediated activation of
ERK (81). One mechanism identified, in response to cell
adhesion and scatter factor treatment, was the stimulation of Src phosphorylation of paxillin Y118 to create
an Erk binding site on paxillin that results in Erk
recruitment to focal adhesions (107). Paxillin then
binds to Raf and MEK to activate Erk in these dynamic
focal adhesions. Subsequently, Erk phosphorylation of
paxillin then increases the association of FAK with
paxillin to potentiate cell spreading and motility (158).
A second mechanism involves the paxillin-binding proPhysiol Rev • VOL
tein GIT1 functioning as a MEK scaffold to facilitate
ERK activation in focal adhesions (319).
B. p21-GTPase Regulation and Migration
The process of directed migration can be broken
down into several discrete steps, namely, protrusion/polarization, attachment/adhesion assembly, traction/translocation, and adhesion disassembly/retraction (12, 98,
252, 300, 301). After cellular stimulation with a chemotactic growth factor, the cell extends a protrusion resulting
from the polymerization of a dense network of actin
toward the chemoattractant in the form of fingerlike
filopodial projections and thin lamellipodial sheets. This
occurs in a Cdc42- and Rac p21GTPase-dependent manner, respectively (84). How are these GTPases activated?
Recent studies have implicated heterotrimeric G protein ␤␥-subunits liberated from activated receptors (145).
They bind to the Ser/Thr kinase PAK that stimulates the
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FIG. 5. Physical and functional connections between paxillin and p21 GTPase regulators. Major remodeling of the
actin cytoskeleton is required during cell migration. The Rho family of small GTPases (Cdc42, Rac, and Rho) is an
essential regulator of this process contributing to cell polarity, lamellipodial extension, and cell contractility. The p21
ARF GTPase family also contributes significantly to these processes. Their activity is positively regulated by guanine
nucleotide exchange factors (GEFs) and negatively by GTPase activating proteins (GAPs). As a result of its localization
to focal adhesions, sites of integrin attachment to the extracellular matrix, paxillin is exquisitely positioned to contribute
to the regulation of p21 GTPase activity and function. Indeed, as illustrated here, paxillin interacts either directly or
indirectly with several GEF and GAP proteins and in so doing likely contributes to the tight spatiotemporal regulation
of GTPase activity that is a prerequisite for productive cell motility.
PAXILLIN ADAPTING TO CHANGE
Physiol Rev • VOL
encing GEFH1 activity (133), is currently an area of active
investigation.
In addition to the requisite primary initiation of polarized actin assembly for protrusion and lamellipodial
extension, a global reorientation of the cell to face the
chemotactic agent must occur for efficient directional
motility in many cell types. This is thought to function in
part by orienting the microtubule cytoskeleton towards
the lamellipodium and facilitating membrane cycling
(181). One measure of cellular polarization is the reorientation of the Golgi towards the leading edge. This process
is independent of actin polymerization, but dependent on
integrin ligation and Cdc42 activation (60, 199). Interestingly, expression of a paxillin molecule that lacks LD4
results in a profound inhibition of the capacity to reorient
the Golgi in a scratch wound assay (Fig. 6), the generation
of multiple randomly oriented protrusions, and loss of
directed cell migration (303). Thus paxillin plays a significant role in cell polarization.
Cdc42 and Arf1 activities are essential for integrity of
the Golgi and microtubule organizing center (MTOC) (59).
Importantly, Arf1-dependent paxillin shuttling between
the Golgi and focal adhesions has been described (191),
with the ArfGAPs, GIT2short, and ASAP2/PAP␣ implicated in this process (128, 175). Other critical mediators
of Golgi reorientation/polarization are Src (120) and
p120RasGAP (135), which are paxillin binding partners
(112, 302). p120RasGAP is essential for polarization likely
through the temporal-spatial regulation of Rho activities
(135). Paxillin, via pY31/118-p120RasGAP binding and resulting dissociation of p190RhoGAP from p120RasGAP,
has been proposed as a mechanism to regulate
p190RhoGAP activity (112). Through this exchange paxillin may contribute both to the inhibition of Rho function
at the leading edge to promote lamellipodial extension
and activation of Cdc42 in the context of cell polarity.
Roles for PYK2 or FAK binding to the PKL/GIT and ASAP
families of ArfGAPs and the PSGAP/GRAF family of
RhoGAPs in influencing Golgi/polarization are currently
unknown, although FAK regulation of diaphanous and
FAK phosphorylation by cdk5 have been implicated in
microtubule stabilization and maintenance of the leading
edge (197, 198, 309).
The lipid microenvironment also plays an important
role in cell polarization, with activation of PI3K by ␤␥subunits and/or Ras (145) at the leading edge being important in the localized generation of phosphoinositides
that can activate the Cdc42 and Rac GEF activities of Vav
and PIX (87, 161, 321). In this regard paxillin, tyrosine
phosphorylated in response to integrin engagement with
the extracellular matrix, can function to bind p85 PI3K
(273), thereby positioning it as a potential adaptor for
3⬘-phosphoinositide function. Paxillin also binds to talin,
an integrin-binding protein that recruits phosphatidylinositol 4-phosphate 5-kinase (148) to produce localized
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GEF function of the PAK interacting exchange factor PIX
to generate active Cdc42 at the leading edge. This activated Cdc42 can then induce PAK kinase activity to regulate actomyosin function (20) as well as stimulate the
function of WASP to activate Arp2/3 actin nucleation and
branching to trigger polarized protrusion (215, 256). Paxillin and Nck are critical for the appropriate localization
and function of PAK and PIX; thus it will be of interest to
further examine the role for these interactions in protrusion (20, 28, 171). The demonstration that PIX binds to the
actin and paxillin-binding protein actopaxin/parvin, which
regulates cell adhesion and spreading, provides an additional paxillin connection to the initial events of protrusion (183, 217, 278).
The mechanism by which paxillin contributes to PAK
function is likely through binding of the Arf-GAP PKL to
paxillin LD4 (28, 303). Activation of the PAK scaffold
function, perhaps by G protein ␤␥-subunit binding as well
as by localized Rac activation by integrins (49), stimulates
the capacity of PKL (bound to PAK through PIX) to
effectively associate with paxillin at focal adhesions. This
provides a regulated mechanism for the transient targeting of PAK to focal adhesions (28), where it triggers the
formation of Rac focal adhesions and the turnover of Rho
focal adhesions (28, 329) to facilitate directed protrusion
and lamellipodial extension. PAK function may be differentially regulated by the paxillin-binding Arf-GAPs PKL/
GIT2 and GIT1 in that PKL is excluded from focal adhesions formed by constitutively active PAK (28), whereas
GIT1 potentiates the localization of active PAK to focal
adhesions (169). Further analyses will determine the differential temporal-spatial regulation of PKL and GIT at the
level of PAK activation and in the context of the status of
the focal adhesion.
The coordination of protrusion stabilization and forward extension is a fine balance of many components
(139, 300). After generation of a productive protrusion,
the cell attaches and anchors the membrane with focal
complexes. Importantly, paxillin is among the first proteins to localize to these sites (139). Following lamellipodial extension these complexes remodel with paxillin loss
from these (now) Rho focal adhesions correlating temporally with the formation of new paxillin-containing peripheral Cdc42/Rac focal complexes at the leading edge of the
new protrusion (139). Perturbation of paxillin by germline
knockout or introduction of paxillin lacking LD4 causes
an inhibition of this focal adhesion turnover, loss of polarization, and therefore a loss of directional motility (81,
299, 303). Interestingly, a similar phenotype is observed
upon overexpression of kinase dead PAK (125, 239). In
addition to a role for PAK in focal adhesion turnover,
microtubule targeting of focal adhesions promotes their
turnover and potentiates protrusion. The role for paxillin
binding to tubulin (94) in this process, possibly via influ-
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MICHAEL C. BROWN AND CHRISTOPHER E. TURNER
phosphatidylinositol bisphosphate, which inhibits actin
capping proteins in addition to stimulating Arp2/3 binding
to vinculin (50, 51), another paxillin-binding protein (277).
However, a role for paxillin adaptor function in these
processes has not been established. Whereas polarization
of lipid is important, it similarly has been found that
sequestration of lipid phosphatases, including the 3⬘-phosphoinositide phosphatase PTEN, away from the lamellipodia is important for maintaining polarity (145). Interestingly, an ILK-actopaxin complex regulates PTEN
partitioning to the membrane (9) likely in a paxillin-dependent manner (183–185). 3⬘-Phosphoinositides also regulate the activities of ArfGAPs like PKL and GIT1 (292),
which may be important in the regulation of their Arf6
GAP activity and consequently the delivery of Rac and
integrins to the leading edge of motile cells (55a, 207).
These proteins are also likely important in recycling of
membrane proteins (169), with GIT1 in Rab11 recycling
endosomal and PKL in EEA1 early endosomal compartments (200).
Finally, retraction/tail release, which pulls the rear of
the cell forward, is essential for efficient directional motility. This occurs through a combination of myosin IIB
contractility (8, 127, 271), microtubule targeting of attachment sites, and calpain-mediated proteolysis of adhesion
proteins including paxillin (18, 157, 312). Paxillin knockout or overexpression of paxillin lacking LD4 causes the
formation of long retraction fibers, further implicating
paxillin in the regulation of this process (303). The MAP
kinase Erk is involved in the regulation of cell migration
at several levels. Its activity is required for calpain activation, in part through the formation of a ternary complex
with FAK (33, 44). Erk also phosphorylates and activates
calpain (71) as well as myosin light-chain kinase to proPhysiol Rev • VOL
mote contractility (22). Importantly Erk, which is in part
regulated via PAK activation (251), binds paxillin and
localizes to focal adhesions (107). Furthermore, Erk activation is defective in paxillin null cells as well as in
Chinese hamster ovary cells expressing paxillin lacking
LD4 (81; unpublished observations), suggesting that paxillin directly impacts on Erks’ contribution to cell migration. This is consistent with the recent report detailing the
importance of Erk activation in focal adhesion turnover
(299).
C. Integrin-Actin Linkages and Muscle Contraction
Physical linkage between the extracellular matrix,
via integrin to the actin-based cytoskeleton, is critical for
the transduction of force generated during contraction of
skeletal, smooth, and cardiac muscle. The molecular organization of these structures is remarkably similar to
that of focal adhesion architecture within fibroblasts and
other more motile cells. Indeed, both the structural and
regulatory proteins including paxillin, FAK, vinculin, etc.
are highly enriched in the dense plaques of smooth muscle tissue (280), the intercalated discs of cardiac muscle,
and the myotendinous junctions of skeletal muscle (280).
Detailed studies in tracheal smooth muscle tissue
have questioned the general perception that such physical
linkages are robust, stable points of contact and suggest
instead that these are quite dynamic structures that serve
as important signal transduction centers. As with focal
adhesion signaling, paxillin phosphorylation appears to
play a critical role. Not only is paxillin tyrosine phosphorylation on residues Y31 and 118 stimulated during acetylcholine-induced muscle contraction, but overexpression
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FIG. 6. Paxillin LD4 motif is required for cell
polarity. Following scrape wounding of a cell monolayer, the cells at the wound edge produce a dominant lamellipodium to initiate migration into the
wound and subsequent wound closure. The cells also
typically reorient the Golgi apparatus and microtubule organizing center (MTOC) to face the wound,
thereby contributing to polarization of the cell. This
process requires, among other things, activation of
the p21 GTPase Cdc42. Here we show that cells
expressing a mutant of paxillin, lacking the LD4 motif
(which couples to the PKL-PIX-PAK complex, key
components in p21 GTPase signaling; right panels)
fail to effectively reorient their Golgi (labeled in red
with a marker for ␣-mannosidase II) compared with
cells expressing wild-type paxillin (left panels), suggesting paxillin may perform an important role in
regulating Cdc42 activity and thereby cell polarity.
PAXILLIN ADAPTING TO CHANGE
Physiol Rev • VOL
diac muscle (30), although its function in the early development of this tissue is unclear.
D. Gene Expression
Historically, LIM domains have been shown to function in protein-protein interactions rather than DNA binding (48). However, a growing body of evidence detailing
the capacity of LIM domain containing proteins to undergo regulated nucleocytoplasmic shuttling has refocused some attention towards the potential nuclear function for these proteins (297) (Fig. 7). The first demonstration of this level of regulation was in an analysis of zyxin
(189, 190) and later confirmed for its paralogs LPP (204),
trip6 (296), and Ajuba (119). Similar to that described for
the zyxin family, expression of the paxillin or Hic-5 LIM
domains as GFP-fusions results in a protein that localizes
both to focal adhesions and the nucleus (266). For the
zyxin family, a nuclear export sequence (NES) mediates
the regulated shuttling. Interestingly, the paxillin family
LD motifs resemble NES sequences, thus they may act as
de facto NES sequences in addition to their protein recognition function.
The function of NES sequences can be negated by
treatment with the nuclear export inhibitor leptomycin B.
Such treatment of fibroblasts causes a retention of paxillin in the nucleus (307), providing evidence that paxillin
normally cycles between the nucleus and cytoplasm. The
capacity to sequester Hic-5 in the nucleus following leptomycin B inhibition of nuclear export machinery has also
been shown (4, 245). For Hic-5, a redox-sensitive mechanism for regulating localization to the nucleus has been
identified (245), providing the first compelling evidence
for the normophysiological function of nucleocytoplasmic shuttling of group 3 LIM proteins. This switch mechanism may be regulated in part through Hic-5 dimerization
(245). A potential role for PYK2 in the nuclear localization
of paxillin and Hic-5 has also been reported (4). The
localization of paxillin and Hic-5 to the nucleus in normally growing cells would provide additional assurance
for a relevant role for these proteins in this compartment,
and in fact, ⬃10% of cellular paxillin and Hic-5 can be
recovered in nuclear matrix fractions (121, 315). Hic-5
association with the nuclear matrix is mediated through
the LIM domains (315), whereas for paxillin, the amino
terminus is required in addition to the LIM domains (121).
Once in the nucleus what is the function for paxillin
family members? One possibility is that the nucleus provides an additional means of compartmentalizing and
thereby restricting access to paxillin binding sites and in
turn allowing for assembly and modification of a unique
paxillin scaffold. Second, paxillin may function to assist in
the translocation from the nucleus to focal adhesions for
proteins such as Abl (142, 143) and STAT3 (250). How-
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of a paxillin Y31F/118F mutant in isolated muscle fibers
inhibits tension development (261), suggesting phosphorylation of paxillin may be important in regulating actin
filament dynamics. This may occur via p21 Rho family
GTPase signaling since, as seen in other cell types, phosphorylation of paxillin on Y31/118 stimulates CrkII binding thereby potentially linking paxillin to Rac activation
via a Crk-Cas-DOCK180 signaling pathway (29, 126). Interestingly, paxillin is actively recruited from the cytosol
in response to acetylcholine stimulation, and this dynamic
translocation is essential for tension development (261,
262). Paxillin recruitment to the membrane is necessary
for a similar recruitment of vinculin, a paxillin and actin
binding protein. Again, a clear parallel can be drawn with
the focal adhesion and cytoskeletal reorganization that
occurs in nonmuscle cells responding to mechanical
stretching of the extracellular matrix caused either by the
cell itself or external forces. In response to increased
tension, both paxillin (229) and vinculin (11, 213) translocate from the cytosol and become enriched in focal
adhesions, which in turn likely contributes to cytoskeletal
stabilization and the activation of intracellular signaling
cascades (37).
As noted earlier, paxillin interacts directly with the
actin binding actopaxin family members as well as ILK
(183, 184). Both of these proteins have emerged as evolutionarily conserved mediators of integrin-actin linkages
in muscle tissue. Together with the LIM-only adaptor
protein PINCH and possibly Nck2, ILK and actopaxin are
required for normal muscle development and integrinactin attachment in organisms as diverse as flies and
worms (147, 164, 327). Although a role for paxillin in
mammalian muscle cytoarchitecture is clear, the contribution played by this protein in lower organisms remains
to be determined. It should be noted, however, that paxillin and many of its binding partners, such as PKL, vinculin, and FAK, are highly conserved between mammals
and flies (104, 304), while the existence of a full-length
worm ortholog of paxillin remains unresolved (54, 147).
Paxillin family members may also play a pivotal role
in muscle differentiation. Forced overexpression of paxillin in cultured quail myoblasts blocks differentiation,
instead promoting continued cell proliferation and signaling through the MAP kinase pathway (227). Phosphorylation of paxillin on amino acids Y31/118 and S188/190,
which have each been shown to be regulated via integrin
signaling, is necessary to maintain the undifferentiated
phenotype (227). The role for Hic-5 is less clear. Forced
expression Hic-5 in C2C12 myoblasts inhibits their fusion
and conversion to myotubes, instead inducing apoptosis
suggesting a negative role in muscle differentiation (100).
In contrast, Shibanuma et al. (244), also using C2C12 cells,
reported a proactive role for Hic-5 in early myogenic
differentiation. Hic-5 is also expressed in embryonic car-
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MICHAEL C. BROWN AND CHRISTOPHER E. TURNER
ever, the capacity of both Hic-5 and paxillin to bind steroid receptors provides a more direct path to nuclear
function. They contribute to the transactivation of androgen, glucocorticoid, and progesterone receptors, but not
the estrogen receptor (63, 121, 315). The level of transactivation is approximately fivefold stimulation, with paxillin and Hic-5 capable of additive transactivation. The
mechanism in which this is accomplished differs between
the two molecules. While both paxillin and Hic-5 bind to
steroid receptors through their carboxy-terminal LIM domains (121, 315), the paxillin transactivation domain is
Physiol Rev • VOL
confined to the carboxy terminus (121), whereas this
activity resides solely within the amino terminus of Hic-5
(315). In addition, Hic-5 binding to steroid receptors may
be ligand dependent (63) while paxillin binding does not
require ligand-bound steroid receptor (121). The transactivating potential of Hic-5␤ can be suppressed by PYK2
phosphorylation of Y43 (295), providing further evidence
for an important role as a coregulator. Evidence that Hic-5
is critical for androgen signaling was provided upon the
identification of a Hic-5 ␤-mutant, A413T, that blocks
androgen receptor function in a dominant negative fash-
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FIG. 7. Cytoplasmic-nuclear shuttling of paxillin family members. Paxillin nucleocytoplasmic translocation may
regulate its function as well as influence a diverse number of cellular signaling pathways. Although it has been
established that in addition to paxillin and other LIM family proteins a number of proteins including PKC, Pyk2 and Abl,
phospholipase C, and actin transit the nucleus, the role(s) ascribed has not been fully elucidated. Segregation of paxillin
family members within the nucleus may allow for the assembly of a unique signaling apparatus that may function within
the nucleus or be specifically translocated to the cytosol and plasma membrane. Thus nucleocytoplasmic shuttling may
function to temporally regulate paxillin access to focal adhesions. A further intriguing possibility is that transit through
the nucleus results in a modification event that is important for its localization to focal adhesions. In considering these
possibilities, it is appealing to equate paxillin with the yeast MAPK scaffold protein Ste5. Ste5 nucleates the assembly of
a MEKK/ERK/PAK/G␤/Cdc42GEF/Cdc42 signaling complex at sites of polarized growth in response to pheromone (56),
analogous to the scaffold formed in response to G protein-coupled activation of directional motility in metazoan cells
(145, 310). Ste5 as well as the Cdc42GEF cdc24p cycle through the nucleus where Ste5 undergoes an as yet undefined
modification to make it competent to localize to the site of polarized growth (167). The discovery of paxillin directly
binding to steroid receptor transcription factors provided a direct link to function. In fact, paxillin and Hic-5 have been
shown to function as transactivators for androgen and glucocorticoid receptors. Much work has been performed
characterizing the mechanisms and identifying genes regulated by Hic-5 that include collagenase, fibronectin, the
transcription factor c-fos, and cell cycle regulator p21CIP1/WAF1 (246, 248). In turn, these changes in gene expression
likely impact on cell migration, invasion, survival, and proliferation. Interestingly, Hic-5 also regulates the mRNA splicing
pattern of fibronectin. Paxillin has been demonstrated to bind to the poly(A) binding protein-1 (307) and speculated to
regulate the splicing and movement of specific mRNAs into the cytosol and to the leading edge to facilitate their
translation. Furthermore, paxillin has been identified in complex with the translation factor EF-1␣, a protein that
localizes to the leading edge of motile cells to support efficient protein production at these dynamic sites (151). In total,
these observations position the paxillin family in a fully integrated role of responding to extracellular cues through
immediate changes in the existing adhesion and signaling machinery, as well as to regulating gene expression and the
global profile of protein expression within the cell. AR, androgen receptor; GCR, glucocorticoid receptor; PABP, poly(A)
binding proteins; Pax, paxillin.
PAXILLIN ADAPTING TO CHANGE
VI. DEVELOPMENT AND DISEASE
A. Paxillin Family Member Expression
At the organismal level, paxillin exhibits complex
developmental regulation. In early murine gastrulation
paxillin mRNA is expressed only in extraembryonic tissue, whereas Hic-5 and leupaxin are expressed in embryonic tissue (81). Later in midgastrulation, paxillin is expressed in mesodermal and endodermal tissue. Structures
that are positive include the endocardium, dorsal aorta,
and notochord (81). Although highly motile neural crest
cells express paxillin (81), little neural expression is detectable, consistent with previous studies of protein expression in chick (275). Similar expression patterns have
been reported in zebrafish (43) and Drosophila (304, 311).
The ␣-paxillin isoform is first expressed followed by upregulation of the ␤-isoform in midgestation (177). Interestingly, during renal development in mouse, paxillin is
expressed at high levels, whereas postpartum, paxillin is
downregulated and a 43- to 47-kDa immunoreactive band,
potentially Hic-5 but more likely paxillin ␦, is upregulated
and maintained (255). The expression and tyrosine phosphorylation profile of paxillin changes during the process
of embryogenesis, closely mirroring the expression and
activation profile of FAK (275, 284).
In analyses of mRNA expression profiles of adult
human tissues, paxillin is expressed abundantly in most
tissues with the exception of brain (176, 221). Hic-5 also
demonstrates widespread expression (247), although it is
largely absent from leukocyte cell-rich tissues like spleen
and thymus (328), whereas leupaxin is restricted to leukocyte populations (150). The genomic promoter elePhysiol Rev • VOL
ments of paxillin, Hic-5, and leupaxin have not been characterized; however, similar to Hic-5 (247), paxillin mRNA
is induced by TGF-␤ as determined using a retrovirusmediated gene trap screen (2). Hic-5 mRNA expression is
induced by retinoic acid, with Hic-5 then potentiating
retinoic acid-dependent differentiation processes (249).
Expression of paxillin family members in hematopoeitic cells shows a tremendous amount of regulation.
The paxillin ␥-isoform appears to be restricted to cells of
the myeloid lineage, with differentiation of promonocytic
cells leading to an upregulation of both the ␤- and ␥-isoforms (176). Leupaxin is similarly upregulated in differentiating lymphocytes (150), while Hic-5 is specifically
upregulated during the transition of megakaryocytes to
platelets with a concomitant downregulation of paxillin
(82).
B. Potential Roles in Disease
Hic-5 is upregulated during cellular senescence and
downregulated during Ras transformation or spontaneous
immortalization (247). Forced expression of Hic-5 in immortalized human fibroblasts induces senescence and
blocks colony formation (248) in a FAK-dependent manner (109). These data suggest the exciting possibility that
Hic-5 has tumor-suppressor functions. However, although
it has been noted that Hic-5 expression is largely absent
from immortalized and/or transformed cell lines (328),
Hic-5 is widely expressed in prostate cancer lines and
clinical samples (63) as well as in epithelial carcinomas
(328) and in many breast cancer cell lines (166). The
known roles for Hic-5 in scaffolding and steroid receptor
coactivation positions this protein as a potential major
mediator of carcinogenesis in these tissues.
The critical importance of paxillin was confirmed
upon generation of a mouse knockout of the paxillin gene
(81). The resulting embryonic lethal nature, despite the
expression of Hic-5 and leupaxin, revealed the lack of
compensation by these paralogs and thus the specific
requirement for paxillin. Although no definitive human
diseases thus far have been linked to the expression of a
mutant paxillin, chromosome 12q24 duplications (the location of paxillin and PKL) have been reported which
result in severe musculoskeletal, cardiovascular, and central nervous system malformation syndromes (162, 179,
214). In addition, amplification of 12q24 was found in
many bladder tumors in a comparative genomic hybridization and cytogenetic analysis screen (130), whereas
chromosome 12q24 was identified as containing tumor
suppressors, in a micro cell-mediated chromosome transfer screen in prostate cancer cells (105). Interestingly, in
this respect paxillin binds to the antiapoptosis protein
bcl-2 (254, 255). Induction of apoptosis targets paxillin for
caspase cleavage (36, 173) with amino acid residues D102
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ion (208). This mutation is within LIM4 where an androgen receptor-binding FXXLF motif, which is conserved
among paxillin family members, also resides (92). RNA
interference-mediated silencing of Hic-5 ␤ has provided
further support for the importance of this molecule in
normal androgen signaling (208).
Insofar as target genes are concerned, several reports
have implicated Hic-5 in regulating c-fos expression (244,
245, 248). Hic-5 regulation of the cdk inhibitor p21WAF1/
CIP1 promoter through Sp1 binding sites has also been
established (246). The coregulator function was localized
to LIM4 and suggested to involve Hic-5 binding to smad3
and CBP/p300. Because an androgen receptor-Sp1 complex regulates p21WAF1/CIP1 expression (159), further
studies will be required to determine the precise mechanism of Hic-5 action. Nonetheless, these studies provide
persuasive evidence for an important role of focal adhesion proteins in gene expression and merit considerable
attention as relates to function of paxillin family members
in cell, tissue, and organism development and function.
1329
1330
MICHAEL C. BROWN AND CHRISTOPHER E. TURNER
Physiol Rev • VOL
illin in cellular function. Further research including
molecular profiling (149) to identify the interrelationships
between the many proteins required for normal function of
signaling networks will surely illuminate our understanding
of the many roles for paxillin.
VII. FUTURE DIRECTIONS
With the identification of so many specific paxillin binding partners, several of which bind to overlapping domains,
determining how and when these proteins associate, and
what the consequence is for other binding partners will be
critical to understanding how the cell effectively utilizes the
paxillin adaptor function to respond to changes in the external environment and thereby derives the appropriate
functional outcome. Several lines of evidence point to the
LD-PBS association as one that is a potentially important
point of such regulation. For instance, the paxillin LD-binding domain(s) on FAK contains Y925, a site that when phosphorylated functions as a Grb2 SH2 domain-binding site (7,
234). Importantly, a paxillin LD4-FAK association would
likely sterically antagonize the capacity to target Y925 for
phosphorylation and subsequent binding to Grb2 (96),
thereby potentially regulating FAK-mediated activation of
the Ras-MAPK pathway. With respect to vinculin binding,
the face of the vinculin H2/5 helix, which corresponds to
H1/4 of FAK and could potentially function as a second
paxillin LD binding site, is blocked. Evidence exists, however, that this region may be regulatable by acidic phospholipids and/or other vinculin binding partners (10, 96, 114).
Whether access is controlled and the proper hydrophobic
interface is available for paxillin LD interactions under physiological conditions to perhaps stabilize this association and
provide a platform for actin assembly remains to be determined. Similarly, the capacity of PKL to associate with the
LD4 motif of paxillin is regulated, requiring a stepwise activation cascade involving the p21-activated kinase PAK transmitting a signal through the PKL binding partner PIX to
elaborate, by an uncertain mechanism, the PBS domain to
facilitate an interaction with paxillin LD4 (28, 329). Such
modulation in paxillin binding partners combined with phosphorylation of paxillin itself to impart structural changes
within the LD motifs will presumably be important in determining whether FAK, PKL, or actopaxin, for instance, associates with the LD4 motif and in so doing determine whether
the downstream response is primarily of a structural readout, e.g., LD-actopaxin-actin, or primarily signaling via LDFAK or LD-PKL-PIX-PAK. Clearly, further studies will shed
more light on this exciting arena of dynamic reciprocity
between paxillin and its LD binding partners.
As noted, paxillin is a remarkably frequent target of
phosphorylation in response to cellular stimulation (230).
Interestingly, Y118 may be the preferred target following
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and D301 identified as major caspase cleavage sites (36).
Importantly, expression of a paxillin molecule lacking
these cleavage sites protected cells from apoptosis offering compelling support for an important role for paxillin
in survival. Another direct link to cancer was identified
with the finding that paxillin binding to the NF2 tumor
suppressor (Merlin/Schwannomin) may mediate pathogenesis of neurofibromatosis type 2 in humans with exon
2 mutations (61). A role for paxillin in papillomavirusmediated cell transformation associated with cervical
cancer is postulated to be mediated in part through an
association of paxillin with the E6 protein (47).
Additional roles for paxillin in cancer have been reasoned primarily based on the propensity of paxillin to be
phosphorylated by integrin and growth factor receptor
ligation (228, 230, 277) and the known roles for these
transmembrane receptors in tumorigenesis and invasion
(97, 201, 236). Studies in which paxillin function in normal
and cancer cells is directly manipulated followed by examination of the consequences on cell function have identified clear roles for paxillin as addressed above. Profiling
of the expression and activities of paxillin family members in various cancers is further illuminating the potential role for these proteins in cancer and disease.
Paxillin is transcriptionally upregulated by heregulin
treatment of breast cancer cells, and the increased expression of paxillin directly correlates with HER2/3 receptor expression in both aggressive breast cancer lines and
grade III human breast tumors (287). Paxillin tyrosine
phosphorylation changes were not observed in conjunction with increases in paxillin levels (287) consistent with
increased invasiveness being associated with the formation of a novel signaling invadopodia complex that includes paxillin, cortactin, and PKC␮ but not FAK (21).
However, a decrease in paxillin levels and an increase in
p130cas levels have been associated with metastatic
breast cancers in canine and feline models (237). This is
consistent with the demonstration of a functional antagonism between paxillin and p130cas with respect to epithelial-mesenchymal transition and motility in a murine
mammary cell model (316).
In lung cancers, a decrease in paxillin expression and
tyrosine phosphorylation has been correlated with generation of metastatic cancer (115, 222). However, upregulation
of paxillin expression has been observed in proliferative
prostate epithelium and correlated with increased metastatic potential (272), with increases in paxillin tyrosine
phosphorylation proposed to be important (5). Similarly,
paxillin upregulation has been reported in experimental nephritic syndrome (132), in glomerular injury (131), and in
metastatic renal carcinoma (117). The apparent contradictions regarding a direct correlation between paxillin expression/phosphorylation and cancer aggressiveness likely represents the tissue-specific and context-specific roles for pax-
PAXILLIN ADAPTING TO CHANGE
Studies in the authors’ laboratory are supported by grants
from the National Institutes of Health.
Address for reprint requests and other correspondence:
C. E. Turner, Dept. of Cell and Developmental Biology, SUNY
Upstate Medical University, 750 East Adams St., Syracuse, NY
13210 (E-mail: [email protected]).
Physiol Rev • VOL
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