REVIEWS

FOCAL ADHESION KINASE:

IN COMMAND AND CONTROL
OF CELL MOTILITY
Satyajit K. Mitra, Daniel A. Hanson and David D. Schlaepfer
Abstract | A central question in cell biology is how membrane-spanning receptors transmit
extracellular signals inside cells to modulate cell adhesion and motility. Focal adhesion kinase
(FAK) is a crucial signalling component that is activated by numerous stimuli and functions as a
biosensor or integrator to control cell motility. Through multifaceted and diverse molecular
connections, FAK can influence the cytoskeleton, structures of cell adhesion sites and
membrane protrusions to regulate cell movement.

INTEGRINS
A large family of heterodimeric
transmembrane proteins that
function as receptors for cell-
adhesion molecules.
EXTRACELLULAR MATRIX
(ECM). The complex, multi-
molecular material that
surrounds cells. The ECM
comprises a scaffold on which
tissues are organized, it provides
cellular microenvironments and
it regulates various cellular
functions.
The Scripps Research
Institute, Department of
Immunology, IMM21 10550
North Torrey Pines Road,
La Jolla, California 92037,
USA.
Correspondence to D.D.S.
e-mail: dschlaep@scripps.edu
doi:10.1038/nrm1549
Cell migration is a coordinated process that involves
rapid changes in the dynamics of actin filaments,
together with the formation and disassembly of cell
adhesion sites1. A complex interplay between the actin
cytoskeleton and cell adhesion sites leads to the genera-
tion of membrane protrusions and traction forces2.
External stimuli that control cell migration are trans-
duced into intracellular biochemical signals through the
interactions of transmembrane INTEGRINS that bind to
EXTRACELLULAR MATRIX (ECM) proteins, growth factors
that bind to their cognate cell-surface receptors, or
mechanical stimuli such as shear stress that promote
deformation of the actin cytoskeleton. For a cell to
process these different environmental motility-pro-
moting stimuli correctly, there must be essential intra-
cellular signalling proteins that function as ‘integrators’
— that is, proteins that are stimulated by multiple
extracellular inputs and that function to regulate
multiple signalling pathway outputs3. Here, we
describe the unique molecular connections of focal
adhesion kinase (FAK) that allow this tyrosine kinase
to function as an important receptor-proximal regula-
tor of cell shape, adhesion and motility.
The complexities of FAK
FAK was independently identified in 1992 by Steve
Hanks, Jun-Lin Guan and Michael Schaller as a sub-
strate of the viral Src oncogene and, in normal cells, as a
highly tyrosine-phosphorylated protein that localized
to integrin-enriched cell adhesion sites that are known
as focal contacts (BOX 1). Focal contacts are formed at
ECM–integrin junctions that bring together cytoskele-
tal and signalling proteins during the processes of cell
adhesion, spreading and migration. Early studies found
that FAK could be activated by either ECM or growth
factors, and that tyrosine phosphorylation of FAK was a
rapid event that was associated with the formation of
focal contacts4. Subsequent studies using knockout
mice revealed that null mutation of FAK resulted in
defective developmental morphogenesis5. As FAK-null
fibroblasts show excessive, rather than decreased (as was
initially predicted), formation of focal contacts, FAK
signalling has been associated with the disassembly of
integrin-based adhesion sites6. The loss of FAK expres-
sion also disrupts microtubule polarization within
cells7, and this phenotype, as well as the defect in focal
contact turnover, has been linked to the FAK-mediated
regulation of RHO-FAMILY GTPases in cells8. Rho-family
GTPases are molecular ‘switches’ within cells, which
control the formation and disassembly of actin
cytoskeletal structures (STRESS FIBRES, LAMELLIPODIA and
filopodia) and that function to provide the molecular
framework that supports directed cell motility.
In both normal and transformed cells, FAK signalling
can promote increased cell motility. The genomic desig-
nation of human FAK is protein-tyrosine kinase-2

56 | JANUARY 2005 | VOLUME 6 www.nature.com/reviews/molcellbio

© 2005 Nature Publishing Group

 REVIEWS

many studies have shown that FAK inhibition blocks the

Box 1 | Molecular architecture of focal contacts
response to cell motility cues18, recent and provocative
studies have shown that inhibition of FAK expression or
activity resulted in increased carcinoma cell migration
through the dissolution of N-cadherin-mediated cell–cell
Actin stress fibres
contacts in HeLa CELLS19.
It is possible that this latter observation might be a
cell-type-specific signalling event. However, we specu-
late that the ability of FAK to promote both the matura-

in
tion and turnover of focal contacts is related to its role as

os
My
both a signalling kinase and as an adaptor/scaffold pro-
in

in
os

os
tein, which places FAK in a position to modulate various
My

My
intracellular signalling pathways (FIG. 1). Namely, it is the
association of FAK with both activators and/or
Zyxin
α -A
inhibitors of various small GTPase proteins (Rho, Rac,
c tin α -A Cdc42 and Ras) that enables changes in FAK activity to
in c tin
Vi
nc

in be connected to alterations in the polymerization or sta-

Vi

α -A
ul

nc

α -A
in

ul

c tin bilization of actin and microtubule filaments.

in

in c tin
in Focal contact Additionally, because migrating cells experience changes
proteins
Vi

FAK FAK in forces through integrin contacts that link the ECM
nc
ul

illin P ax with the cytoskeleton, FAK is important in the ‘sensing’

in

Pax in p130 p130 ill

Tali in
Plasma Tl
a Src Cas Cas Src n of mechanical forces that are either generated internally
membrane
or exerted on cells20. FAK activation is therefore involved
in modulating ‘corrective’ cell responses to environmen-
tal stimuli. FAK does this through signal-mediated
effects on actin polymerization, the assembly or disas-
Integrins sembly of focal contacts, and the regulation of protease
activation or secretion16,21,22.
α β β α
The FERM and FAT domains of FAK
Extracellular matrix
FAK is a ubiquitously expressed 125-kDa protein tyro-
sine kinase that is composed of an N-terminal FERM
(protein 4.1, ezrin, radixin and moesin homology)
The extracellular matrix, integrins (α- and β-transmembrane heterodimeric proteins)
domain, a central kinase domain, proline-rich regions
and the cell cytoskeleton interact at sites called focal contacts. Focal contacts are dynamic
groups of structural and regulatory proteins that transduce external signals to the cell and a C-terminal focal-adhesion targeting (FAT)
interior and can also relay intracellular signals to generate an activated integrin state at domain (FIG. 2). The FERM domain of FAK facilitates a
the cell surface113. The integrin-binding proteins paxillin and talin recruit focal adhesion signalling linkage from receptor tyrosine kinases such as
kinase (FAK) and vinculin to focal contacts (see figure). α-Actinin is a cytoskeletal the epidermal growth factor receptor (EGFR) and the
protein that is phosphorylated by FAK, binds to vinculin and crosslinks actomyosin platelet-derived growth factor receptor (PDGFR)23. In
stress fibres and tethers them to focal contacts. Zyxin is an α-actinin- and stress-fibre- analysing cell-motility-promoting signals that are initi-
binding protein that is present in mature contacts. Although the aforementioned proteins ated by G-PROTEIN-COUPLED RECEPTORS (GPCRs), overexpres-
are found in most focal contacts, the membrane-associated protein tyrosine kinase Src sion of the FAK FERM domain blocked FAK activation
and the ADAPTOR PROTEIN p130Cas associate with focal contacts following integrin and resulted in the inhibition of G-protein-stimulated
clustering. Integrin-mediated FAK activation is mediated in part by matrix binding or by cell migration24. It is the FAK FERM domain that can
force-dependent changes in cytoskeletal linkages. Several other proteins such as bind to and promote the integrin- and FAK-mediated
extracellular signal-regulated kinase 2 (ERK2) and calpain are known to be transiently activation of other non-receptor tyrosine kinases such as
present at focal contacts (not shown). The composition of a focal contact is therefore ETK25. Additionally, actin- and membrane-associated
constantly varying depending on external cues and cellular responses. adaptor proteins such as ezrin can bind to the FAK
FERM domain and facilitate increased FAK activation in
an integrin-independent manner26.
(PTK2) and it is located at human chromosome How the FAK FERM domain associates with various
RHO-FAMILY GTPases 8q24term, a locus that is subject to amplification in targets is an active area of research. FAK can become
A subfamily of small (~21 kDa) human cancer cells9. Furthermore, elevated levels of post-translationally modified by the covalent addition
GTP-binding proteins that are
PTK2 mRNA have been found in studies of human car- of a small ubiquitin-related modifier (SUMO) at the
related to Ras and that regulate
the cytoskeleton. The cinoma tumours and in acute lymphoblastic leukaemias, ε-amino position of Lys152 (REF. 27). In most instances,
nucleotide-bound state is as detected by large-scale gene expression profiling10,11. sumoylation is associated with the nuclear import of
regulated by GTPase-activating FAK protein expression is elevated in many highly proteins and, correspondingly, sumoylated FAK was
proteins, which catalyse malignant human cancers12, and studies have shown enriched in the nuclear fraction of cells27. Although
hydrolysis of the bound GTP,
and guanine nucleotide-
that FAK signalling can promote changes in cell shape13,14 blocking nuclear export using leptomycin B promotes
exchange factors, which catalyse and the formation of podosomes or invadopodia15, the nuclear accumulation of FAK, and exogenous
GDP–GTP exchange. which leads to an invasive cell phenotype16,17. Whereas expression of the FAK FERM domain exhibits strong

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY VOLUME 6 | JANUARY 2005 | 5 7

© 2005 Nature Publishing Group

REVIEWS

directly to the cytoplasmic tails of integrins33, accu-

Extracellular
matrix Growth
α β factor receptors
Integrins
Assembly
Cadherins
Focal FAK
contacts
Disassembly
?
Rho-family GTPases
mDia RhoA Rac
Microtubule Stress Lamellipodia
stabilization fibres
Cell migration
Figure 1 | Focal adhesion kinase integrates signals to promote cell migration. Focal
adhesion kinase (FAK) is activated by growth factors and integrins during migration, and functions as
a receptor-proximal regulator of cell motility. At contacts between cells and the extracellular matrix,
FAK functions as an adaptor protein to recruit other focal contact proteins or their regulators, which
affects the assembly or disassembly of focal contacts. FAK activity and downstream signalling can
promote changes in actin and microtubule structures, and FAK signalling can affect the formation
and disassembly of cell–cell (cadherin-based) contacts. The Rho-family GTPases (RhoA, Rac and
Cdc42) direct local actin assembly into stress fibres, lamellipodia and filopodia, respectively. FAK can
influence the activity of Rho-family GTPases through a direct interaction with, or phosphorylation of,
protein activators or inhibitors of Rho GTPases. RhoA can also influence the stability of microtubules
through its effector Diaphanous (mDia).
nuclear localization28, it is not known whether these
events are dependent on sumoylation. As sumoylated
FAK showed elevated activity27, and FAK signalling has
been linked to enhanced gene transcription29 and cell-
STRESS FIBRES
cycle progression30, it is possible that sumoylation of
Also termed ‘actin- FAK might facilitate a direct signalling route between
microfilament bundles’, these are focal contacts and the nucleus.
bundles of parallel filaments that The C-terminal domain of FAK contains two
contain F-actin and other
proline-rich regions that function as binding sites for
contractile molecules, and often
SRC-HOMOLOGY (SH)3-DOMAIN-containing proteins (FIG. 2).
stretch between cell attachments
as if under stress. SH3-domain-mediated binding of the adaptor protein
p130Cas to FAK is important in promoting cell migra-
LAMELLIPODIA tion through the coordinated activation of Rac at mem-
Broad, flat protrusions at the
leading edge of a moving cell
brane extensions31,32. The SH3-mediated binding of
that are enriched with a other proteins, such as GRAF (GTPase regulator associ-
branched network of actin ated with FAK) and ASAP1 (Arf GTPase-ACTIVATING PROTEIN
filaments. (GAP) containing SH3, ankyrin repeat and pleckstrin
homology (PH) domains-1), connects FAK to the regu-
HeLa CELLS
An established tissue-culture lation of cytoskeletal dynamics and focal contact assem-
strain of human epidermoid bly. However, the downstream connections of GRAF
carcinoma cells, containing and ASAP1 remain undefined4.
70–80 chromosomes per cell. The C-terminal domain of FAK also encompasses
These cells were originally
derived from tissue taken from a
the FAT region, which promotes the colocalization of
patient named Henrietta Lacks FAK with integrins at focal contacts (BOX 1). Whereas
in 1951. it was first hypothesized that FAK might bind
mulated evidence supports an indirect association of
FAK with integrins through binding to integrin-
associated proteins such as paxillin and talin18. The
FAK FAT domain also binds directly to an activator
of Rho-family GTPases that is known as p190
RhoGEF, and FAK-mediated tyrosine phosphoryla-
tion of p190 RhoGEF might be a direct link to RhoA
activation34.
FAK activation and phosphorylation
The best-characterized FAK phosphorylation event is
AUTOPHOSPHORYLATION at Tyr397, which can occur in
either cis or trans 35. Phosphorylation of FAK at Tyr397
creates a motif that is recognized by various SH2-DOMAIN-
containing proteins, such as SRC-FAMILY KINASES (SFKs),
Cdc42 phospholipase Cγ (PLCγ), suppressor of cytokine sig-
nalling (SOCS), growth-factor-receptor-bound protein-7
(GRB7), the Shc adaptor protein, p120 RasGAP, and
Filopodia
the p85 subunit of phosphatidylinositol 3-kinase
(PI3K)4,18,31,33 (FIG. 2). It is not known whether these
different signalling proteins differentially bind to
Tyr397-phosphorylated FAK in response to particular
cell stimuli or whether simultaneously there are differ-
ent complexes with a larger pool of activated FAK. In
this respect, we favour a sequential association model
whereby the binding of cellular Src (hereafter referred to
as Src) to FAK initiates signalling (discussed below) and
the association of SOCS with FAK is a terminal event
that leads to ubiquitin-mediated degradation of FAK36.
For integrin-, growth factor- and G-protein-linked
stimuli that promote cell motility, it is the transient
recruitment of SFKs into a signalling complex with FAK
that is one of the first events associated with FAK activa-
tion18. Proline-rich tyrosine kinase-2 (PYK2) is related
to FAK and shares a similar domain structure (FERM,
kinase, proline-rich and FAT domains) as well as com-
mon phosphorylation sites (BOX 2). The binding of SFKs
to PYK2 that is phosphorylated at Tyr402 is also associ-
ated with PYK2 activation. However, FAK and PYK2
possess distinct signalling roles in cells, partly owing to
differential binding of target proteins to the FERM and
FAT domains of FAK and PYK2, respectively.
Additionally, PYK2 is preferentially expressed in cells of
the endothelium, central nervous system and
haematopoietic lineages; PYK2 activation is sensitive
to intracellular Ca2+ signals; and PYK2 is only weakly
activated in response to the binding of α5β1-integrin
to fibronectin, whereas FAK is strongly activated37.
The difference in α5β1-mediated activation of FAK
versus PYK2 is directly related to the FAT-mediated
localization of FAK at focal contacts compared with a
perinuclear distribution of PYK2 in cells38. Although
PYK2 and FAK can bind SFKs and can activate com-
mon signalling pathways, the differential binding
activities of the FERM and FAT domains might limit
the functional redundancy of these PTKs in cells.
The activity of FAK is dependent on integrin-
mediated cell adhesion. Models of integrin-mediated
intermolecular FAK activation are based on the fact that
FAK mutants can compete for integrin association and

58 | JANUARY 2005 | VOLUME 6 www.nature.com/reviews/molcellbio

© 2005 Nature Publishing Group

 REVIEWS

p120 RasGAP, thereby reinforced the role of integrins in the regu-

GRB7, Shc, PLCγ, GRB2, p190 RhoGEF, lation of FAK signalling. Results showing that FAK
p85, Src, SOCS talin, paxillin
Ezrin
catalytic activity can be modulated by either post-
PDGF receptor, translational or mutational changes in activation-loop
ETK, P P P P P residues are consistent with crystal structure analysis of
EGF receptor
Tyr397 Tyr576 Tyr577 Tyr861 Tyr925 the ATP-bound kinase domain of FAK, which shows a
disordered activation-loop conformation41. As the
FAK FERM Kinase domain FAT
crystal structure of the kinase domain of FAK also
Lys152 PRR1 FIP200 PRR2 PRR3 showed the presence of an unusual disulphide bond
SUMO between Cys456 and Cys459 in a regulatory region,
p130Cas, ASAP1, GRAF
conformational changes or protein-binding interac-
Figure 2 | Focal adhesion kinase domain structure and phosphorylation sites. Focal tions might also function to modulate the activation
adhesion kinase (FAK) contains a FERM (protein 4.1, ezrin, radixin and moesin homology) state of FAK.
domain, a kinase domain and a focal adhesion targeting (FAT) domain. The FERM domain This model is supported by findings that cellular
mediates interactions of FAK with the epidermal growth factor (EGF) receptor, platelet-derived
proteins such as FAK-interacting protein of 200 kDa
growth factor (PDGF) receptor, the ETK tyrosine kinase and ezrin, and the FERM domain can be
conjugated to SUMO (small ubiquitin-related modifier) at Lys152. The FAT domain recruits FAK to
(FIP200) bind to the kinase domain of FAK and inhibit
focal contacts by associating with integrin-associated proteins such as talin and paxillin. It also FAK activity42. Additionally, evidence is accumulating
links FAK to the activation of Rho GTPases by binding to guanine nucleotide-exchange factors that intramolecular constraints also have a role in the
(GEFs) such as p190 RhoGEF. FAK contains three proline-rich regions (PRR1–3), which bind Src- regulation of FAK activity. There are alternatively spliced
homology-3 (SH3) domain-containing proteins such as p130Cas, the GTPase regulator isoforms of FAK in which amino-acid additions sur-
associated with FAK (GRAF) and the Arf-GTPase-activating protein ASAP1. FAK is rounding the Tyr397 site promote a change in the
phosphorylated (P) on several tyrosine residues, including Tyr397, 407, 576, 577, 861 and 925.
kinetics of FAK activation (as measured by Tyr397
Tyrosine phosphorylation on Tyr397 creates a Src-homology-2 (SH2) binding site for Src,
phospholipase Cγ (PLCγ), suppressor of cytokine signalling (SOCS), growth-factor-receptor- phosphorylation) from a primarily trans-intermolecular
bound protein 7 (GRB7), the Shc adaptor protein, p120 RasGAP and the p85 subunit of reaction to a cis-intramolecular reaction35. Although
phosphatidylinositol 3-kinase (PI3K). Phosphorylation of Tyr576 and Tyr577 within the kinase alternative splicing of FAK does not alter the FERM
domain is required for maximal FAK catalytic activity, whereas the binding of FAK-family domain residues of FAK, truncation or removal of the
interacting protein of 200 kDa (FIP200) to the kinase region inhibits FAK catalytic activity. FAK FAK FERM domain does result in enhanced FAK cat-
phosphorylation at Tyr925 creates a binding site for GRB2.
alytic activity35. As binding of proteins such as ezrin or
the GUANINE NUCLEOTIDE-EXCHANGE FACTOR (GEF) TRIO to
the FAK FERM domain result in enhanced FAK activ-
can inhibit endogenous FAK activity4 and that kinase- ity26,43, and as the FAK FERM domain can bind in trans
inactive FAK can become transphosphorylated on to the FAK catalytic domain, resulting in the inhibition
Tyr397 in cells23. FAK Tyr397 phosphorylation pro- of FAK activity44, it is possible that binding interactions
motes Src binding, which leads to the conformational or conformational changes in the FAK FERM domain
activation of Src and results in a dual-activated FAK–Src might function to release cis-inhibitory constraints on
signalling complex18. Within this FAK–Src complex, Src FAK catalytic activation.
ADAPTOR PROTEINS
Proteins that augment cellular phosphorylates FAK at Tyr861, and this is associated The activity of FAK can also be modulated posi-
responses by recruiting other with an increase in SH3-domain-mediated binding of tively45 or negatively 46 by the action of protein-tyrosine
proteins to a complex. They p130Cas to the FAK C-terminal proline-rich regions39. phosphatases (PTPs). Studies using PTPα-deficient
usually contain several Activated Src also phosphorylates FAK at Tyr925, which fibroblasts showed that this phosphatase was required
protein–protein interaction
domains.
creates an SH2-binding site for the GRB2 adaptor pro- for maximal stimulation of Src catalytic activity by
tein. GRB2 binding to FAK is one of several connections β1-integrins, and that PTPα functioned as an upstream
G-PROTEIN-COUPLED that lead to the activation of Ras and the extracellular regulator of FAK Tyr397 phosphorylation45. This result
RECEPTOR signal-regulated kinase-2 (ERK2)/mitogen-activated is consistent with the potential intermolecular activa-
A seven-helix membrane-
protein kinase (MAPK) cascade18. ERK2 phosphoryla- tion of FAK by Src. As Src can also become activated
spanning cell-surface receptor
that signals through tion and the subsequent activation of myosin light chain through direct interaction with the cytoplasmic
heterotrimeric GTP-binding and kinase can modulate focal contact dynamics in motile domains of β-integrins47, these types of result reinforce
GTP-hydrolysing G-proteins to cells3, as well as generate both proliferative and survival the fact that Tyr397 phosphorylation of FAK might not
stimulate or inhibit the activity signals inside cells31. always reflect FAK catalytic activity that is mediated by
of a downstream enzyme.
autophosphorylation.
SRC-HOMOLOGY (SH)3-DOMAIN Regulation of FAK catalytic activity
A protein sequence of 50 amino Src-mediated transphosphorylation of FAK within the p130Cas and paxillin as targets of FAK
acids that recognizes and binds kinase domain ACTIVATION LOOP at Tyr576 and Tyr577 In addition to promoting maximal FAK activation, the
sequences that are rich in proline.
promotes maximal FAK catalytic activation31. Mutation recruitment of Src into a FAK–Src signalling complex
GTPase-ACTIVATING PROTEIN
of FAK within this loop produces FAK mutants with functions to facilitate the phosphorylation of various
(GAP). A protein that stimulates either enhanced or refractory activities. One such FAK-associated proteins, as many FAK targets are also
the intrinsic ability of a GTPase mutant, ‘superFAK’, contains a Lys to Glu substitution independent binding partners and phosphorylation tar-
to hydrolyse GTP to GDP. at residues 578 and 581, and results in a FAK protein gets of Src. Two of the best-characterized target proteins
Therefore, GAPs negatively
regulate GTPases by converting
with adhesion-independent activity40. However, the of FAK–Src-mediated phosphorylation are p130Cas and
them from active (GTP-bound) phosphorylation of downstream targets in superFAK- paxillin31,32,48. SH3-mediated binding of p130Cas to FAK
to inactive (GDP-bound). expressing cells remained adhesion dependent, which is linked to enhanced tyrosine phosphorylation of

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY VOLUME 6 | JANUARY 2005 | 5 9

© 2005 Nature Publishing Group

REVIEWS

of FAK with β1-integrin and the localization of FAK to

Box 2 | The FAK-related kinase PYK2
focal contacts38. Paxillin binding is mediated by two
Src GRB2 leucine-rich peptide regions in paxillin that are known
↑ ↑
Tyr397 Tyr576 Tyr577 Tyr861 Tyr925 as LD MOTIFS, which interface with hydrophobic surface
grooves on the FAT domain54,55. Interestingly, the SH2
FAK FERM Kinase domain FAT
binding site for GRB2 at FAK Tyr925 partially overlaps
PRR1 PRR2 PRR3 with one of the two paxillin LD-motif binding sites in
Sequence the FAT domain54, and localization studies of phospho-
similarity <10% ~40% ~60% ~40%
rylated FAK have shown that Tyr925-phosphorylated
FAK might be selectively excluded from focal contact
PYK2 FERM Kinase domain FAT sites56. Overexpression of a Tyr925Phe mutant of FAK
PRR1 Tyr402 Tyr579 Tyr580 PRR2 PRR3 Tyr881 resulted in strong focal contact distribution56, and in
↓ ↓ activated Src-expressing cells, Tyr925Phe FAK blocks
Src GRB2 the turnover of focal contacts (V. Brunton, personal
Proline-rich tyrosine kinase-2 (PYK2) shares a similar domain arrangement with focal communication; see note added in proof). As NMR
adhesion kinase (FAK) (see figure), with 60% sequence identity in the central kinase analyses have shown that the FAT domain can
domain, conservation of proline-rich regions (PRRs), and identical positions of four undergo conformational rearrangements that might
tyrosine phosphorylation sites. PYK2 tyrosines 402, 579, 580 and 881 correspond to FAK selectively promote either Tyr925 phosphorylation
tyrosines 397, 576, 577 and 925, respectively. Phosphorylation of PYK2 Tyr402 and and/or paxillin binding57, it is possible that Src-medi-
Tyr881 create Src-homology-2 (SH2) binding sites for Src and growth-factor-receptor- ated phosphorylation of FAK on Tyr925, and subse-
bound-2 (GRB2), respectively. PYK2 contains a C-terminal focal adhesion targeting quent GRB2 binding, could displace paxillin, promote
(FAT) domain that binds to paxillin53. However, PYK2 shows perinuclear distribution the dissociation of FAK from focal contacts, and sub-
and is not strongly localized to focal contacts in many cells37. The substitution of the FAK sequently lead to focal contact turnover through
C-terminal domain to PYK2 facilitated the colocalization of this PYK2–FAK chimaera to undefined mechanisms (FIG. 3).
β1-integrin-containing focal contacts38, which indicates that there are biologically relevant Ser910 within the FAT domain is phosphorylated
binding differences between FAK and PYK2. For instance, the FAK C-terminal domain during mitosis58 and after growth factor stimulation of
uniquely binds the integrin-associated protein talin114, and PYK2 — but not FAK — cells. Ser910 is phosphorylated by ERK2 and this is also
binds the actin-associated protein gelsolin115. Although PYK2 can be activated by associated with reduced paxillin binding to FAK59. So,
integrins, this is dependent on integrin-mediated activation of Src-family kinases116,117. Src-mediated phosphorylation of Tyr925 on FAK and
The 40% sequence similarity between the N-terminal FERM (protein 4.1, ezrin, radixin GRB2 binding leading to ERK2 activation, coupled
and moesin homology) domains of PYK2 and FAK also accounts for differential association
with the feedback of ERK2-mediated Ser910 phospho-
with target proteins118.What remains unknown is why PYK2 activity is highly dependent
rylation, could potentiate the release of FAK from focal
on intracellular Ca2+ levels and how PYK2 associates with members of the Janus kinase
contacts. Alternatively, FAK–Src-mediated phosphory-
family37,119 — properties that are not shared by FAK.As PYK2 regulates several signalling
lation of paxillin at Tyr118 promotes the binding of
events that are crucial for macrophage120 and monocyte morphology121, and the Pyk2-null
phenotype results in a MARGINAL ZONE B-cell developmental defect122, there is probably a ERK2 to paxillin60, and ERK2-mediated phosphoryla-
unique role for PYK2 in mediating haematopoietic cell responses to chemokine stimuli. tion of paxillin can facilitate FAK binding to paxillin
and can enhance FAK activation60,61. Therefore, we
speculate that there might be a regulatory cycle in
which FAK–Src activation and signalling to ERK2 can
p130Cas at multiple sites, which promotes SH2-medi- function first to promote FAK release from existing
ated binding of the Crk adaptor protein to p130Cas. focal contacts and then, through ERK2-mediated phos-
Signalling downstream of p130Cas results in increased phorylation of paxillin, to promote FAK re-binding
activity of Rac, enhanced MEMBRANE RUFFLING or lamel- and activation at new or different focal contacts in a
lipodia formation, and the promotion of cell motility or migrating cell (FIG. 3).
invasion17,49,50 (FIG. 3). Paxillin is phosphorylated by
AUTOPHOSPHORYLATION
The transfer of a phosphate FAK–Src on Tyr31 and Tyr118, and this can also pro- Regulation of focal contact dynamics
group by a protein kinase either mote SH2-mediated binding of Crk to paxillin48,51. Lessons from FAK–/– and SHP2 –/– cells. In analyses mon-
to a residue in the same kinase Overexpressing paxillin that is mutated at these phos- itoring the formation of focal contacts, FAK was found
molecule (cis) or to a residue in a phorylation sites inhibits the turnover of focal contacts6 to be one of the first signalling proteins to be recruited to
different kinase molecule but of
the same type (trans).
and cell motility52, which therefore supports the pres- these sites62. Although FAK recruitment to focal con-
ence of multiple routes for FAK–Src-mediated sig- tacts is associated with increased FAK tyrosine phos-
SH2 DOMAIN nalling in modulating the dynamics of cell adhesion phorylation63, focal contacts readily form in FAK-null
A protein motif that recognizes sites. (FAK–/–) fibroblasts, which indicates that FAK activity is
and binds tyrosine-
not essential for the process of focal-adhesion forma-
phosphorylated sequences, and
thereby has a key role in relaying Regulated targeting of FAK to focal contacts tion5. However, focal contacts in FAK–/– cells form pri-
cascades of signal transduction. It is the C-terminal FAT domain of FAK that facilitates marily around the cell periphery, enmeshed in a cortical
the linkage to integrins and focal contacts. The FAT actin ring, and do not undergo a normal maturation
SRC-FAMILY KINASES domain adopts a four-helix bundle structure that con- cycle64. In normal fibroblasts, peripheral immature focal
Kinases that belong to the Src
family of tyrosine kinases, the
tains binding sites for integrin-associated proteins such contacts become connected to longitudinal stress fibres
largest of the non-receptor- as paxillin53. Point mutations in the FAT domain of FAK in cells and undergo actin contractility-mediated matu-
tyrosine-kinase families. that disrupt paxillin binding also prevent the association ration during cell polarization63. FAK re-expression in

60 | JANUARY 2005 | VOLUME 6 www.nature.com/reviews/molcellbio

© 2005 Nature Publishing Group

 REVIEWS

phosphatase SHP2 (SH2-domain-containing protein

α β
Migration Integrins
tyrosine phosphatase 2). SHP2–/– cells have increased
Membrane
FAK activity, but they also show an accumulation of
Rac immature focal contacts and similar refractory migra-
tion defects to FAK–/– cells67. Hyperactive FAK in SHP2–/–
Src
Crk cells results in an increase in the levels of Tyr12-phos-
Focal contacts phorylated α-actinin, which thereby reduces the
SH2 p130Cas P
crosslinking of stress fibres and prevents the maturation
P P P SH3
of focal contacts68. In SHP2–/– cells, there is a high level of
Tyr397 Tyr576 Tyr577 P-X-X-P Tyr925
Paxillin focal contact turnover68, whereas in FAK–/– cells, focal
FAK FERM Kinase domain FAT ERK2 contact turnover and maturation are inhibited8. So, both
P Tyr118 SHP2–/– and FAK–/– cells show an accumulation of
P immature focal contacts, but through different mecha-
nisms. These studies support the importance of FAK
Release expression and the precise temporal regulation of
ERK2
FAK activity as important factors that control the
dynamics of focal contacts.
P GRB2
Tyr925 Paxillin–/–, p130Cas–/– and Src –/– cells. In addition to
FAK FERM Kinase domain FAT FAK–/– and SHP2–/– cells, fibroblasts that contain null
mutations for various other focal-contact-associated
Ser910
proteins also show altered dynamics of focal contact
P
maturation, cell spreading defects and refractory cell
motility responses (TABLE 1). Time-lapse analyses showed
Figure 3 | Focal adhesion kinase (FAK)–Src signals that regulate cell motility and focal
contact localization. Integrin clustering promotes FAK autophosphorylation (P) at Tyr397, which
that the incorporation of labelled paxillin into focal con-
creates a binding site for the Src-homology (SH)2 domain of Src. Src-mediated phosphorylation tacts of FAK–/–, paxillin–/–, p130Cas–/– or SYF–/– (Src–/–,
of FAK at Tyr576 and Tyr577 promotes maximal FAK catalytic activity. Active FAK–Src facilitates Yes–/– and Fyn–/–) cells did not significantly differ com-
SH3-mediated binding of p130Cas to FAK and its subsequent phosphorylation. Crk binding to pared to normal fibroblasts6. However, the rate of focal
phosphorylated p130Cas facilitates Rac activation, lamellipodia formation and cell migration. contact disassembly was much slower in these null cells.
Paxillin binding to the FAK focal adhesion targeting (FAT) domain is important for FAK focal Analyses of FAK–/– cells showed that disassembly was
contact localization. Src-mediated phosphorylation of FAK at Tyr925 creates an SH2 binding site
for the growth-factor-receptor-bound protein 2 (GRB2) adaptor protein, which leads to the
dependent on FAK Tyr397 phosphorylation; SYF–/– cells
activation of Ras and the extracellular signal-regulated kinase-2 (ERK2) cascade. The GRB2 and showed that Src kinase activity was required; and studies
paxillin binding sites within the FAT domain overlap and Tyr925-phosphorylated FAK might be with paxillin–/– cells indicated that the integrity of the
selectively released from focal contacts. ERK2 activation promotes FAK phosphorylation at Tyr31 and Tyr118 paxillin phosphorylation sites were
Ser910, which is also associated with decreased paxillin binding to FAK. Within focal contacts, needed to promote focal contact turnover6. As the
FAK–Src-mediated phosphorylation of paxillin at Tyr118 promotes ERK2 binding. ERK2-mediated expression of constitutively active Src in FAK–/– cells can
phosphorylation of paxillin can facilitate FAK binding to paxillin and enhances FAK activation. So,
promote focal contact turnover and increased cell
there might be a cycle whereby Src- and ERK2-mediated phosphorylation of FAK promotes its
release from focal contacts and ERK2-mediated phosphorylation of paxillin promotes the motility17,69, these combined analyses support the con-
association of unphosphorylated FAK with paxillin at new or growing focal contact sites. clusion that, in normal cells, integrin- and FAK-medi-
ated control of Src activity is a key event that promotes
focal contact dynamics.

MARGINAL ZONE
A region in the spleen in which
white blood cell precursors such
as B-cells, granulocytes,
macrophages and plasma-cells
reside or transit through during
primary or secondary immune
responses.
ACTIVATION LOOP
A conserved structural motif in
kinase domains, which needs to
be phosphorylated for full
activation of the kinase.
GUANINE NUCLEOTIDE-
EXCHANGE FACTOR
A protein that facilitates the
exchange of GDP for GTP in the
nucleotide-binding pocket of a
GTP-binding protein.
FAK–/– cells promotes the reorganization of the ‘imma-
ture’ focal contacts, which allows for their connection to
actin stress fibres, therefore mediating cell contractility
and cell polarization64.
Mechanistically, these alterations in focal contacts
and actin structures involve the regulation of the activity
of α-actinin, a protein that promotes actin crosslinking
and that has an important role in maintaining the link-
age between focal contacts and stress fibres1,65 (FIG. 4).
FAK phosphorylates α-actinin at Tyr12, which results in
reduced α-actinin binding to actin66. α-Actinin is not
phosphorylated in FAK–/– cells, so this FAK signalling
linkage to α-actinin might underlie some of the matu-
ration defects, as well as turnover dynamics, of focal
contacts in FAK–/– cells66. It is possible that the lack of
α-actinin phosphorylation might be associated with the
presence of focal contacts enmeshed in a cortical actin
ring at the FAK–/– cell periphery. This hypothesis is fur-
ther supported by studies of cells that lack the tyrosine
FAK–Src and proteolysis. In addition to signalling events
that are associated with the phosphorylation of α-actinin,
p130Cas or paxillin, FAK–Src signalling can affect focal
contact dynamics through the regulation of both
extracellular and intracellular proteolytic events.
Inhibition of FAK activity in human carcinoma cells or
Src-transformed cells, or stable FAK re-expression in
FAK–/– cells, can alter the expression and activation of
MATRIX METALLOPROTEINASES (MMPs)
16,17,22
. The influence
of FAK on MMP regulation is associated with sig-
nalling from Ras to ERK2 and from Rac to Jun N-ter-
minal kinase (JNK). Activation of MMPs at the LEADING
EDGE of migrating cells functions to promote matrix
proteolysis, which leads to the extracellular release of
integrin–matrix contacts and thereby facilitates focal
contact turnover70.
The intracellular linkage of focal contacts to the
actin cytoskeleton is also regulated by calpain-mediated
proteolysis71. Calpain can cleave constituents of focal

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY VOLUME 6 | JANUARY 2005 | 6 1

© 2005 Nature Publishing Group

REVIEWS
MEMBRANE RUFFLE
A process that is formed by the
movement of lamellipodia that
are in the dynamic process of
folding back onto the cell body
from which they previously
extended.
LD MOTIF
A short sequence found within
proteins that has the consensus
sequence LDXLLXXL and
functions as a protein-binding
interface.
MATRIX METALLOPROTEINASES
Proteolytic enzymes that
degrade the extracellular matrix
and have important roles in
tissue remodelling and tumour
metastasis.
LEADING EDGE
The thin margin of a
lamellipodium that spans the
area of the cell from the plasma
membrane to a depth of about
1 µm into the lamellipodium.
62 | JANUARY 2005 | VOLUME 6
Actin stress fibres
Actin stress fibres
Myosin Myosin
Assembly
Myosin Myosin
MLCK ↑
ARP2/3 Reduced
α-actinin
Increased MLC crosslinking
α-actinin phosphatase
crosslinking in
ctin
α-A
in ROCK
Cdc42
P ctin
α-A Tyr12
Tyr256 P
in Tyr256
ctin Rho GRAF Rho
Cdc42 N-WASP α-A p190 p190
RhoGEF N-WASP RhoGEF
FAK FAK
P
Tyr256
N-WASP Focal contact Focal contact
Nucleus
Figure 4 | Focal adhesion kinase promotes cytoskeletal fluidity. Stress fibres and cortical actin are continuously
destabilized/stabilized by focal adhesion kinase (FAK)-regulated processes. Normally, the actin cytoskeleton exists in a semi-solid
state, owing to a high degree of α-actinin-mediated crosslinking of stress fibres, which are tethered and exert tension at focal
contacts (left panel). Conversion to a more soluble state (right panel) is promoted by FAK phosphorylation (P) on Tyr12 of α-actinin,
which results in reduced crosslinking and the release of actin stress fibres from focal contacts. Cytoskeletal fluidity is also regulated
by the effects of FAK on Rho-family GTPases and on the neuronal Wiskott–Aldrich syndrome protein (N-WASP). FAK
phosphorylates Cdc42-activated N-WASP at Tyr256, thereby retaining phosphorylated N-WASP in the cytoplasm where it can
affect ARP2/3-mediated actin polymerization. Through associations with Rho GTPase-activating proteins (GAPs) and Rho guanine
nucleotide-exchange factors (GEFs), FAK can regulate actomyosin stress fibre polymerization. Reduced tension can be attributed in
part to increased RhoGAP activity of GTPase regulator associated with FAK (GRAF). Conversely, FAK can promote cytoskeletal
tension through phosphorylation and activation of p190 RhoGEF. Subsequent Rho activation indirectly regulates myosin light chain
(MLC) phosphorylation through Rho-associated kinase (ROCK) phosphorylation of MLC phosphatase, which leads to increased
MLC kinase (MLCK) activity through the downregulation of MLC phosphatase activity.
contacts, such as talin and FAK, and calpain-4–/– cells FAK effects on GTPases and actin
have an increased number of peripheral focal contacts72. The activity of Ras and the Rho-family GTPases Rho,
Calpain is not appropriately activated in FAK–/– cells21, Rac and Cdc42 is positively regulated by GEFs and neg-
and this defect might be due in part to ERK2 activa- atively regulated by GAPs. As mentioned above, a num-
tion being required for calpain function73. Notably, ber of studies have shown that FAK–Src-mediated
FAK re-expression in FAK–/– cells promotes the forma- phosphorylation events can lead to the activation of
tion of a complex between calpain, ERK2 and activated Ras–ERK2 and Rac–JNK signalling cascades to pro-
Src21. Restoration of calpain activity in FAK–/– cells mote increased cell migration and invasion18. In a
requires specific FAK phosphorylation events: a form recently discovered mechanism, FAK overexpression
of FAK that is mutated at several phosphorylation sites facilitated the SH2-mediated binding and sequestering
can form a complex with calpain and ERK2, but it of p120 RasGAP, which diminished the association of
does not restore full calpain activity in contrast to cells p120 RasGAP with active Ras76 and thereby led to Ras
in which wild-type FAK is re-expressed74. So, FAK sig- activation.
nalling is connected to the increased turnover of focal In FAK –/– cells, the intrinsic GTPase activity of
contacts through calpain activation. As calpain is a RhoA is elevated8, and pharmacological inhibitors of
Ca2+-dependent protease, and FAK activation is associ- Rho-associated kinase (ROCK) — a substrate of Rho
ated with local Ca2+-flux-induced disassembly of focal — partially reverse the polarization defects of FAK–/–
contacts75, the FAK–calpain linkage might be selec- cells77. Integrin signalling can suppress RhoA activity
tively activated at either cell protrusions or tail retrac- by tyrosine phosphorylation of p190 RhoGAP
tion sites in motile cells. (which increases its GAP activity)78. Likewise, stable
www.nature.com/reviews/molcellbio
© 2005 Nature Publishing Group
 REVIEWS

Table 1 | Phenotypes associated with null mutations in focal contact proteins

Cell Embryonic Focal contact Focal contact Integrin-stimulated FAK tyrosine Reference
phenotypes (lethality) day formation turnover migration phosphorylation
FAK–/– (p53–/–) 8.5 Increased immature Inhibited Inhibited NA 5
SYF–/– 9.5 No change Inhibited Reduced pTyr397 reduced 123
p130Cas–/– 11.5–12.5 No change Inhibited Reduced No difference 124
Paxillin–/– 9.5 Increased size Decreased Inhibited pTyr397 reduced 125
Vinculin–/– 10.0 Decreased size ND Stimulated Increased activity 126
–/–
PTPα None Delayed ND Reduced pTyr397 reduced 45
SHP2–/– 8.5–10.5 Increased immature Elevated Inhibited Increased activity 67,68
in suspension
Calpain-4–/– 10.0 Larger Reduced Decreased No change 72
This table summarizes the phenotypes of fibroblasts that are derived from mice that are null for various proteins associated with focal contacts. FAK and paxillin are involved in
the formation of focal contacts, whereas FAK, Src-family kinases (Src, Yes and Fyn; SYF), p130Cas and calpain are also involved in focal contact turnover. Except for vinculin-
null cells, the lack of any of the above proteins results in impaired integrin-stimulated cell migration. FAK, focal adhesion kinase; NA, not applicable; ND, not determined;
PTPα, protein tyrosine phosphatase-α; pTyr, phosphotyrosine; SHP2, Src-homology (SH)2-containing phosphotyrosine phosphatase-2.

ARP2/3 COMPLEX
A complex that consists of two
actin-related proteins ARP2 and
ARP3, along with five smaller
proteins. When activated, the
ARP2/3 complex binds to the
side of an existing actin filament
and nucleates the assembly of a
new actin filament. The resulting
branch structure is Y-shaped.
GANGLIOSIDE
An anionic glycosphingolipid
that carries, in addition to other
sugar residues, one or more sialic
acid residues.
LIPID RAFTS
Lateral aggregates of cholesterol
and sphingomyelin that are
thought to occur in the plasma
membrane.
FAK re-expression in FAK–/– cells decreased RhoA
activity 8 and enhanced p190 RhoGAP tyrosine phos-
phorylation17. In other cell types, FAK activation and
tyrosine phosphorylation are associated with RhoA
activation and the formation of stress fibres18. This con-
nection could be mediated by FAK binding to, and
phosphorylating, p190 RhoGEF34. In neuronal develop-
ment, FAK signalling through p190 RhoGEF controls
axonal branching and synapse formation79. Although
FAK-mediated activation of p190 RhoGEF is a direct
route to RhoA activation, the formation of distinct sig-
nalling complexes will probably influence whether FAK
activation leads to increased or decreased RhoA activity
in cells (FIG. 4).
In addition to affecting the activity of Ras, Rac and
Rho, FAK can influence the function of Cdc42 through
binding and phosphorylation of the Cdc42 effector
Wiskott–Aldrich syndrome protein N-WASP (neuronal
WASP)80. N-WASP, which, in contrast to its name, is
ubiquitously expressed, regulates the actin cytoskeleton
through activation of the ARP2/3 COMPLEX3. Interestingly,
FAK only associates with Cdc42-activated N-WASP, and
does not itself activate N-WASP. Although FAK
phosphorylation of N-WASP at Tyr256 does not
affect N-WASP activity towards ARP2/3, it does seem
important for maintaining a cytoplasmic distribution of
N-WASP and for promoting cell motility80. As Cdc42
regulates actin dynamics in cellular projections, the
interaction of FAK with Cdc42-activated N-WASP
might couple actin polymerization with membrane
protrusion during cell motility (FIG. 4).
FAK and microtubules
Integrating factors coordinate the regulation of micro-
tubule structures and the actin cytoskeleton during cell
motility. Microtubules are important in the establishment
and maintenance of cell polarity, and the Rho effector
Diaphanous (mDia) functions to stabilize microtubules
at the leading edge of migrating cells81. Integrin-mediated
activation of FAK is required for microtubule stabiliza-
tion by the Rho–mDia signalling pathway7 (FIG. 5). This is
partly the result of the FAK-regulated localization of a
lipid-raft marker, GANGLIOSIDE GM1, to the leading edge
of motile cells. It is hypothesized that the lipid environ-
ment at the leading edge preferentially localizes micro-
tubule capping or bridging proteins, which stabilize the
association of microtubules with cortical receptors82.
This regulation of a distinct membrane lipid environ-
ment by FAK or integrin signalling83 also functions to
promote Rac signalling by maintaining a suitable lipid
environment that facilitates the interaction of Rac and
effectors such as p21-activated kinase (PAK)84.
Interestingly, FAK-stimulated phosphorylation of
Ser298 in MAPK/ERK kinase-1 (MEK1, also known as
MAPK kinase-1) by PAK is a secondary route that leads
to ERK2/MAPK activation85.
In neuronal cells, a fraction of FAK colocalizes with
a distinct microtubule structure that arises from
microtubule-organizing centres and that extends
around the nucleus in a branched fork-like form
termed a microtubule fork86. Microtubule forks are
believed to promote nuclear re-positioning in the
direction of cell movement. Cyclin-dependent kinase-5
(CDK5) phosphorylates FAK at Ser732 in post-mitotic
neurons, and antibodies that recognize Ser732-phospho-
rylated FAK specifically stain microtubule fork structures
near the nucleus86. Neurons that are devoid of CDK5 or
that express a FAK mutant in which Ser732 cannot be
phosphorylated show a malformed microtubule fork,
impaired nuclear movement and altered neuronal devel-
opment positioning in vivo 86. Whereas the molecular
mechanisms that link FAK Ser732 phosphorylation to the
localization and organization of microtubule fork struc-
tures remain to be defined, this observation is the first of
its kind and supports studies that link FAK phosphoryla-
tion to enhanced neuronal cell migration87.
FAK and membrane composition
The polarization of migrating cells requires membrane
modification as well as changes in the underlying
cytoskeleton. In addition to the role of FAK in the
translocation of LIPID RAFT components7, FAK–Src sig-
nalling is involved in the modification of phosphatidyli-
nositol lipids, and differentially phosphorylated lipid

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY VOLUME 6 | JANUARY 2005 | 6 3

© 2005 Nature Publishing Group

REVIEWS

phosphorylated by a FAK–Src complex, which facilitates

increased PIPKIγ activity (and, therefore, increased pro-
duction of PtdIns(4,5)P2) and increased PIPKIγ associa-
tion with talin91. In this manner, FAK signalling is con-
nected to the formation of focal contacts and the spatial
PtdIns Formation of α regulation of PtdIns(4,5)P2 generation. However, the
membrane Integrins β integrin and PIPKIγ binding sites within the talin
proximal contacts
PtdIns(4)P FERM domain overlap, which implies that PIPKIγ
Talin Vinculin binding might displace talin from integrin tails92. To this
PIPKIγ
end, FAK-enhanced Src-mediated phosphorylation of
PtdIns(4,5)P2 FAK
Paxillin PIPKIγ on Tyr644 creates a high affinity binding site for
PI3K p85 the talin FERM domain, which displaces β-integrin
PtdIns(3,4,5)P3 binding from talin FERM93. So, although FAK–Src
New Membrane activity could promote the production of PtdIns(4,5)P2
membranes component mDia GM1 and the formation of focal contacts by enhancing the
activity of PIPKIγ, subsequent phosphorylation of
Phospholipid
PIPKIγ by activated Src might break the talin–integrin
Rac

modification by Stable
phosphorylation Lipid lamellipodia linkage and promote the turnover of focal contacts.
rafts
Rac

FAK and intercellular contacts

Increased Another biological context in which FAK signalling
Microtubule
membrane has been associated with the formation or turnover of
fluidity
stabilization contacts is cadherin-based cell–cell junctions.
+
Cadherins are transmembrane proteins that mediate
+
Ca2+-dependent homophilic protein–protein attach-
+
ments between cells and that are also linked to the
actin cytoskeleton through interaction with α- or β-
catenins94. Downregulation of E-cadherin-based
ADHERENS JUNCTIONS is a hallmark of malignant and

Figure 5 | Focal adhesion kinase influences phospholipid and microtubule structures. invasive carcinomas, and the activity of the FAK–Src
The phospholipid kinases that have a role in the modification of phosphatidylinositol (PtdIns) complex promotes the disruption of colon carcinoma
cooperate with focal adhesion kinase (FAK) at several levels. Type I PtdIns phosphate kinase-γ cell homotypic adhesions95. Importantly, expression of
(PIPKIγ) associates with FAK and talin, and promotes the conversion of PtdIns-4-phosphate a FAK protein that is mutated at five tyrosine phospho-
(PtdIns(4)P) to PtdIns-4,5-bisphosphate (PtdIns(4,5)P2). PIPKIγ is phosphorylated by FAK, which
rylation sites (Tyr407, 576, 577, 861 and 925) blocked
leads to increased PIPKIγ activity and increased generation of PtdIns(4,5)P2. The binding of
PtdIns(4,5)P2 to talin and vinculin is associated with the formation of focal contacts. PtdIns(4,5)P2 the Src-mediated disruption of colon carcinoma E-cad-
can be converted to PtdIns-3,4,5-trisphosphate (PtdIns(3,4,5)P3) by PtdIns 3-kinase (PI3K). The herin-based contacts, thereby implying that phosphory-
regulatory p85 subunit of PI3K binds to FAK at Tyr397, which leads to PI3K activation by FAK112. lation-dependent signalling through FAK was
Directional motility requires the generation of phospholipid components such as PtdIns(4,5)P2 and required96. In an opposite manner, overexpression of a
PtdIns(3,4,5)P3. Integrin and FAK signalling also promote the translocation of specific kinase-defective mutant of FAK blocked the accumula-
components of lipid rafts to membranes. The stabilization of lipid rafts through integrin signalling
tion of peripheral E-cadherin in endothelial cells that
facilitates the coupling of Rac to target proteins. FAK-mediated translocation of the lipid
ganglioside GM1 to the membrane, which is mediated through the activation of the Rho GTPase
were subjected to a hyperosmolar challenge (a stimu-
effector Diaphanous (mDia), regulates microtubule polarity at the leading edge of motile cells. lus that promotes increased E-cadherin-based TIGHT-
97
Microtubule polarization and Rac activation contribute to the formation of membrane ruffles and JUNCTION barrier formation) . These results imply that
stable lamellipodia. FAK signalling has a role in both the formation and
turnover of E-cadherin-based contacts.
As opposed to E-cadherin function, N-cadherin
expression in carcinoma cells is generally associated
intermediates function as binding sites for signalling with a scattered morphology and a migratory or inva-
proteins that are involved in the formation of focal con- sive phenotype. Antisense and DOMINANT-NEGATIVE inhibi-
tacts (FIG. 5). Phosphatidylinositol-4,5-bisphosphate tion of FAK showed that FAK expression and activity
ADHERENS JUNCTION (PtdIns(4,5)P2) binds to and controls the assembly of were needed for the formation of N-cadherin-based
A cell–cell adhesion complex proteins such as α-actinin, vinculin and talin into focal cell–cell contacts in HeLa cells19. However, in contradic-
that contains classical cadherins contacts2. As the binding of the talin FERM domain to tion, the above study also found that cells with less FAK
and catenins that are attached to
β-integrin cytoplasmic tails is enhanced by expression and reduced N-cadherin-mediated cell–cell
cytoplasmic actin filaments.
PtdIns(4,5)P2 (REF. 88), and the talin rod domain binds contacts exhibited ‘increased’ motility when plated as
TIGHT JUNCTION vinculin and actin89, a link between integrins, focal individual cells on a collagen matrix. Whereas much
A circumferential ring at the contact formation and the actin cytoskeleton is estab- remains to be determined about the molecular role of
apex of epithelial cells that seals lished. The type I phosphatidylinositol phosphate FAK in either the dissolution or formation of cadherin-
adjacent cells to one another.
Tight junctions regulate solute
kinase-γ (PIPKIγ) is an enzyme that makes based contacts, it is intriguing that the findings so far are
and ion flux between adjacent PtdIns(4,5)P2 and it is targeted to focal contacts by an somewhat similar to the bi-functional role of FAK in
epithelial cells. association with the talin FERM domain90. PIPKIγ is focal contact dynamics.

64 | JANUARY 2005 | VOLUME 6 www.nature.com/reviews/molcellbio

© 2005 Nature Publishing Group


DOMINANT NEGATIVE
A defective protein that retains
interaction capabilities and so
competes with normal proteins,
thereby impairing protein
function.
TANGENTIAL FLUID SHEAR
STRESS
A planar force exerted by the
friction of a flowing substance
— for example, forces
experienced by endothelial cells
as blood flows through
capillaries.
CYTOTROPHOBLAST
The inner trophoblastic layer of
cells that give rise to the
syncytiotrophoblast facing the
maternal circulation and
constitute a layer through which
all substances must pass from
the mother to the fetus.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
FAK as a biosensor
Early studies with integrins found that they ‘sensed’
environmental cues and functioned to control anchorage-
dependent cell proliferation and survival98. Integrins are
also intimately involved in the conversion of physical
signals, such as contractile forces or external mechanical
perturbations, into chemical signalling events, and FAK
activation is an important component of this
‘mechanosensing’ by cells20. Early observations showed
that FAK could be activated by TANGENTIAL FLUID SHEAR
STRESS of endothelial cells and that this was associated
with the formation of a FAK–Src signalling complex,
FAK Tyr925 phosphorylation, and the downstream acti-
vation of ERK2 and JNK18. In a process known as
mechanotaxis, endothelial cells that encounter shear
stress will initiate focal contact remodelling and cell
migration in the direction of the flow. Under these con-
ditions, phosphorylation of FAK is enhanced at the
leading edge of motile cells99.
Another means of ‘mechanoperception’ is the ability
of cells to sense the ‘rigidity’ of the surrounding ECM, as
cells will preferentially migrate towards areas of higher
substrate rigidity in a process known as durotaxis.
Studies have shown that this response requires FAK
expression and that FAK–/– cells are insensitive to
changes in substrate flexibility100. Interestingly, whereas
expression of a Tyr397Phe mutant of FAK does not res-
cue the overall migration speed or directional motility
persistence defects of FAK–/– cells23,64,101, cells expressing
this mutant exhibited similar durotaxis responses to
wild-type cells100. Although it remains to be determined
whether intrinsic FAK catalytic activity or the role of
FAK as a scaffolding protein at focal contacts is the
determining factor for the durotaxis response, this
observation remains one of the few examples in which
Tyr397 FAK phosphorylation was not required for a sig-
nalling response.
Moving in three dimensions
As adherent cells in culture readily make focal contacts,
these points of cell adhesion undergo a maturation
process to form fibrillar adhesions and they will form
three-dimensional (3D) adhesions when cells are placed
in a 3D environment102. FAK–/– fibroblasts fail to prop-
erly remodel focal contacts into fibrillar adhesions103
and FAK–/– endothelial cells do not form tubule struc-
tures when grown in a 3D matrix environment13.
Interfering with FAK activity prevents tubule forma-
tion in human brain microvascular endothelial cells14,
and gain-of-function experiments with FAK–/– fibrob-
lasts show that the formation of fibrillar adhesions
requires phosphorylation of Tyr397, FAK catalytic
activity and FAK scaffolding functions103. Similar to the
maturation process of fibrillar and 3D adhesions, FAK-
dependent matrix organization is also observed during
dorsal forebrain development in mice, as glial cells that
lack FAK exhibit basement-membrane formation
defects104.
Although immunostaining of cells that were grown
in 3D did not reveal enhanced Tyr397 phosphorylation
of FAK compared with the levels found in cells forming
© 2005 Nature Publishing Group
REVIEWS
2D focal contacts105, functional experiments with
human lung fibroblasts106, breast carcinoma cells107 or
normal CYTOTROPHOBLASTS108 have supported the impor-
tance of FAK Tyr397 phosphorylation in promoting cell
survival, cell proliferation and cell invasion, respectively,
in 3D cell culture. As increased FAK phosphorylation of
Tyr397 is associated with the elevated rigidity of a 3D
collagen matrix107, it is likely that factors such as integrin
engagement and cell tension are needed to promote
FAK activity in 3D. Integrin signalling is also important
in the formation of tumour cell invadopodia — 3D cell
extensions that are enriched in MMPs and that promote
tumour cell invasion through matrix barriers109.
FAK–Src signalling has an important role in promoting
invadopodia formation110 and lung carcinoma cell inva-
sion22, in part through the induction of Rac activity17,49.
FAK–Src signalling leads to the elevated expression and
secretion of MMPs and is associated with a metastatic
tumour cell phenotype16. As FAK expression is corre-
lated with increased tumour cell malignancy12, the
importance of FAK catalytic activity in promoting cell
motility and invasion17,64 make it an attractive target for
potential therapeutic intervention.
Conclusions and perspectives
The number of different signalling connections that
have been characterized for FAK has expanded greatly
over the past 5 years. In this review, we have high-
lighted the diversity of FAK-signalling inputs and out-
puts and the molecular mechanisms that connect FAK
to both the assembly and disassembly of focal contacts.
It is in this unique signalling position that FAK can exert
control over cytoskeletal or cell adhesion dynamics and,
therefore, cell motility.
Continued efforts will be needed to understand the
regulatory factors that influence whether FAK–Src sig-
nalling is coupled to the assembly or disassembly of cell
adhesion sites in 2D and 3D, and how FAK targeting to
different cellular locations influences these processes. As
such, it is likely that the many interactions and phospho-
rylation events that are associated with FAK are tran-
sient events and possibly occur in a defined sequence as
adhesions transit from assembly to disassembly during
cell migration. Additionally, as structural studies have
provided important information about the function of
the FAT domain of FAK, similar studies are needed to
understand the role of the FAK FERM domain. The
finding that exogenous overexpression of the FAK
FERM domain can inhibit cell motility24 might be asso-
ciated with FERM-mediated inhibition of FAK catalytic
activity44, so it is also possible that the FAK FERM
domain functions to target FAK to distinct cellular sites
that are involved in growth factor signalling or GPCR
signalling events23,24. To this end, sumoylation can
occur within the FAK FERM domain, which promotes
FAK activation27, a fraction of FAK can translocate to
the nucleus28,111, and FAK signalling can influence the
expression of transcription factors30. How — if at all —
these events are related to the ability of FAK to promote
cell polarization and motility is the subject of active
investigation.
VOLUME 6 | JANUARY 2005 | 6 5
REVIEWS

It is also notable that the FERM domain of FAK can
localize to cell–cell junctions111, whereas the FAT
domain is associated with focal contacts in epithelial
cells. It is possible that the FAK-mediated regulation of
cadherin-based cell contacts might be distinguished
through the differential FERM- or FAT-mediated target-
ing of FAK to distinct intracellular sites. As many of the
phenotypes that are associated with FAK have been elu-
cidated using overexpression studies, the development
of pharmacological inhibitors to FAK and analyses of
catalytically-defective FAK mutants in FAK–/– cells will
probably yield valuable insights into the role of FAK as
an essential scaffolding protein or as an active contribu-
tor to the formation of a FAK–Src signalling complex.
Note added in proof
V. Brunton’s personal communication has now been
accepted for publication: Brunton, V. G. et al.
Identification of Src-specific phosphorylation site on
FAK: dissection of the role of Src SH2 and catalytic
functions and their consequences for tumour cell
behaviour. Cancer Res. (in the press).

1. Brakebusch, C. & Fassler, R. The integrin–actin
connection, an eternal love affair. EMBO J. 22,
2324–2333 (2003).
2. DeMali, K. A., Wennerberg, K. & Burridge, K. Integrin
signaling to the actin cytoskeleton. Curr. Opin. Cell Biol.
15, 572–582 (2003).
3. Ridley, A. J. et al. Cell migration: integrating signals from
front to back. Science 302, 1704–1709 (2003).
4. Parsons, J. T. Focal adhesion kinase: the first ten years.
J. Cell Sci. 116, 1409–1416 (2003).
Provides a good overview of the early studies on
FAK.
5. Ilic, D. et al. Reduced cell motility and enhanced focal
adhesion contact formation in cells from FAK-deficient
mice. Nature 377, 539–544 (1995).
Shows that null mutation of FAK results in defects in
embryonic morphogenesis, and that FAK-null cells
show enhanced focal-contact formation and cell
motility defects in culture.
6. Webb, D. J. et al. FAK–Src signalling through paxillin, ERK
and MLCK regulates adhesion disassembly. Nature Cell
Biol. 6, 154–161 (2004).
7. Palazzo, A. F., Eng, C. H., Schlaepfer, D. D., Marcantonio,
E. E. & Gundersen, G. G. Localized stabilization of
microtubules by integrin- and FAK-facilitated Rho
signaling. Science 303, 836–839 (2004).
Provides evidence that FAK promotes cell
polarization through the stabilization of
microtubules at leading edges of motile cells.
8. Ren, X. et al. Focal adhesion kinase suppresses Rho
activity to promote focal adhesion turnover. J. Cell Sci.
113, 3673–3678 (2000).
9. Agochiya, M. et al. Increased dosage and amplification of
the focal adhesion kinase gene in human cancer cells.
Oncogene 18, 5646–5653 (1999).
10. Bhattacharjee, A. et al. Classification of human lung
carcinomas by mRNA expression profiling reveals distinct
adenocarcinoma subclasses. Proc. Natl Acad. Sci. USA
98, 13790–13795 (2001).
11. Yeoh, E. J. et al. Classification, subtype discovery, and
prediction of outcome in pediatric acute lymphoblastic
leukemia by gene expression profiling. Cancer Cell 1,
133–143 (2002).
12. Cance, W. G. et al. Immunohistochemical analyses of
focal adhesion kinase expression in benign and malignant
human breast and colon tissues: correlation with
preinvasive and invasive phenotypes. Clin. Cancer Res. 6,
2417–2423 (2000).
13. Ilic, D. et al. Focal adhesion kinase is required for blood
vessel morphogenesis. Circ. Res. 92, 300–307 (2003).
14. Haskell, H. et al. Focal adhesion kinase is expressed in
the angiogenic blood vessels of malignant astrocytic
tumors in vivo and promotes capillary tube formation of
brain microvascular endothelial cells. Clin. Cancer Res. 9,
2157–2165 (2003).
15. Hauck, C. R., Hsia, D. A., Ilic, D. & Schlaepfer, D. D. v-Src
SH3-enhanced interaction with focal adhesion kinase at
β1 integrin-containing invadopodia promotes cell invasion.
J. Biol. Chem. 277, 12487–12490 (2002).
16. Hauck, C. R., Hsia, D. A., Puente, X. S., Cheresh, D. A. &
Schlaepfer, D. D. FRNK blocks v-Src-stimulated invasion
and experimental metastases without effects on cell
motility or growth. EMBO J. 21, 6289–6302 (2002).
17. Hsia, D. A. et al. Differential regulation of cell motility and
invasion by FAK. J. Cell Biol. 160, 753–767 (2003).
This reference, together with reference 69, shows
that constitutively active Src can bypass the need
for FAK in promoting the turnover of focal contacts.
18. Schlaepfer, D. D., Mitra, S. K. & Ilic, D. Control of motile
and invasive cell phenotypes by focal adhesion kinase.
Biochim. Biophys. Acta 1692, 77–102 (2004).
Provides a solid review on the role of FAK during
embryonic development.
19. Yano, H. et al. Roles played by a subset of integrin
signaling molecules in cadherin-based cell–cell adhesion.
J. Cell Biol. 166, 283–295 (2004).
20. Katsumi, A., Orr, A. W., Tzima, E. & Schwartz, M. A.
Integrins in mechanotransduction. J. Biol. Chem. 279,
12001–12004 (2004).
21. Carragher, N. O., Westhoff, M. A., Fincham, V. J., Schaller,
M. D. & Frame, M. C. A novel role for FAK as a protease-
targeting adaptor protein. Regulation by p42 ERK and
Src. Curr. Biol. 13, 1442–1450 (2003).
22. Hauck, C. R. et al. Inhibition of focal adhesion kinase
expression or activity disrupts epidermal growth factor-
stimulated signaling promoting the migration of invasive
human carcinoma cells. Cancer Res. 61, 7079–7090
(2001).
23. Sieg, D. J. et al. FAK integrates growth-factor and integrin
signals to promote cell migration. Nature Cell Biol. 2,
249–256 (2000).
24. Streblow, D. N. et al. Human cytomegalovirus chemokine
receptor US28-induced smooth muscle cell migration is
mediated by focal adhesion kinase and Src. J. Biol.
Chem. 278, 50456–50465 (2003).
Together with reference 23, this paper shows that
the FAK FERM domain has important roles in
promoting growth-factor-stimulated and G-protein-
stimulated cell motility.
25. Chen, R. et al. Regulation of the PH-domain-containing
tyrosine kinase Etk by focal adhesion kinase through
the FERM domain. Nature Cell Biol. 3, 439–444 (2001).
26. Poullet, P. et al. Ezrin interacts with focal adhesion
kinase and induces its activation independently of
cell–matrix adhesion. J. Biol. Chem. 276, 37686–37691
(2001).
27. Kadare, G. et al. PIAS1-mediated sumoylation of focal
adhesion kinase activates its autophosphorylation. J. Biol.
Chem. 278, 47434–47440 (2003).
Shows that sumoylation of FAK within the FERM
domain is associated with catalytic activation and
preferential nuclear localization.
28. Jones, G. & Stewart, G. Nuclear import of N-terminal FAK
by activation of the FcεRI receptor in RBL-2H3 cells.
Biochem. Biophys. Res. Comm. 314, 39–45 (2004).
29. McKean, D. M. et al. FAK induces expression of Prx1 to
promote tenascin-C-dependent fibroblast migration.
J. Cell Biol. 161, 393–402 (2003).
30. Zhao, J. et al. Identification of transcription factor KLF8 as
a downstream target of focal adhesion kinase in its
regulation of cyclin D1 and cell cycle progression.
Mol. Cell 11, 1503–1515 (2003).
31. Hanks, S. K., Ryzhova, L., Shin, N. Y. & Brabek, J. Focal
adhesion kinase signaling activities and their implications
in the control of cell survival and motility. Front. Biosci. 8,
982–996 (2003).
32. Chodniewicz, D. & Klemke, R. L. Regulation of integrin-
mediated cellular responses through assembly of a
CAS/Crk scaffold. Biochim. Biophys. Acta. 1692, 63–76
(2004).
33. Schaller, M. D. Biochemical signals and biological
responses elicited by the focal adhesion kinase. Biochim.
Biophys. Acta. 1540, 1–21 (2001).
34. Zhai, J. et al. Direct interaction of focal adhesion kinase
with p190RhoGEF. J. Biol. Chem. 278, 24865–24873
(2003).
Together with reference 79, shows that FAK can
directly activate Rho through binding and
phosphorylation of a GEF, and that this activation
regulates axonal branching.
35. Toutant, M. et al. Alternative splicing controls the
mechanisms of FAK autophosphorylation. Mol. Cell. Biol.
22, 7731–7743 (2002).
36. Liu, E., Cote, J. F. & Vuori, K. Negative regulation of FAK
signaling by SOCS proteins. EMBO J. 22, 5036–5046
(2003).
This paper established a link between FAK
activation, phosphorylation of Tyr397 and
subsequent degradation of FAK.
37. Avraham, H., Park, S. Y., Schinkmann, K. & Avraham, S.
RAFTK/Pyk2-mediated cellular signalling. Cell. Signal 12,
123–133 (2000).
38. Klingbeil, C. K. et al. Targeting Pyk2 to β1-integrin-
containing focal contacts rescues fibronectin-stimulated
signaling and haptotactic motility defects of focal
adhesion kinase-null cells. J. Cell Biol. 152, 97–110 (2001).
39. Lim, Y. et al. Phosphorylation of focal adhesion kinase at
tyrosine 861 is crucial for Ras transformation of
fibroblasts. J. Biol. Chem. 279, 29060–29065 (2004).
40. Gabarra-Niecko, V., Keely, P. J. & Schaller, M. D.
Characterization of an activated mutant of focal adhesion
kinase: ‘SuperFAK’. Biochem. J. 365, 591–603 (2002).
41. Nowakowski, J. et al. Structures of the cancer-related
Aurora-A, FAK, and EphA2 protein kinases from
nanovolume crystallography. Structure (Camb.) 10,
1659–1667 (2002).
42. Abbi, S. et al. Regulation of focal adhesion kinase by a
novel protein inhibitor FIP200. Mol. Biol. Cell 13,
3178–3191 (2002).
43. Medley, Q. G. et al. Signaling between focal adhesion
kinase and Trio. J. Biol. Chem. 278, 13265–13270
(2003).
44. Cooper, L. A., Shen, T. L. & Guan, J. L. Regulation of focal
adhesion kinase by its amino-terminal domain through an
autoinhibitory interaction. Mol. Cell. Biol. 23, 8030–8041
(2003).
45. Zeng, L. et al. PTPα regulates integrin-stimulated FAK
autophosphorylation and cytoskeletal rearrangement in
cell spreading and migration. J. Cell Biol. 160, 137–146
(2003).
46. Chiarugi, P. et al. Reactive oxygen species as essential
mediators of cell adhesion: the oxidative inhibition of a
FAK tyrosine phosphatase is required for cell adhesion.
J. Cell Biol. 161, 933–944 (2003).
47. Arias-Salgado, E. G. et al. Src kinase activation by direct
interaction with the integrin β cytoplasmic domain.
Proc. Natl Acad. Sci. USA 100, 13298–13302 (2003).
Shows that selected β-integrin subunits can bind
and activate Src in the absence of a contribution
from FAK.
48. Turner, C. E. Paxillin and focal adhesion signalling. Nature
Cell Biol. 2, 231–236 (2000).
49. Cho, S. Y. & Klemke, R. L. Purification of pseudopodia
from polarized cells reveals redistribution and activation of
Rac through assembly of a CAS/Crk scaffold. J. Cell Biol.
156, 725–736 (2002).
50. Brabek, J. et al. CAS promotes invasiveness of
Src-transformed cells. Oncogene 23, 7406–7415
(2004).
51. Schaller, M. D. Paxillin: a focal adhesion-associated
adaptor protein. Oncogene 20, 6459–6472 (2001).
52. Subauste, M. C. et al. Vinculin modulation of paxillin–FAK
interactions regulates ERK to control survival and motility.
J. Cell Biol. 165, 371–381 (2004).

66 | JANUARY 2005 | VOLUME 6 www.nature.com/reviews/molcellbio

© 2005 Nature Publishing Group


53. Hayashi, I., Vuori, K. & Liddington, R. C. The focal
adhesion targeting (FAT) region of focal adhesion kinase is
a four-helix bundle that binds paxillin. Nature Struct. Biol.
9, 101–106 (2002).
54. Liu, G., Guibao, C. D. & Zheng, J. Structural insight into
the mechanisms of targeting and signaling of focal
adhesion kinase. Mol. Cell. Biol. 22, 2751–2760 (2002).
55. Gao, G. et al. NMR solution structure of the focal
adhesion targeting domain of focal adhesion kinase in
complex with a paxillin LD peptide: evidence for a two-site
binding model. J. Biol. Chem. 279, 8441–8451 (2004).
56. Katz, B. Z. et al. Targeting membrane-localized focal
adhesion kinase to focal adhesions: roles of tyrosine
phosphorylation and SRC family kinases. J. Biol. Chem.
278, 29115–29120 (2003).
57. Prutzman, K. C. et al. The focal adhesion targeting
domain of focal adhesion kinase contains a hinge region
that modulates tyrosine 926 phosphorylation. Structure
(Camb.) 12, 881–891 (2004).
References 53, 54, 55 and 57 provide structural
analyses of the FAK FAT domain and the paxillin LD
peptide binding, and show that Tyr925
phosphorylation might require conformational
alterations in the FAT domain.
58. Ma, A., Richardson, A., Schaefer, E. M. & Parsons, J. T.
Serine phosphorylation of focal adhesion kinase in
interphase and mitosis: a possible role in modulating
binding to p130Cas. Mol. Biol. Cell 12, 1–12 (2001).
59. Hunger-Glaser, I., Fan, R. S., Perez-Salazar, E. &
Rozengurt, E. PDGF and FGF induce focal adhesion
kinase (FAK) phosphorylation at Ser-910: dissociation
from Tyr-397 phosphorylation and requirement for ERK
activation. J. Cell Physiol. 200, 213–222 (2004).
60. Liu, Z. X., Yu, C. F., Nickel, C., Thomas, S. & Cantley, L. G.
Hepatocyte growth factor induces ERK-dependent
paxillin phosphorylation and regulates paxillin–focal
adhesion kinase association. J. Biol. Chem. 277,
10452–10458 (2002).
61. Ishibe, S., Joly, D., Zhu, X. & Cantley, L. G.
Phosphorylation-dependent paxillin–ERK association
mediates hepatocyte growth factor-stimulated epithelial
morphogenesis. Mol. Cell 12, 1275–1285 (2003).
62. Kirchner, J., Kam, Z., Tzur, G., Bershadsky, A. D. &
Geiger, B. Live-cell monitoring of tyrosine phosphorylation
in focal adhesions following microtubule disruption.
J. Cell Sci. 116, 975–986 (2003).
63. Zaidel-Bar, R., Ballestrem, C., Kam, Z. & Geiger, B. Early
molecular events in the assembly of matrix adhesions at
the leading edge of migrating cells. J. Cell Sci. 116,
4605–4613 (2003).
64. Sieg, D. J., Hauck, C. R. & Schlaepfer, D. D. Required role
of focal adhesion kinase (FAK) for integrin-stimulated cell
migration. J. Cell Sci. 112, 2677–2691 (1999).
65. Rajfur, Z., Roy, P., Otey, C., Romer, L. & Jacobson, K.
Dissecting the link between stress fibres and focal
adhesions by CALI with EGFP fusion proteins. Nature Cell
Biol. 4, 286–293 (2002).
66. Izaguirre, G. et al. The cytoskeletal/non-muscle isoform of
α-actinin is phosphorylated on its actin-binding domain by
the focal adhesion kinase. J. Biol. Chem. 276,
28676–28685 (2001).
67. Yu, D. H., Qu, C. K., Henegariu, O., Lu, X. & Feng, G. S.
Protein-tyrosine phosphatase Shp-2 regulates cell
spreading, migration, and focal adhesion. J. Biol. Chem.
273, 21125–21131 (1998).
68. Von Wichert, G., Haimovich, B., Feng, G. S. &
Sheetz, M. P. Force-dependent integrin–cytoskeleton
linkage formation requires downregulation of focal
complex dynamics by Shp2. EMBO J. 22, 5023–5035
(2003).
Together with reference 67, this study shows that
null mutation of SHP2 results in FAK
hyperactivation, elevated α-actinin phosphorylation,
and the failure to promote the maturation of
integrin–cytoskeletal linkages.
69. Moissoglu, K. & Gelman, I. H. v-Src rescues actin-based
cytoskeletal architecture and cell motility and induces
enhanced anchorage independence during oncogenic
transformation of focal adhesion kinase-null fibroblasts.
J. Biol. Chem. 278, 47946–47959 (2003).
70. Visse, R. & Nagase, H. Matrix metalloproteinases and
tissue inhibitors of metalloproteinases: structure,
function, and biochemistry. Circ. Res. 92, 827–839
(2003).
71. Bhatt, A., Kaverina, I., Otey, C. & Huttenlocher, A.
Regulation of focal complex composition and
disassembly by the calcium-dependent protease calpain.
J. Cell Sci. 115, 3415–3425 (2002).
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
72. Dourdin, N. et al. Reduced cell migration and disruption of
the actin cytoskeleton in calpain-deficient embryonic
fibroblasts. J. Biol. Chem. 276, 48382–48388 (2001).
73. Cuevas, B. D. et al. MEKK1 regulates calpain-dependent
proteolysis of focal adhesion proteins for rear-end
detachment of migrating fibroblasts. EMBO J. 22,
3346–3355 (2003).
74. Westhoff, M. A., Serrels, B., Fincham, V. J., Frame, M. C.
& Carragher, N. O. Src-mediated phosphorylation of focal
adhesion kinase couples actin and adhesion dynamics to
survival signaling. Mol. Cell. Biol. 24, 8113–8133 (2004).
75. Giannone, G. et al. Calcium rises locally trigger focal
adhesion disassembly and enhance residency of focal
adhesion kinase at focal adhesions. J. Biol. Chem. 279,
28715–28723 (2004).
76. Hecker, T. P., Ding, Q., Rege, T. A., Hanks, S. K. &
Gladson, C. L. Overexpression of FAK promotes Ras
activity through the formation of a FAK/p120RasGAP
complex in malignant astrocytoma cells. Oncogene 23,
3962–3971 (2004).
77. Chen, B. H., Tzen, J. T., Bresnick, A. R. & Chen, H. C.
Roles of Rho-associated kinase and myosin light chain
kinase in morphological and migratory defects of focal
adhesion kinase-null cells. J. Biol. Chem. 277,
33857–33863 (2002).
78. Arthur, W. T., Petch, L. A. & Burridge, K. Integrin
engagement suppresses RhoA activity via a c-Src-
dependent mechanism. Curr. Biol. 10, 719–722 (2000).
79. Rico, B. et al. Control of axonal branching and synapse
formation by focal adhesion kinase. Nature Neurosci. 7,
1059–1069 (2004).
80. Wu, X., Suetsugu, S., Cooper, L. A., Takenawa, T. &
Guan, J. L. Focal adhesion kinase regulation of N-WASP
subcellular localization and function. J. Biol. Chem. 279,
9565–9576 (2004).
81. Palazzo, A. F., Cook, T. A., Alberts, A. S. & Gundersen, G. G.
mDia mediates Rho-regulated formation and orientation
of stable microtubules. Nature Cell Biol. 3, 723–729
(2001).
82. Gundersen, G. G., Gomes, E. R. & Wen, Y. Cortical
control of microtubule stability and polarization. Curr.
Opin. Cell Biol. 16, 106–112 (2004).
83. del Pozo, M. A. et al. Integrins regulate Rac targeting by
internalization of membrane domains. Science 303,
839–842 (2004).
84. del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D. &
Schwartz, M. A. Adhesion to the extracellular matrix
regulates the coupling of the small GTPase Rac to its
effector PAK. EMBO J. 19, 2008–2014 (2000).
85. Slack-Davis, J. K. et al. PAK1 phosphorylation of MEK1
regulates fibronectin-stimulated MAPK activation. J. Cell
Biol. 162, 281–291 (2003).
86. Xie, Z. et al. Serine 732 phosphorylation of FAK by Cdk5
is important for microtubule organization, nuclear
movement, and neuronal migration. Cell 114, 469–482
(2003).
87. Ivankovic-Dikic, I., Gronroos, E., Blaukat, A., Barth, B.-U.
& Dikic, I. Pyk2 and FAK regulate neurite outgrowth
induced by growth factors and integrins. Nature Cell Biol.
2, 574–581 (2000).
88. Calderwood, D. A. Integrin activation. J. Cell Sci. 117,
657–666 (2004).
89. Papagrigoriou, E. et al. Activation of a vinculin-binding site
in the talin rod involves rearrangement of a five-helix
bundle. EMBO J. 23, 2942–2951 (2004).
90. Di Paolo, G. et al. Recruitment and regulation of
phosphatidylinositol phosphate kinase type 1γ by the
FERM domain of talin. Nature 420, 85–89 (2002).
91. Ling, K., Doughman, R. L., Firestone, A. J., Bunce, M. W.
& Anderson, R. A. Type Iγ phosphatidylinositol phosphate
kinase targets and regulates focal adhesions. Nature 420,
89–93 (2002).
92. Barsukov, I. L. et al. Phosphatidylinositol phosphate
kinase type 1γ and β1-integrin cytoplasmic domain bind to
the same region in the talin FERM domain. J. Biol. Chem.
278, 31202–31209 (2003).
93. Ling, K. et al. Tyrosine phosphorylation of type Iγ
phosphatidylinositol phosphate kinase by Src regulates an
integrin–talin switch. J. Cell Biol. 163, 1339–1349 (2003).
Together with references 91 and 92, this reference
shows that FAK–Src phosphorylation events
function to control the composition of membrane
lipids and the dynamics of focal contacts.
94. Wheelock, M. J. & Johnson, K. R. Cadherins as
modulators of cellular phenotype. Ann. Rev. Cell Dev. Biol.
19, 207–235 (2003).
95. Irby, R. B. & Yeatman, T. J. Increased Src activity disrupts
cadherin/catenin-mediated homotypic adhesion in human
© 2005 Nature Publishing Group
REVIEWS
colon cancer and transformed rodent cells. Cancer Res.
62, 2669–2674 (2002).
96. Avizienyte, E. et al. Src-induced de-regulation of
E-cadherin in colon cancer cells requires integrin
signalling. Nature Cell Biol. 4, 632–638 (2002).
97. Quadri, S. K., Bhattacharjee, M., Parthasarathi, K.,
Tanita, T. & Bhattacharya, J. Endothelial barrier
strengthening by activation of focal adhesion kinase.
J. Biol. Chem. 278, 13342–13349 (2003).
98. Miranti, C. K. & Brugge, J. S. Sensing the environment:
a historical perspective on integrin signal transduction.
Nature Cell Biol. 4, E83–E90 (2002).
99. Li, S. et al. The role of the dynamics of focal adhesion
kinase in the mechanotaxis of endothelial cells. Proc. Natl
Acad. Sci. USA 99, 3546–3551 (2002).
100. Wang, H. B., Dembo, M., Hanks, S. K. & Wang, Y. Focal
adhesion kinase is involved in mechanosensing during
fibroblast migration. Proc. Natl Acad. Sci. USA 98,
11295–11300 (2001).
Shows that FAK functions as an important
environmental biosensor in promoting directional
motility signals in response to changes in substrate
flexibility.
101. Owen, J. D., Ruest, P. J., Fry, D. W. & Hanks, S. K.
Induced focal adhesion kinase (FAK) expression in
FAK-null cells enhances cell spreading and migration
requiring both auto- and activation loop phosphorylation
sites and inhibits adhesion-dependent tyrosine
phosphorylation of Pyk2. Mol. Cell. Biol. 19, 4806–4818
(1999).
102. Cukierman, E., Pankov, R. & Yamada, K. M. Cell
interactions with three-dimensional matrices. Curr. Opin.
Cell Biol. 14, 633–639 (2002).
103. Ilic, D. et al. FAK promotes organization of fibronectin
matrix and fibrillar adhesions. J. Cell Sci. 117, 177–187
(2004).
104. Beggs, H. E. et al. FAK deficiency in cells contributing to
the basal lamina results in cortical abnormalities
resembling congenital muscular dystrophies. Neuron 40,
501–514 (2003).
References 103 and 104 show that FAK has crucial
roles in promoting 3D-matrix assembly and/or
remodelling during development and in cell culture
model systems.
105. Cukierman, E., Pankov, R., Stevens, D. R. & Yamada, K. M.
Taking cell–matrix adhesions to the third dimension.
Science 294, 1708–1712 (2001).
106. Xia, H., Nho, R. S., Kahm, J., Kleidon, J. & Henke, C. A.
Focal adhesion kinase is upstream of
phosphatidylinositol 3-kinase/Akt in regulating
fibroblast survival in response to contraction of
type I collagen matrices via a β1 integrin viability
signaling pathway. J. Biol. Chem. 279, 33024–33034
(2004).
107. Wozniak, M. A., Desai, R., Solski, P. A., Der, C. J. & Keely,
P. J. ROCK-generated contractility regulates breast
epithelial cell differentiation in response to the physical
properties of a three-dimensional collagen matrix.
J. Cell Biol. 163, 583–595 (2003).
108. Ilic, D. et al. Plasma membrane-associated
pY397FAK is a marker of cytotrophoblast invasion
in vivo and in vitro. Am. J. Pathol. 159, 93–108
(2001).
109. Bowden, E. T., Coopman, P. J. & Mueller, S. C.
Invadopodia: unique methods for measurement of
extracellular matrix degradation in vitro. Methods Cell Biol.
63, 613–627 (2001).
110. Hauck, C. R., Hunter, T. & Schlaepfer, D. D. The v-Src
SH3 domain facilitates a cell adhesion-independent
association with focal adhesion kinase. J. Biol. Chem.
276, 17653–17662 (2001).
111. Stewart, A., Ham, C. & Zachary, I. The focal adhesion
kinase amino-terminal domain localises to nuclei and
intercellular junctions in HEK 293 and MDCK cells
independently of tyrosine 397 and the carboxy-terminal
domain. Biochem. Biophys. Res. Comm. 299, 62–73
(2002).
112. Chen, H. C., Appeddu, P. A., Isoda, H. & Guan, J. L.
Phosphorylation of tyrosine 397 in focal adhesion
kinase is required for binding phosphatidylinositol 3-
kinase. J. Biol. Chem. 271, 26329–26334
(1996).
113. Calderwood, D. A. & Ginsberg, M. H. Talin forges the links
between integrins and actin. Nature Cell Biol. 5, 694–697
(2003).
114. Zheng, C. et al. Differential regulation of Pyk2 and focal
adhesion kinase (FAK). J. Biol. Chem. 273, 2384–2389
(1998).
VOLUME 6 | JANUARY 2005 | 6 7
REVIEWS
115. Wang, Q. et al. Regulation of the formation of
osteoclastic actin rings by proline-rich tyrosine kinase 2
interacting with gelsolin. J. Cell Biol. 160, 565–575
(2003).
116. Sieg, D. J. et al. Pyk2 and Src-family protein-tyrosine
kinases compensate for the loss of FAK in fibronectin-
stimulated signaling events but Pyk2 does not fully
function to enhance FAK– cell migration. EMBO J. 17,
5933–5947 (1998).
117. Lakkakorpi, P. T., Bett, A. J., Lipfert, L., Rodan, G. A. &
Duong, L. T. Pyk2 autophosphorylation, but not kinase
activity, is necessary for adhesion-induced association
with c-Src, osteoclast spreading, and bone resorption.
J. Biol. Chem. 278, 11502–11512 (2003).
118. Lev, S. et al. Identification of a novel family of targets
of Pyk2 related to Drosophila retinal degeneration B
(rdgB) protein. Mol. Cell. Biol. 19, 2278–2288
(1999).
119. Benbernou, N., Muegge, K. & Durum, S. K. Interleukin
(IL)-7 induces rapid activation of Pyk2, which is bound to
Janus kinase 1 and IL-7Rα. J. Biol. Chem. 275,
7060–7065 (2000).
120. Okigaki, M. et al. Pyk2 regulates multiple signaling
events crucial for macrophage morphology and
migration. Proc. Natl Acad. Sci. USA 100, 10740–10745
(2003).
68 | JANUARY 2005 | VOLUME 6
Shows that null mutation of the FAK-related
kinase PYK2 results in integrin and chemokine-
stimulated motility defects of macrophages
that are not functionally compensated by FAK
expression.
121. Watson, J. M. et al. Inhibition of the calcium-dependent
tyrosine kinase (CADTK) blocks monocyte spreading and
motility. J. Biol. Chem. 276, 3536–3542 (2001).
122. Guinamard, R., Okigaki, M., Schlessinger, J. & Ravetch, J. V.
Absence of marginal zone B cells in Pyk2-deficient mice
defines their role in the humoral response. Nature
Immunol. 1, 31–36 (2000).
123. Klinghoffer, R. A., Sachsenmaier, C., Cooper, J. A. &
Soriano, P. Src family kinases are required for integrin but
not PDGFR signal transduction. EMBO J. 18, 2459–2471
(1999).
124. Honda, H. et al. Cardiovascular anomaly, impaired actin
bundling and resistance to Src- induced transformation in
mice lacking p130Cas. Nature Genet. 19, 361–365 (1998).
125. Hagel, M. et al. The adaptor protein paxillin is essential for
normal development in the mouse and is a critical
transducer of fibronectin signaling. Mol. Cell. Biol. 22,
901–915 (2002).
126. Xu, W., Baribault, H. & Adamson, E. D. Vinculin knockout
results in heart and brain defects during embryonic
development. Development 125, 327–337 (1998).
© 2005 Nature Publishing Group
Acknowledgements
S. Mitra is supported by a fellowship from the California
Tobacco-Related Disease Research Program and D. Schlaepfer
is supported by grants from the National Cancer Institute. This is
manuscript 16827-IMM from The Scripps Research Institute.
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
PTK2
Swiss-Prot: http://www.expasy.ch
calpain | CDK5 | Diaphanous | E-cadherin | ezrin | FAK | FIP200 |
Fyn | N-cadherin | N-WASP | p130Cas | paxillin | PTPα | RhoA |
SHP2 | Src | talin| TRIO | Yes
FURTHER INFORMATION
The Schlaepfer laboratory:
http://www.scripps.edu/imm/schlaepfer/index1.htm
Access to this interactive links box is free online.
www.nature.com/reviews/molcellbio