Download FOCAL ADHESION KINASE: IN COMMAND AND CONTROL OF

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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 celladhesion molecules.
EXTRACELLULAR MATRIX
(ECM). The complex, multimolecular 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: [email protected]
doi:10.1038/nrm1549
56
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 generation of membrane protrusions and traction forces2.
External stimuli that control cell migration are transduced 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-promoting stimuli correctly, there must be essential intracellular 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 regulator 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 substrate of the viral Src oncogene and, in normal cells, as a
| JANUARY 2005 | VOLUME 6
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 cytoskeletal 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 expression 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 designation of human FAK is protein-tyrosine kinase-2
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Box 1 | Molecular architecture of focal contacts
α -A
c tin
in
c tin
in
in
in
FAK
p130 p130
Src Cas Cas Src
in
ul
nc
Plasma
membrane
illin
Pax in
a
Tl
α -A
Vi
FAK
c tin
in
ul
nc
α -A
Vi
in
ul
nc
Vi
Zyxin
α -A
c tin
in
My
My
os
os
in
My
os
in
Actin stress fibres
Focal contact
proteins
P ax
ill
Tali in
n
Integrins
α β
β α
Extracellular matrix
The extracellular matrix, integrins (α- and β-transmembrane heterodimeric proteins)
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
interior and can also relay intracellular signals to generate an activated integrin state at
the cell surface113. The integrin-binding proteins paxillin and talin recruit focal adhesion
kinase (FAK) and vinculin to focal contacts (see figure). α-Actinin is a cytoskeletal
protein that is phosphorylated by FAK, binds to vinculin and crosslinks actomyosin
stress fibres and tethers them to focal contacts. Zyxin is an α-actinin- and stress-fibrebinding protein that is present in mature contacts. Although the aforementioned proteins
are found in most focal contacts, the membrane-associated protein tyrosine kinase Src
and the ADAPTOR PROTEIN p130Cas associate with focal contacts following integrin
clustering. Integrin-mediated FAK activation is mediated in part by matrix binding or by
force-dependent changes in cytoskeletal linkages. Several other proteins such as
extracellular signal-regulated kinase 2 (ERK2) and calpain are known to be transiently
present at focal contacts (not shown). The composition of a focal contact is therefore
constantly varying depending on external cues and cellular responses.
RHO-FAMILY GTPases
A subfamily of small (~21 kDa)
GTP-binding proteins that are
related to Ras and that regulate
the cytoskeleton. The
nucleotide-bound state is
regulated by GTPase-activating
proteins, which catalyse
hydrolysis of the bound GTP,
and guanine nucleotideexchange factors, which catalyse
GDP–GTP exchange.
(PTK2) and it is located at human chromosome
8q24term, a locus that is subject to amplification in
human cancer cells9. Furthermore, elevated levels of
PTK2 mRNA have been found in studies of human carcinoma tumours and in acute lymphoblastic leukaemias,
as detected by large-scale gene expression profiling10,11.
FAK protein expression is elevated in many highly
malignant human cancers12, and studies have shown
that FAK signalling can promote changes in cell shape13,14
and the formation of podosomes or invadopodia15,
which leads to an invasive cell phenotype16,17. Whereas
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many studies have shown that FAK inhibition blocks the
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
contacts in HeLa CELLS19.
It is possible that this latter observation might be a
cell-type-specific signalling event. However, we speculate that the ability of FAK to promote both the maturation and turnover of focal contacts is related to its role as
both a signalling kinase and as an adaptor/scaffold protein, which places FAK in a position to modulate various
intracellular signalling pathways (FIG. 1). Namely, it is the
association of FAK with both activators and/or
inhibitors of various small GTPase proteins (Rho, Rac,
Cdc42 and Ras) that enables changes in FAK activity to
be connected to alterations in the polymerization or stabilization of actin and microtubule filaments.
Additionally, because migrating cells experience changes
in forces through integrin contacts that link the ECM
with the cytoskeleton, FAK is important in the ‘sensing’
of mechanical forces that are either generated internally
or exerted on cells20. FAK activation is therefore involved
in modulating ‘corrective’ cell responses to environmental stimuli. FAK does this through signal-mediated
effects on actin polymerization, the assembly or disassembly of focal contacts, and the regulation of protease
activation or secretion16,21,22.
The FERM and FAT domains of FAK
FAK is a ubiquitously expressed 125-kDa protein tyrosine kinase that is composed of an N-terminal FERM
(protein 4.1, ezrin, radixin and moesin homology)
domain, a central kinase domain, proline-rich regions
and a C-terminal focal-adhesion targeting (FAT)
domain (FIG. 2). The FERM domain of FAK facilitates a
signalling linkage from receptor tyrosine kinases such as
the epidermal growth factor receptor (EGFR) and the
platelet-derived growth factor receptor (PDGFR)23. In
analysing cell-motility-promoting signals that are initiated by G-PROTEIN-COUPLED RECEPTORS (GPCRs), overexpression of the FAK FERM domain blocked FAK activation
and resulted in the inhibition of G-protein-stimulated
cell migration24. It is the FAK FERM domain that can
bind to and promote the integrin- and FAK-mediated
activation of other non-receptor tyrosine kinases such as
ETK25. Additionally, actin- and membrane-associated
adaptor proteins such as ezrin can bind to the FAK
FERM domain and facilitate increased FAK activation in
an integrin-independent manner26.
How the FAK FERM domain associates with various
targets is an active area of research. FAK can become
post-translationally modified by the covalent addition
of a small ubiquitin-related modifier (SUMO) at the
ε-amino position of Lys152 (REF. 27). In most instances,
sumoylation is associated with the nuclear import of
proteins and, correspondingly, sumoylated FAK was
enriched in the nuclear fraction of cells27. Although
blocking nuclear export using leptomycin B promotes
the nuclear accumulation of FAK, and exogenous
expression of the FAK FERM domain exhibits strong
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Extracellular
matrix
directly to the cytoplasmic tails of integrins33, accumulated evidence supports an indirect association of
FAK with integrins through binding to integrinassociated 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 phosphorylation of p190 RhoGEF might be a direct link to RhoA
activation34.
Growth
factor receptors
α β
Integrins
Assembly
Cadherins
Focal
contacts
FAK
Disassembly
?
Rho-family GTPases
mDia
Microtubule
stabilization
RhoA
Rac
Cdc42
Stress
fibres
Lamellipodia
Filopodia
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).
STRESS FIBRES
Also termed ‘actinmicrofilament bundles’, these are
bundles of parallel filaments that
contain F-actin and other
contractile molecules, and often
stretch between cell attachments
as if under stress.
LAMELLIPODIA
Broad, flat protrusions at the
leading edge of a moving cell
that are enriched with a
branched network of actin
filaments.
HeLa CELLS
An established tissue-culture
strain of human epidermoid
carcinoma cells, containing
70–80 chromosomes per cell.
These cells were originally
derived from tissue taken from a
patient named Henrietta Lacks
in 1951.
58
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 cellcycle progression30, it is possible that sumoylation of
FAK might facilitate a direct signalling route between
focal contacts and the nucleus.
The C-terminal domain of FAK contains two
proline-rich regions that function as binding sites for
SRC-HOMOLOGY (SH)3-DOMAIN-containing proteins (FIG. 2).
SH3-domain-mediated binding of the adaptor protein
p130Cas to FAK is important in promoting cell migration through the coordinated activation of Rac at membrane extensions31,32. The SH3-mediated binding of
other proteins, such as GRAF (GTPase regulator associated with FAK) and ASAP1 (Arf GTPase-ACTIVATING PROTEIN
(GAP) containing SH3, ankyrin repeat and pleckstrin
homology (PH) domains-1), connects FAK to the regulation of cytoskeletal dynamics and focal contact assembly. However, the downstream connections of GRAF
and ASAP1 remain undefined4.
The C-terminal domain of FAK also encompasses
the FAT region, which promotes the colocalization of
FAK with integrins at focal contacts (BOX 1). Whereas
it was first hypothesized that FAK might bind
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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-DOMAINcontaining proteins, such as SRC-FAMILY KINASES (SFKs),
phospholipase Cγ (PLCγ), suppressor of cytokine signalling (SOCS), growth-factor-receptor-bound protein-7
(GRB7), the Shc adaptor protein, p120 RasGAP, and
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 different 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 activation18. 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 common phosphorylation sites (BOX 2). The binding of SFKs
to PYK2 that is phosphorylated at Tyr402 is also associated 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 common 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 integrinmediated cell adhesion. Models of integrin-mediated
intermolecular FAK activation are based on the fact that
FAK mutants can compete for integrin association and
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p120 RasGAP,
GRB7, Shc, PLCγ,
p85, Src, SOCS
Ezrin
PDGF receptor,
ETK,
EGF receptor
P
Tyr397
FERM
FAK
Lys152
GRB2, p190 RhoGEF,
talin, paxillin
P
P
P
P
Tyr861 Tyr925
Tyr576 Tyr577
Kinase domain
PRR1
FIP200
SUMO
FAT
PRR2
PRR3
p130Cas, ASAP1, GRAF
Figure 2 | Focal adhesion kinase domain structure and phosphorylation sites. Focal
adhesion kinase (FAK) contains a FERM (protein 4.1, ezrin, radixin and moesin homology)
domain, a kinase domain and a focal adhesion targeting (FAT) domain. The FERM domain
mediates interactions of FAK with the epidermal growth factor (EGF) receptor, platelet-derived
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
focal contacts by associating with integrin-associated proteins such as talin and paxillin. It also
links FAK to the activation of Rho GTPases by binding to guanine nucleotide-exchange factors
(GEFs) such as p190 RhoGEF. FAK contains three proline-rich regions (PRR1–3), which bind Srchomology-3 (SH3) domain-containing proteins such as p130Cas, the GTPase regulator
associated with FAK (GRAF) and the Arf-GTPase-activating protein ASAP1. FAK is
phosphorylated (P) on several tyrosine residues, including Tyr397, 407, 576, 577, 861 and 925.
Tyrosine phosphorylation on Tyr397 creates a Src-homology-2 (SH2) binding site for Src,
phospholipase Cγ (PLCγ), suppressor of cytokine signalling (SOCS), growth-factor-receptorbound protein 7 (GRB7), the Shc adaptor protein, p120 RasGAP and the p85 subunit of
phosphatidylinositol 3-kinase (PI3K). Phosphorylation of Tyr576 and Tyr577 within the kinase
domain is required for maximal FAK catalytic activity, whereas the binding of FAK-family
interacting protein of 200 kDa (FIP200) to the kinase region inhibits FAK catalytic activity. FAK
phosphorylation at Tyr925 creates a binding site for GRB2.
ADAPTOR PROTEINS
Proteins that augment cellular
responses by recruiting other
proteins to a complex. They
usually contain several
protein–protein interaction
domains.
G-PROTEIN-COUPLED
RECEPTOR
A seven-helix membranespanning cell-surface receptor
that signals through
heterotrimeric GTP-binding and
GTP-hydrolysing G-proteins to
stimulate or inhibit the activity
of a downstream enzyme.
SRC-HOMOLOGY (SH)3-DOMAIN
A protein sequence of 50 amino
acids that recognizes and binds
sequences that are rich in proline.
GTPase-ACTIVATING PROTEIN
(GAP). A protein that stimulates
the intrinsic ability of a GTPase
to hydrolyse GTP to GDP.
Therefore, GAPs negatively
regulate GTPases by converting
them from active (GTP-bound)
to inactive (GDP-bound).
can inhibit endogenous FAK activity4 and that kinaseinactive FAK can become transphosphorylated on
Tyr397 in cells23. FAK Tyr397 phosphorylation promotes Src binding, which leads to the conformational
activation of Src and results in a dual-activated FAK–Src
signalling complex18. Within this FAK–Src complex, Src
phosphorylates FAK at Tyr861, and this is associated
with an increase in SH3-domain-mediated binding of
p130Cas to the FAK C-terminal proline-rich regions39.
Activated Src also phosphorylates FAK at Tyr925, which
creates an SH2-binding site for the GRB2 adaptor protein. GRB2 binding to FAK is one of several connections
that lead to the activation of Ras and the extracellular
signal-regulated kinase-2 (ERK2)/mitogen-activated
protein kinase (MAPK) cascade18. ERK2 phosphorylation and the subsequent activation of myosin light chain
kinase can modulate focal contact dynamics in motile
cells3, as well as generate both proliferative and survival
signals inside cells31.
thereby reinforced the role of integrins in the regulation of FAK signalling. Results showing that FAK
catalytic activity can be modulated by either posttranslational or mutational changes in activation-loop
residues are consistent with crystal structure analysis of
the ATP-bound kinase domain of FAK, which shows a
disordered activation-loop conformation41. As the
crystal structure of the kinase domain of FAK also
showed the presence of an unusual disulphide bond
between Cys456 and Cys459 in a regulatory region,
conformational changes or protein-binding interactions might also function to modulate the activation
state of FAK.
This model is supported by findings that cellular
proteins such as FAK-interacting protein of 200 kDa
(FIP200) bind to the kinase domain of FAK and inhibit
FAK activity42. Additionally, evidence is accumulating
that intramolecular constraints also have a role in the
regulation of FAK activity. There are alternatively spliced
isoforms of FAK in which amino-acid additions surrounding the Tyr397 site promote a change in the
kinetics of FAK activation (as measured by Tyr397
phosphorylation) from a primarily trans-intermolecular
reaction to a cis-intramolecular reaction35. Although
alternative splicing of FAK does not alter the FERM
domain residues of FAK, truncation or removal of the
FAK FERM domain does result in enhanced FAK catalytic 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 activity26,43, and as the FAK FERM domain can bind in trans
to the FAK catalytic domain, resulting in the inhibition
of FAK activity44, it is possible that binding interactions
or conformational changes in the FAK FERM domain
might function to release cis-inhibitory constraints on
FAK catalytic activation.
The activity of FAK can also be modulated positively45 or negatively 46 by the action of protein-tyrosine
phosphatases (PTPs). Studies using PTPα-deficient
fibroblasts showed that this phosphatase was required
for maximal stimulation of Src catalytic activity by
β1-integrins, and that PTPα functioned as an upstream
regulator of FAK Tyr397 phosphorylation45. This result
is consistent with the potential intermolecular activation of FAK by Src. As Src can also become activated
through direct interaction with the cytoplasmic
domains of β-integrins47, these types of result reinforce
the fact that Tyr397 phosphorylation of FAK might not
always reflect FAK catalytic activity that is mediated by
autophosphorylation.
Regulation of FAK catalytic activity
Src-mediated transphosphorylation of FAK within the
kinase domain ACTIVATION LOOP at Tyr576 and Tyr577
promotes maximal FAK catalytic activation31. Mutation
of FAK within this loop produces FAK mutants with
either enhanced or refractory activities. One such
mutant, ‘superFAK’, contains a Lys to Glu substitution
at residues 578 and 581, and results in a FAK protein
with adhesion-independent activity40. However, the
phosphorylation of downstream targets in superFAKexpressing cells remained adhesion dependent, which
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
p130Cas and paxillin as targets of FAK
In addition to promoting maximal FAK activation, the
recruitment of Src into a FAK–Src signalling complex
functions to facilitate the phosphorylation of various
FAK-associated proteins, as many FAK targets are also
independent binding partners and phosphorylation targets of Src. Two of the best-characterized target proteins
of FAK–Src-mediated phosphorylation are p130Cas and
paxillin31,32,48. SH3-mediated binding of p130Cas to FAK
is linked to enhanced tyrosine phosphorylation of
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Box 2 | The FAK-related kinase PYK2
Src
↑
Tyr397 Tyr576 Tyr577
FAK
FERM
Kinase domain
PRR1
Sequence
<10%
similarity
PYK2
GRB2
↑
Tyr861 Tyr925
~40%
FERM
FAT
PRR2
~60%
PRR3
~40%
Kinase domain
FAT
PRR1 Tyr402 Tyr579 Tyr580 PRR2
↓
Src
PRR3 Tyr881
↓
GRB2
Proline-rich tyrosine kinase-2 (PYK2) shares a similar domain arrangement with focal
adhesion kinase (FAK) (see figure), with 60% sequence identity in the central kinase
domain, conservation of proline-rich regions (PRRs), and identical positions of four
tyrosine phosphorylation sites. PYK2 tyrosines 402, 579, 580 and 881 correspond to FAK
tyrosines 397, 576, 577 and 925, respectively. Phosphorylation of PYK2 Tyr402 and
Tyr881 create Src-homology-2 (SH2) binding sites for Src and growth-factor-receptorbound-2 (GRB2), respectively. PYK2 contains a C-terminal focal adhesion targeting
(FAT) domain that binds to paxillin53. However, PYK2 shows perinuclear distribution
and is not strongly localized to focal contacts in many cells37. The substitution of the FAK
C-terminal domain to PYK2 facilitated the colocalization of this PYK2–FAK chimaera to
β1-integrin-containing focal contacts38, which indicates that there are biologically relevant
binding differences between FAK and PYK2. For instance, the FAK C-terminal domain
uniquely binds the integrin-associated protein talin114, and PYK2 — but not FAK —
binds the actin-associated protein gelsolin115. Although PYK2 can be activated by
integrins, this is dependent on integrin-mediated activation of Src-family kinases116,117.
The 40% sequence similarity between the N-terminal FERM (protein 4.1, ezrin, radixin
and moesin homology) domains of PYK2 and FAK also accounts for differential association
with target proteins118.What remains unknown is why PYK2 activity is highly dependent
on intracellular Ca2+ levels and how PYK2 associates with members of the Janus kinase
family37,119 — properties that are not shared by FAK.As PYK2 regulates several signalling
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
unique role for PYK2 in mediating haematopoietic cell responses to chemokine stimuli.
AUTOPHOSPHORYLATION
The transfer of a phosphate
group by a protein kinase either
to a residue in the same kinase
molecule (cis) or to a residue in a
different kinase molecule but of
the same type (trans).
SH2 DOMAIN
A protein motif that recognizes
and binds tyrosinephosphorylated sequences, and
thereby has a key role in relaying
cascades of signal transduction.
SRC-FAMILY KINASES
Kinases that belong to the Src
family of tyrosine kinases, the
largest of the non-receptortyrosine-kinase families.
60
p130Cas at multiple sites, which promotes SH2-mediated binding of the Crk adaptor protein to p130Cas.
Signalling downstream of p130Cas results in increased
activity of Rac, enhanced MEMBRANE RUFFLING or lamellipodia formation, and the promotion of cell motility or
invasion17,49,50 (FIG. 3). Paxillin is phosphorylated by
FAK–Src on Tyr31 and Tyr118, and this can also promote SH2-mediated binding of Crk to paxillin48,51.
Overexpressing paxillin that is mutated at these phosphorylation sites inhibits the turnover of focal contacts6
and cell motility52, which therefore supports the presence of multiple routes for FAK–Src-mediated signalling in modulating the dynamics of cell adhesion
sites.
Regulated targeting of FAK to focal contacts
It is the C-terminal FAT domain of FAK that facilitates
the linkage to integrins and focal contacts. The FAT
domain adopts a four-helix bundle structure that contains binding sites for integrin-associated proteins such
as paxillin53. Point mutations in the FAT domain of FAK
that disrupt paxillin binding also prevent the association
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of FAK with β1-integrin and the localization of FAK to
focal contacts38. Paxillin binding is mediated by two
leucine-rich peptide regions in paxillin that are known
as LD MOTIFS, which interface with hydrophobic surface
grooves on the FAT domain54,55. Interestingly, the SH2
binding site for GRB2 at FAK Tyr925 partially overlaps
with one of the two paxillin LD-motif binding sites in
the FAT domain54, and localization studies of phosphorylated FAK have shown that Tyr925-phosphorylated
FAK might be selectively excluded from focal contact
sites56. Overexpression of a Tyr925Phe mutant of FAK
resulted in strong focal contact distribution56, and in
activated Src-expressing cells, Tyr925Phe FAK blocks
the turnover of focal contacts (V. Brunton, personal
communication; see note added in proof). As NMR
analyses have shown that the FAT domain can
undergo conformational rearrangements that might
selectively promote either Tyr925 phosphorylation
and/or paxillin binding57, it is possible that Src-mediated phosphorylation of FAK on Tyr925, and subsequent GRB2 binding, could displace paxillin, promote
the dissociation of FAK from focal contacts, and subsequently lead to focal contact turnover through
undefined mechanisms (FIG. 3).
Ser910 within the FAT domain is phosphorylated
during mitosis58 and after growth factor stimulation of
cells. Ser910 is phosphorylated by ERK2 and this is also
associated with reduced paxillin binding to FAK59. So,
Src-mediated phosphorylation of Tyr925 on FAK and
GRB2 binding leading to ERK2 activation, coupled
with the feedback of ERK2-mediated Ser910 phosphorylation, could potentiate the release of FAK from focal
contacts. Alternatively, FAK–Src-mediated phosphorylation of paxillin at Tyr118 promotes the binding of
ERK2 to paxillin60, and ERK2-mediated phosphorylation 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
function first to promote FAK release from existing
focal contacts and then, through ERK2-mediated phosphorylation of paxillin, to promote FAK re-binding
and activation at new or different focal contacts in a
migrating cell (FIG. 3).
Regulation of focal contact dynamics
Lessons from FAK–/– and SHP2 –/– cells. In analyses monitoring the formation of focal contacts, FAK was found
to be one of the first signalling proteins to be recruited to
these sites62. Although FAK recruitment to focal contacts is associated with increased FAK tyrosine phosphorylation63, focal contacts readily form in FAK-null
(FAK–/–) fibroblasts, which indicates that FAK activity is
not essential for the process of focal-adhesion formation5. However, focal contacts in FAK–/– cells form primarily around the cell periphery, enmeshed in a cortical
actin ring, and do not undergo a normal maturation
cycle64. In normal fibroblasts, peripheral immature focal
contacts become connected to longitudinal stress fibres
in cells and undergo actin contractility-mediated maturation during cell polarization63. FAK re-expression in
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α β
Migration
Membrane
Integrins
Rac
Crk
Src
Focal contacts
FAK
SH2
P
P
P
Tyr397 Tyr576 Tyr577
FERM
p130Cas P
SH3
P-X-X-P
Kinase domain
Tyr925
Paxillin
ERK2
FAT
P
Tyr118
P
Release
ERK2
P GRB2
Tyr925
FAK
FERM
Kinase domain
FAT
Ser910
P
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
creates a binding site for the Src-homology (SH)2 domain of Src. Src-mediated phosphorylation
of FAK at Tyr576 and Tyr577 promotes maximal FAK catalytic activity. Active FAK–Src facilitates
SH3-mediated binding of p130Cas to FAK and its subsequent phosphorylation. Crk binding to
phosphorylated p130Cas facilitates Rac activation, lamellipodia formation and cell migration.
Paxillin binding to the FAK focal adhesion targeting (FAT) domain is important for FAK focal
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
activation of Ras and the extracellular signal-regulated kinase-2 (ERK2) cascade. The GRB2 and
paxillin binding sites within the FAT domain overlap and Tyr925-phosphorylated FAK might be
selectively released from focal contacts. ERK2 activation promotes FAK phosphorylation at
Ser910, which is also associated with decreased paxillin binding to FAK. Within focal contacts,
FAK–Src-mediated phosphorylation of paxillin at Tyr118 promotes ERK2 binding. ERK2-mediated
phosphorylation of paxillin can facilitate FAK binding to paxillin and enhances FAK activation. So,
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
association of unphosphorylated FAK with paxillin at new or growing focal contact sites.
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 NUCLEOTIDEEXCHANGE 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 ‘immature’ 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 linkage 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 maturation 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 further supported by studies of cells that lack the tyrosine
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
phosphatase SHP2 (SH2-domain-containing protein
tyrosine phosphatase 2). SHP2–/– cells have increased
FAK activity, but they also show an accumulation of
immature focal contacts and similar refractory migration defects to FAK–/– cells67. Hyperactive FAK in SHP2–/–
cells results in an increase in the levels of Tyr12-phosphorylated α-actinin, which thereby reduces the
crosslinking of stress fibres and prevents the maturation
of focal contacts68. In SHP2–/– cells, there is a high level of
focal contact turnover68, whereas in FAK–/– cells, focal
contact turnover and maturation are inhibited8. So, both
SHP2–/– and FAK–/– cells show an accumulation of
immature focal contacts, but through different mechanisms. These studies support the importance of FAK
expression and the precise temporal regulation of
FAK activity as important factors that control the
dynamics of focal contacts.
Paxillin–/–, p130Cas–/– and Src –/– cells. In addition to
FAK–/– and SHP2–/– cells, fibroblasts that contain null
mutations for various other focal-contact-associated
proteins also show altered dynamics of focal contact
maturation, cell spreading defects and refractory cell
motility responses (TABLE 1). Time-lapse analyses showed
that the incorporation of labelled paxillin into focal contacts of FAK–/–, paxillin–/–, p130Cas–/– or SYF–/– (Src–/–,
Yes–/– and Fyn–/–) cells did not significantly differ compared to normal fibroblasts6. However, the rate of focal
contact disassembly was much slower in these null cells.
Analyses of FAK–/– cells showed that disassembly was
dependent on FAK Tyr397 phosphorylation; SYF–/– cells
showed that Src kinase activity was required; and studies
with paxillin–/– cells indicated that the integrity of the
Tyr31 and Tyr118 paxillin phosphorylation sites were
needed to promote focal contact turnover6. As the
expression of constitutively active Src in FAK–/– cells can
promote focal contact turnover and increased cell
motility17,69, these combined analyses support the conclusion that, in normal cells, integrin- and FAK-mediated control of Src activity is a key event that promotes
focal contact dynamics.
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
16,17,22
MATRIX METALLOPROTEINASES (MMPs)
. The influence
of FAK on MMP regulation is associated with signalling from Ras to ERK2 and from Rac to Jun N-terminal 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
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© 2005 Nature Publishing Group
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Actin stress fibres
Actin stress fibres
Myosin
Myosin
Assembly
Myosin
Myosin
ARP2/3
in
ctin
ROCK
in
ctin
α-A
N-WASP
α-A
in
Cdc42
ctin
FAK
Tyr256
N-WASP
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
Reduced
α-actinin
crosslinking
MLC
phosphatase
Increased
α-actinin
crosslinking
P
Tyr256
MLCK ↑
Rho
p190
RhoGEF
α-A
Cdc42
Tyr12
P
Tyr256
N-WASP
P
Focal contact
GRAF
Rho
p190
RhoGEF
FAK
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
have an increased number of peripheral focal contacts72.
Calpain is not appropriately activated in FAK–/– cells21,
and this defect might be due in part to ERK2 activation being required for calpain function73. Notably,
FAK re-expression in FAK–/– cells promotes the formation of a complex between calpain, ERK2 and activated
Src21. Restoration of calpain activity in FAK–/– cells
requires specific FAK phosphorylation events: a form
of FAK that is mutated at several phosphorylation sites
can form a complex with calpain and ERK2, but it
does not restore full calpain activity in contrast to cells
in which wild-type FAK is re-expressed74. So, FAK signalling is connected to the increased turnover of focal
contacts through calpain activation. As calpain is a
Ca2+-dependent protease, and FAK activation is associated with local Ca2+-flux-induced disassembly of focal
contacts75, the FAK–calpain linkage might be selectively activated at either cell protrusions or tail retraction sites in motile cells.
| JANUARY 2005 | VOLUME 6
FAK effects on GTPases and actin
The activity of Ras and the Rho-family GTPases Rho,
Rac and Cdc42 is positively regulated by GEFs and negatively regulated by GAPs. As mentioned above, a number of studies have shown that FAK–Src-mediated
phosphorylation events can lead to the activation of
Ras–ERK2 and Rac–JNK signalling cascades to promote increased cell migration and invasion18. In a
recently discovered mechanism, FAK overexpression
facilitated the SH2-mediated binding and sequestering
of p120 RasGAP, which diminished the association of
p120 RasGAP with active Ras76 and thereby led to Ras
activation.
In FAK –/– cells, the intrinsic GTPase activity of
RhoA is elevated8, and pharmacological inhibitors of
Rho-associated kinase (ROCK) — a substrate of Rho
— partially reverse the polarization defects of FAK–/–
cells77. Integrin signalling can suppress RhoA activity
by tyrosine phosphorylation of p190 RhoGAP
(which increases its GAP activity)78. Likewise, stable
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Table 1 | Phenotypes associated with null mutations in focal contact proteins
Cell
phenotypes
Embryonic
(lethality) day
Focal contact
formation
Focal contact
turnover
Integrin-stimulated
migration
FAK tyrosine
phosphorylation
FAK–/– (p53–/–)
8.5
Increased immature
Inhibited
Inhibited
NA
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
–/–
Reference
5
PTPα
None
Delayed
ND
Reduced
pTyr397 reduced
45
SHP2–/–
8.5–10.5
Increased immature
Elevated
Inhibited
Increased activity
in suspension
67,68
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 vinculinnull 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.
FAK re-expression in FAK–/– cells decreased RhoA
activity 8 and enhanced p190 RhoGAP tyrosine phosphorylation17. In other cell types, FAK activation and
tyrosine phosphorylation are associated with RhoA
activation and the formation of stress fibres18. This connection could be mediated by FAK binding to, and
phosphorylating, p190 RhoGEF34. In neuronal development, 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 signalling 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
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.
Integrating factors coordinate the regulation of microtubule 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 stabilization by the Rho–mDia signalling pathway7 (FIG. 5). This is
partly the result of the FAK-regulated localization of a
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
lipid-raft marker, GANGLIOSIDE GM1, to the leading edge
of motile cells. It is hypothesized that the lipid environment at the leading edge preferentially localizes microtubule capping or bridging proteins, which stabilize the
association of microtubules with cortical receptors82.
This regulation of a distinct membrane lipid environment 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-phosphorylated 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 development positioning in vivo 86. Whereas the molecular
mechanisms that link FAK Ser732 phosphorylation to the
localization and organization of microtubule fork structures remain to be defined, this observation is the first of
its kind and supports studies that link FAK phosphorylation 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 signalling is involved in the modification of phosphatidylinositol lipids, and differentially phosphorylated lipid
VOLUME 6 | JANUARY 2005 | 6 3
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PtdIns
α
Formation of
membrane
proximal contacts
Integrins
β
PtdIns(4)P
Talin
PIPKIγ
PtdIns(4,5)P2
Vinculin
FAK
PI3K
Paxillin
p85
PtdIns(3,4,5)P3
Membrane
component
mDia
GM1
Phospholipid
modification by
phosphorylation
Stable
lamellipodia
Rac
Lipid
rafts
Rac
New
membranes
FAK and intercellular contacts
Increased
membrane
fluidity
Microtubule
stabilization
+
+
+
Figure 5 | Focal adhesion kinase influences phospholipid and microtubule structures.
The phospholipid kinases that have a role in the modification of phosphatidylinositol (PtdIns)
cooperate with focal adhesion kinase (FAK) at several levels. Type I PtdIns phosphate kinase-γ
(PIPKIγ) associates with FAK and talin, and promotes the conversion of PtdIns-4-phosphate
(PtdIns(4)P) to PtdIns-4,5-bisphosphate (PtdIns(4,5)P2). PIPKIγ is phosphorylated by FAK, which
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
can be converted to PtdIns-3,4,5-trisphosphate (PtdIns(3,4,5)P3) by PtdIns 3-kinase (PI3K). The
regulatory p85 subunit of PI3K binds to FAK at Tyr397, which leads to PI3K activation by FAK112.
Directional motility requires the generation of phospholipid components such as PtdIns(4,5)P2 and
PtdIns(3,4,5)P3. Integrin and FAK signalling also promote the translocation of specific
components of lipid rafts to membranes. The stabilization of lipid rafts through integrin signalling
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
effector Diaphanous (mDia), regulates microtubule polarity at the leading edge of motile cells.
Microtubule polarization and Rac activation contribute to the formation of membrane ruffles and
stable lamellipodia.
ADHERENS JUNCTION
A cell–cell adhesion complex
that contains classical cadherins
and catenins that are attached to
cytoplasmic actin filaments.
TIGHT JUNCTION
A circumferential ring at the
apex of epithelial cells that seals
adjacent cells to one another.
Tight junctions regulate solute
and ion flux between adjacent
epithelial cells.
64
phosphorylated by a FAK–Src complex, which facilitates
increased PIPKIγ activity (and, therefore, increased production of PtdIns(4,5)P2) and increased PIPKIγ association with talin91. In this manner, FAK signalling is connected to the formation of focal contacts and the spatial
regulation of PtdIns(4,5)P2 generation. However, the
integrin and PIPKIγ binding sites within the talin
FERM domain overlap, which implies that PIPKIγ
binding might displace talin from integrin tails92. To this
end, FAK-enhanced Src-mediated phosphorylation of
PIPKIγ on Tyr644 creates a high affinity binding site for
the talin FERM domain, which displaces β-integrin
binding from talin FERM93. So, although FAK–Src
activity could promote the production of PtdIns(4,5)P2
and the formation of focal contacts by enhancing the
activity of PIPKIγ, subsequent phosphorylation of
PIPKIγ by activated Src might break the talin–integrin
linkage and promote the turnover of focal contacts.
intermediates function as binding sites for signalling
proteins that are involved in the formation of focal contacts (FIG. 5). Phosphatidylinositol-4,5-bisphosphate
(PtdIns(4,5)P2) binds to and controls the assembly of
proteins such as α-actinin, vinculin and talin into focal
contacts2. As the binding of the talin FERM domain to
β-integrin cytoplasmic tails is enhanced by
PtdIns(4,5)P2 (REF. 88), and the talin rod domain binds
vinculin and actin89, a link between integrins, focal
contact formation and the actin cytoskeleton is established. The type I phosphatidylinositol phosphate
kinase-γ (PIPKIγ) is an enzyme that makes
PtdIns(4,5)P2 and it is targeted to focal contacts by an
association with the talin FERM domain90. PIPKIγ is
| JANUARY 2005 | VOLUME 6
Another biological context in which FAK signalling
has been associated with the formation or turnover of
contacts is cadherin-based cell–cell junctions.
Cadherins are transmembrane proteins that mediate
Ca2+-dependent homophilic protein–protein attachments between cells and that are also linked to the
actin cytoskeleton through interaction with α- or βcatenins94. Downregulation of E-cadherin-based
ADHERENS JUNCTIONS is a hallmark of malignant and
invasive carcinomas, and the activity of the FAK–Src
complex promotes the disruption of colon carcinoma
cell homotypic adhesions95. Importantly, expression of
a FAK protein that is mutated at five tyrosine phosphorylation sites (Tyr407, 576, 577, 861 and 925) blocked
the Src-mediated disruption of colon carcinoma E-cadherin-based contacts, thereby implying that phosphorylation-dependent signalling through FAK was
required96. In an opposite manner, overexpression of a
kinase-defective mutant of FAK blocked the accumulation of peripheral E-cadherin in endothelial cells that
were subjected to a hyperosmolar challenge (a stimulus that promotes increased E-cadherin-based TIGHT97
JUNCTION barrier formation) . These results imply that
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
with a scattered morphology and a migratory or invasive phenotype. Antisense and DOMINANT-NEGATIVE inhibition of FAK showed that FAK expression and activity
were needed for the formation of N-cadherin-based
cell–cell contacts in HeLa cells19. However, in contradiction, the above study also found that cells with less FAK
expression and reduced N-cadherin-mediated cell–cell
contacts exhibited ‘increased’ motility when plated as
individual cells on a collagen matrix. Whereas much
remains to be determined about the molecular role of
FAK in either the dissolution or formation of cadherinbased contacts, it is intriguing that the findings so far are
somewhat similar to the bi-functional role of FAK in
focal contact dynamics.
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FAK as a biosensor
Early studies with integrins found that they ‘sensed’
environmental cues and functioned to control anchoragedependent 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 activation 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 conditions, 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 rescue 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 signalling response.
Moving in three dimensions
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.
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 properly remodel focal contacts into fibrillar adhesions103
and FAK–/– endothelial cells do not form tubule structures when grown in a 3D matrix environment13.
Interfering with FAK activity prevents tubule formation in human brain microvascular endothelial cells14,
and gain-of-function experiments with FAK–/– fibroblasts 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, FAKdependent 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
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
2D focal contacts105, functional experiments with
human lung fibroblasts106, breast carcinoma cells107 or
normal CYTOTROPHOBLASTS108 have supported the importance 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 invasion22, 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 correlated 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 highlighted the diversity of FAK-signalling inputs and outputs 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 signalling 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 phosphorylation events that are associated with FAK are transient 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 associated 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.
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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 targeting of FAK to distinct intracellular sites. As many of the
phenotypes that are associated with FAK have been elucidated using overexpression studies, the development
of pharmacological inhibitors to FAK and analyses of
catalytically-defective FAK mutants in FAK–/– cells will
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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
© 2005 Nature Publishing Group