Focal adhesion kinase-like
Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic
tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion
in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization.
The regional and cellular distribution of these kinases was studied in adult rat brain and during development. Whereas PYK2/CAKbeta expression
increases with postnatal age and is maximal in the adult, FAK+ levels are stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, are
expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain shows a
widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the
hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta are enriched in the perikarya and growth
cones. FAK+ extends to the periphery of the growth cones tips, whereas PYK2/CAKbeta appear to be excluded from the lamellipodia. During the
establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir can be observed in the growing axon. In most older neurons,
FAK+-ir is confined to the cell bodies, whereas PYK2/CAKbeta-ir is also present in the processes. In vitro and in vivo, a subpopulation of neurons
displays neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly
expressed in the adult brain, with distinctive but overlapping distributions (Menegon, 1999).
pp125 focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase transducing signals initiated by
integrin engagement and G protein-coupled receptors, is highly expressed in brain. FAK from brain has
a higher molecular weight and an increased autophosphorylation activity, as compared to FAK from other tissues. In
addition to a 9-base insertion in the 3'-coding region, which defines FAK+, rat striatal FAK mRNAs
contained several additional short exons, coding for peptides of 28, 6, and 7 residues, respectively
(termed boxes 28, 6, and 7), surrounding the autophosphorylated Tyr-397. In transfected COS 7 cells,
the presence of boxes 6 and 7 confer an increased overall tyrosine phosphorylation, a higher
phosphorylation of Tyr-397 assessed with a phosphorylation state-specific antibody, and a more active
autophosphorylation in immune precipitates. The presence of box 28 does not further alter these
parameters. Two-dimensional phosphopeptide maps of hippocampal FAK are identical to those of
FAK+6,7. The presence of the various exons does not alter the interaction of FAK with c-Src, n-Src, or
Fyn. Thus, several splice isoforms of FAK are preferentially expressed in rat brain, some of which
have an increased autophosphorylation activity, suggesting that FAK may have specific properties in
neurons (Burgaya, 1997).
FAK was originally identified gy its high level of tyrosine phosphorylation in v-src-transformed cells. FAK is also highly phosphorylated during early development. In cultured cells it is localized to focal adhesion contacts and becomes phosphorylated and activated in response to integrin-mediated binding of cells to the extracellular matrix, suggesting an important role in cell adhesion and/or migration. FAK-deficient mice were generated by gene targeting to examine the role of FAK during development. Mutant embryos displayed a general defect of mesoderm development, and cells from these embryos had reduced mobility in vitro. Surprisingly, the number of focal adhesions was increased in FAK-deficient cells, suggesting that FAK may be involved in the turnover of focal adhesion contacts during cell migration (Ilic, 1995).
A characteristic feature of certain integrins is their ability to modulate their affinity for extracellular ligands in response to intracellular signals, a process termed "activation" or inside-out signaling". A Ras/Raf-initiated MAP kinase activity suppresses mammalian integrin activation. Using a screen for suppressors of integrin activation, the small GTP-binding protein H-Ras, and its effector kinase, Raf-1 were identified as negative regulators of integrin activation. HRas inhibits the activation of integrins with three distinct alpha and beta subunit cytoplasmic domains. Suppression is not associated with integrin phosphorylation and is independent of both mRNA transcription and protein synthesis. Furthermore, suppression correlates with activation of the ERK MAP kinase pathway. It is possible that the integrin suppression pathway forms a local negative feedback loop for the regulation of integrin function. Ras activation through integrins might occur via the formation of a complex of FAK, GRB-2 and SOS. Cells derived from FAK-deficient mice show enhanced focal adhesion formation, suggesting that these cells may have lost a negative regulator of integrin function. In addition, dominant negative Ras can enhance focal adhesion formation. Further evidence for the existence of a negative feedback loop comes from observations that integrin occupancy or the expression of isolated beta subunit cytoplasmic domains can suppress the function of other integrins. It is likely that a cytoplasmic substrate of a MAP kinase is involved in suppression (Hughes, 1997).
Integrin-mediated adhesion of cells to extracellular matrix proteins triggers a variety of intracellular
signaling pathways including a cascade of tyrosine phosphorylations. In many cell types, the
cytoplasmic focal adhesion tyrosine kinase, FAK, appears to be the initial protein that becomes
tyrosine-phosphorylated in response to adhesion; however, the molecular mechanisms regulating
integrin-triggered FAK phosphorylation are not understood. Previous studies have shown that the
integrin beta1, beta3, and beta5 (see Drosophila Myospheroid) subunit cytoplasmic domains all contain sufficient information to trigger
FAK phosphorylation when expressed in single-subunit chimeric receptors connected to an
extracellular reporter. In the present study, beta3 cytoplasmic domain deletion and substitution mutants
were constructed to identify amino acids within the integrin beta3 cytoplasmic domain that regulate its
ability to trigger FAK phosphorylation. Cells transiently expressing chimeric receptors containing these
mutant cytoplasmic domains were magnetically sorted and assayed for the tyrosine phosphorylation of
FAK. Analysis of these mutants indicate that structural information in both the membrane-proximal
and C-terminal segments of the beta3 cytoplasmic domain is important for triggering FAK
phosphorylation. In the C-terminal segment of the beta3 cytoplasmic domain, the highly conserved
NPXY motif is found to be required for the beta3 cytoplasmic domain to trigger FAK
phosphorylation. However, the putative FAK binding domain within the N-terminal segment of the
beta3 cytoplasmic domain is found to be neither required nor sufficient for this signaling event. The serine 752 to proline mutation, known to cause a variant of Glanzmann's
thrombasthenia, inhibits the ability of the beta3 cytoplasmic domain to signal FAK phosphorylation,
suggesting that a single mutation in the beta3 cytoplasmic domain can inhibit both "inside-out" and
"outside-in" integrin signaling (Tahiliani, 1997).
Integrin alphaIIbbeta3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction.
Among the events that occur during either "inside-out" or "outside-in" signaling through alphaIIbbeta3 is the phosphorylation of
focal adhesion kinase [pp125(FAK)] and the association of pp125(FAK) with cytoskeletal components. To examine the role
of pp125(FAK) in these integrin-mediated events, pp125(FAK) phosphorylation and association with the cytoskeleton was
determined in cells expressing two mutant forms of alphaIIbbeta3: alphaIIbbeta3(D723A/E726A), a constitutively active
integrin in which the putative binding site for pp125(FAK) is altered, and alphaIIbbeta3(F727A/K729E/F730A), in which the
putative binding site for alpha-actinin is altered. Whereas cells expressing alphaIIbbeta3(D723A/E726A) are able to form focal
adhesions and stress fibers upon adherence to fibrinogen, cells expressing alphaIIbbeta3(F727A/K729E/F730A) adhere to
fibrinogen, but have reduced focal adhesions and stress fibers. pp125(FAK) is recruited to focal adhesions in adherent cells
expressing alphaIIbbeta3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of
fibrinogen. In adherent cells expressing alphaIIbbeta3(F727A/K729E/F730A), pp125(FAK) is phosphorylated despite
reduced formation of focal adhesions and stress fibers. It is concluded that activation of pp125(FAK) can be dissociated from
two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following
ligand occupation (Lyman, 1997).
Because integrin-mediated signals are transferred through a physical architecture and synergistic biochemical network whose properties are not well
defined, quantitative relationships between extracellular integrin-ligand binding events and key intracellular responses are poorly understood. This was addressed by quantifying integrin-mediated FAK and ERK2 responses in CHO cells for varied alpha(5)beta(1) expression level and substratum
fibronectin density. Plating cells on fibronectin-coated surfaces initiates a transient, biphasic ERK2 response, the magnitude and kinetics of which
depended on integrin-ligand binding properties. Whereas ERK2 activity initially increases with a rate proportional to integrin-ligand bond number for low
fibronectin density, the desensitization rate is independent of integrin and fibronectin amount but proportional to the ERK2 activity level with an
exponential decay constant of 0.3 (+/- 0.08) min(-1). Unlike the ERK2 activation time course, FAK phosphorylation follows a superficially disparate
time course. However, analysis of the early kinetics of the two signals reveals them to be correlated. The initial rates of FAK and ERK2 signal generation
exhibits similar dependence on fibronectin surface density, with both rates monotonically increasing with fibronectin amount until saturating at high
fibronectin density. Because of this similar initial rate dependence on integrin-ligand bond formation, the disparity in their time courses is attributed to
differences in feedback regulation of these signals. Whereas FAK phosphorylation increases to a steady-state level as new integrin-ligand bond formation
continues during cell spreading, ERK2 activity is decoupled from the integrin-ligand stimulus and decays back to a basal level. Accordingly, different functional metrics are proposed for representing these two disparate dynamic signals: the steady-state tyrosine phosphorylation level for FAK and the
integral of the pulse response for ERK2. These measures of FAK and ERK2 activity correlate with short term cell-substratum adhesivity,
indicating that signaling via FAK and ERK2 is proportional to the number of integrin-fibronectin bonds (Asthagiri, 1999).
alphaß1 integrins have been implicated in the survival, spreading, and migration of cells and tissues. To explore the underlying biology, conditions were identified where primary ß1 null keratinocytes adhere, proliferate, and display robust alphavß6 integrin-induced, peripheral focal contacts associated with elaborate stress fibers. Mechanistically, this appears to be due to reduced FAK and Src and elevated RhoA and Rock activities. Visualization on a genetic background of GFPactin shows that ß1 null keratinocytes spread, but do so aberrantly, and when induced to migrate from skin explants in vitro, the cells are not able to rapidly reorient their actin cytoskeleton toward the polarized movement. As judged by RFPzyxin/GFPactin videomicroscopy, the alphavß6-actin network does not undergo efficient turnover. Without the ability to remodel their integrin-actin network efficiently, alphaß1-deficient keratinocytes cannot respond dynamically to their environment and polarize movements (Raghavan, 2003).
The results underscore a novel and distinct role for alphaß1 integrins in regulating this equilibrium in focal adhesion dynamics. Not surprisingly, three well-known regulators of focal contacts, FAK, RhoA, and Rock, appear to be at the heart of this regulation. As judged by immunofluorescence with purportedly specific phospho-FAK Abs, activated FAK localizes to the focal contacts of ß1 null keratinocytes. By this criterion, the underlying defects in focal contact turnover and in overall FAK and Src activities are not attributable to a defect in targeting FAK to alphavß6 focal contacts, and indeed, ligand-engaged alphavß6 can bind and activate FAK. Rather, in the absence of ß1, alphavß6 appears unable on its own to activate FAK to the threshold levels needed to properly control focal adhesion-actin cytoskeletal dynamics. Irrespective of the precise underlying mechanism, the consequences to this imbalance are excessive adhesion and inefficient spreading (Raghavan, 2003).
Although tyrosine kinase inhibitors can block focal adhesion formation in some situations, a greater role for tyrosine phosphorylation has been found in focal adhesion turnover and cell motility. Thus, activated FAK negatively regulates RhoA activity, and FAK null fibroblasts express robust actin stress-fiber networks that can be dissipated by Rock inhibition. The ability of Rho and Rock inhibitors to disperse both stress fibers and associated focal contacts in ß1 null keratinocytes provides compelling evidence that a FAK-RhoA imbalance is at the root of the focal adhesion-cytoskeletal imbalance in these cells. Although more complicated mechanisms are possible, the data are consistent with a model whereby in the absence of alphaß1 integrins, FAK/Src activation is not fully achieved, thereby diminishing p190RhoGAP phosphorylation, and yielding elevated RhoA/Rock activities (Raghavan, 2003).
The axon guidance cue netrin is importantly involved in neuronal development. DCC (deleted in colorectal cancer) is a functional receptor for netrin and mediates axon outgrowth and the steering response. Different regions of the intracellular domain of DCC directly interact with the tyrosine kinases Src and focal adhesion kinase (FAK). Netrin activates both FAK and Src and stimulates tyrosine phosphorylation of DCC. Inhibition of Src family kinases reduces DCC tyrosine phosphorylation and blocks both axon attraction and outgrowth of neurons in response to netrin. Mutation of the tyrosine phosphorylation residue in DCC abolishes its function of mediating netrin-induced axon attraction. On the basis of these observations, a model is suggested in which DCC functions as a kinase-coupled receptor, and FAK and Src act immediately downstream of DCC in netrin signaling (Li, 2004).
Although netrins are an important family of neuronal guidance proteins, intracellular mechanisms that mediate netrin function are not well understood. This study shows that netrin-1 induces tyrosine phosphorylation of proteins including focal adhesion kinase (FAK) and the Src family kinase Fyn. Blockers of Src family kinases inhibit FAK phosphorylation and axon outgrowth and attraction by netrin. Dominant-negative FAK and Fyn mutants inhibit the attractive turning response to netrin. Axon outgrowth and attraction induced by netrin-1 are significantly reduced in neurons lacking the FAK gene. These results show the biochemical and functional links between netrin, a prototypical neuronal guidance cue, and FAK, a central player in intracellular signaling that is crucial for cell migration (Liu, 2004).
The signaling mechanisms that lie immediately downstream of netrin receptors remain poorly understood. This study reports that the netrin receptor DCC interacts with the focal adhesion kinase (FAK), a kinase implicated in regulating cell adhesion and migration. FAK is expressed in developing brains and is localized with DCC in cultured neurons. Netrin-1 induces FAK and DCC tyrosine phosphorylation. Disruption of FAK signaling abolishes netrin-1-induced neurite outgrowth and attractive growth cone turning. Taken together, these results indicate a new signaling mechanism for DCC, in which FAK is activated upon netrin-1 stimulation and mediates netrin-1 function; they also identify a critical role for FAK in axon navigation (Ren, 2004).
The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKbeta/FAK2, is a cytoskeleton-associated tyrosine kinase. CADTK regulation was compared with that of the highly homologous focal adhesion tyrosine kinase (FAK). CADTK mutants were generation.
Mutation of Tyr402 eliminates autophosphorylation and significantly decreases kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation
site predicted to bind Grb2, has little effect on CADTK regulation. Src family tyrosine kinases result in CADTK tyrosine phosphorylation even when
co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK
tyrosine-phosphorylates FAK when both are transiently expressed, but FAK does not phosphorylate CADTK. Biochemical experiments confirmed direct
CADTK phosphorylation of FAK. This phosphorylation utilizes tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new
SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Expression of the CADTK carboxyl terminus (CRNK) abolishes
CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibits both FAK and CADTK autophosphorylation,
suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but
not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling (Li, 1999).
Paxillin is a cytoskeletal protein involved in actin-membrane attachment at
sites of cell adhesion to the extracellular matrix. Extensive tyrosine
phosphorylation of this protein occurs during integrin-mediated cell
adhesion, embryonic development, fibroblast transformation and following
stimulation of cells by mitogens that operate through the family of seven
membrane-spanning G-protein-coupled receptors. Paxillin binds in vitro to
the focal adhesion protein vinculin as well as to the SH3 domain of c-src
and, when tyrosine phosphorylated, to the SH2 domain of v-crk. The complementary DNA, and derived amino acid sequence, that
codes for approximately 90% of the paxillin protein is reported. A region in the amino-terminal half of the protein supports the
binding of both vinculin and the focal adhesion tyrosine kinase, pp125Fak.
Although there is no significant overall homology with other identified
proteins, the carboxyl third of paxillin contains one LIM domain and three
LIM-like sequences. The LIM motif is common to a number of transcription
factors and to two other focal adhesion proteins, zyxin and
cysteine-rich protein. In addition to several potential tyrosine
phosphorylation sites there are five tyrosine-containing sequences that
conform to
SH2-binding motifs. The protein also contains a short proline-rich region
indicative of a SH3-binding domain. Taken together, these data suggest
that paxillin is a unique cytoskeletal protein capable of interaction with
a variety of intracellular signalling, and structural, molecules important
in
growth control and the regulation of cytoskeletal organization (Turner, 1994).
Paxillin, a focal-adhesion-associated protein, becomes phosphorylated in
response to a number of stimuli which also induce the tyrosine
phosphorylation of the focal-adhesion-associated protein tyrosine kinase
pp125FAK. On the basis of their colocalization and coordinate
phosphorylation, paxillin is a candidate for a substrate of pp125FAK. Conditions are described under which the phosphorylation of paxillin on
tyrosine is pp125FAK dependent, supporting the hypothesis that paxillin
phosphorylation is regulated by pp125FAK. pp125FAK must localize to
focal adhesions and become autophosphorylated to induce paxillin
phosphorylation. Phosphorylation of paxillin on tyrosine creates binding
sites for
the SH2 domains of Crk, Csk, and Src. Two sites of
phosphorylation have been identified as tyrosine residues 31 and 118, each of which conforms to
the Crk
SH2 domain binding motif, (P)YXXP. These observations suggest that paxillin
serves as an adapter protein, similar to insulin receptor substrate 1,
and that pp125FAK may regulate the formation of signaling complexes by
directing the phosphorylation of paxillin on tyrosine (Schaller, 1995).
Focal adhesion kinase (FAK) and paxillin are focal adhesion-associated, phosphotyrosine-containing proteins that physically interact. Paxillin contains two binding sites for FAK. The
binding affinity of the carboxyl-terminal domain of FAK is the same for each of the two binding sites. The presence of both binding sites increases the
affinity for FAK by 5-10-fold. A conserved paxillin sequence called the LD motif has been implicated in FAK binding. Mutations in the LD
motifs in both FAK-binding sites are required to dramatically impair FAK binding in vitro. A paxillin mutant containing point mutations in both
FAK-binding sites was characterized. The mutant exhibits reduced levels of phosphotyrosine relative to wild type paxillin in subconfluent cells growing in
culture, following cell adhesion to fibronectin and in src-transformed fibroblasts. These results suggest that paxillin must bind FAK for maximal phosphorylation in response to cell adhesion and that FAK may function to direct tyrosine phosphorylation of paxillin in the process of transformation by the src oncogene (Thomas, 1999).
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