why do so many stimuli induce tyrosine phosphorylation of fak?

Why do so many stimuli inducetyrosine phosphorylationof FAK?Jose Luis Rodrıguez-Fernandez

Summary

Engagement of integrins and other adhesion receptors can induce tyrosinephosphorylation of focal adhesion kinase (FAK), a tyrosine kinase present in focaladhesions. Furthermore, in addition to adhesion receptors, a surprising variety ofstimuli, acting either on specific surface receptors or on intracellular molecules,such as PKC or Rho, can induce also tyrosine phosphorylation of FAK. I suggestthat a potential mechanism by which such distinct factors may modulate thetyrosine phosphorylation of FAK is the promotion of integrin or other adhesionreceptor clustering at focal adhesions. BioEssays 21:1069–1075, 1999.r 1999 John Wiley & Sons, Inc.

IntroductionAdhesion of cells to the extracellular matrix (ECM) or to othercells plays a fundamental role in the regulation of cellularmorphology, differentiation and migration.(1) Cell adhesion ismediated by specific cell adhesion receptors responsible ofthe binding to ECM molecules or to counter-receptors onother cells. Cell adhesion receptors can be classified into sixmajor groups: the immunoglobulin gene superfamily, cad-herin, selectins, the mucins (selectin ligands), the CD44family and the integrins.(1) In this review, I will discuss mainlyintegrin receptors because their signalling function has beenstudied more extensively.

Integrin receptors mediate cell attachment to proteins ofthe ECM as well as cell–cell interactions.(2) The integrins thatfunction in cell-substrate adhesion are often localised tospecialised structures on the ventral surface of the cells,termed focal adhesions or focal contacts, where they linkECM proteins with intracellular structures such as compo-nents of the cytoskeleton.(3) Integrins are composed of twomembrane-spanning polypeptides, termed a and b subunits,which are noncovalently associated. The particular a/b pair-ing specifies the ligand binding ability of the integrin het-erodimer.(1,2) The cytoplasmic tails of b integrins are respon-sible of the localization of integrins to focal contacts since

truncation of the b subunits cytoplasmic domain impairsstable cell adhesion and integrin recruitment to these areas.(1)

In addition to mediating adhesive interactions, integrinscan also transmit signals into cells.(4–6) In particular, tyrosinephosphorylation of cellular components has emerged as apotential transducer of integrin-generated signalling. Thecytoplasmic tails of both a and b subunits are responsible forthe tyrosine phosphorylation signalling capabilities of inte-grins. In this regard, b subunit cytoplasmic tails coupled to thetransmembrane and extracellular domain of the IL-2 receptor,not only target these chimeric receptors to the focal contacts,but also stimulate tyrosine phosphorylation.(7) a subunits caninduce tyrosine phosphorylation through the tyrosine kinaseFyn;(8) however, this signalling originates from structures thatdo not contain FAK, the tyrosine kinase considered in thisreview (see below).

Since integrins lack tyrosine kinase activity, the discoveryof the focal adhesion kinase (FAK), a tyrosine kinase presentin focal contacts was of great interest(9–12) (Table 1). Knockoutmice that lack FAK or the ECM protein fibronectin display avery similar phenotype highlighting the role of FAK in transduc-ing signals from integrins. FAK is a structurally distinctnon-receptor protein tyrosine kinase with a centrally locatedcatalytic domain, flanked by amino- and carboxy-terminusnon-catalytic domains of approximately 400 residues.(9–12)

Localisation of FAK in focal contacts depends on the ‘‘focaladhesion targeting’’ (FAT) domain, a 140 amino acid se-quence in the carboxyl termini of FAK, which is necessary andsufficient for targeting this kinase to these structures. FAKdoes not contain SH2 or SH3 domains, but possesses

Departamento de Bioquımica y Biologıa Molecular III, Facultad deMedicina, Universidad Complutense, 28040 Madrid, Spain. E-mail:[email protected]

Hypothesis

BioEssays 21:1069–1075, r 1999 John Wiley & Sons, Inc. BioEssays 21.12 1069

several sites for binding of SH2/SH3 containing signallingproteins. Furthermore, recently it has been reported that theN-termini of FAK contains a band 4.1 domain,(13) which is ableto bind to the cytoplasmic region of transmembrane pro-teins.(13)

Phosphorylated tyrosine residues of FAK play an impor-tant role in the functions of this kinase. Activation of FAKinvolves autophosphorylation at Tyr 397. The motif surround-ing this site is recognized by the SH2 domain of Src-familytyrosine kinase members. Src members can, in turn, phos-phorylate additional tyrosine residues in FAK, which createsbinding sites for signalling proteins such as Grb2. Othersignalling molecules can also bind proline-rich regions pres-ent in FAK through SH3 domains. As a result of theseinteractions, a multicomponent signalling complex is as-sembled that can involve FAK in a variety of important signaltransduction pathways.(9–12) Different studies have suggestedthat signalling through FAK may play an important role in theregulation of several cellular functions, such as cell spreadingand migration, as well as in the control of apoptosis and thecell cycle.(12,14–16) Furthermore, these studies also show thattyrosine phosphorylation of FAK is important in the activity ofthis kinase.(9–12,14–16)

With regard to integrin signalling, expression of b subunittail chimeras is sufficient to induce tyrosine phosphorylationof FAK.(1) Furthermore, peptides that mimic the b integrincytoplasmic domain interacts directly with the N-terminal

portion of FAK in vitro.(10) Truncation or mutation of the b

cytoplasmic domain of integrin receptors may inhibit both thelocalization of this subunit to focal contacts and the phosphor-ylation of FAK.(1,9) In contrast, the a-subunit cytoplasmicdomain of integrins has little effect on the phosphorylation ofFAK.(1)

Integrin-dependent tyrosine phosphorylationof FAK correlates with the location of this kinaseto focal contact or focal contact-like structuresFAK molecules localize, and can be phosphorylated, in focalcontacts.(6,9–12) Furthermore, the overexpression of the FAKrelated non-tyrosine kinase (FRNK), a non-catalytic carboxy-terminus domain of FAK that prevents the localization of FAKto focal adhesion sites, also reduces FAK tyrosine phosphor-ylation.(17) Localization to focal contacts is probably alsorequired for tyrosine phosphorylation of the proline-rich tyro-sine kinase-2 (PYK-2), also termed related adhesion focaltyrosine kinase, (RAFTK), cell adhesion kinase b (CAKb),calcium-dependent tyrosine kinase (CADTK) or FAK2, a newmember of the FAK family of tyrosine kinases present inneural, epithelial and hematopoietic cells.(12). For example,PYK-2 displays b1 integrin-dependent phosphorylation and islocalised in focal contacts in natural killer cells, B lympho-cytes, megakaryocytes, and transfected COS cells.(18–20)

PYK-2 is not tyrosine phosphorylated, however, upon adhe-sion of transfected 3Y1 fibroblasts on fibronectin or integrin

TABLE 1. Summary of Integrin Ligands and Receptors That Induce Tyrosine Phosphorylation of FAK

LigandAb

cross-linkingIntegrinreceptor Cell type Ref.

Fibronectina KB, REF52, CHO, Balbc/3T3, NK cellsNIH 3T3, T cells, Human foreskin fibroblasts (24, 25, 28, 50, 56, 76,78)

1 a4b1, a5b1 NK cells (28)1 a4b1, a5b1, a6b1 B cells (29)1 a5b1 NIH 3T3, Human foreskin fibroblasts (24, 25, 78)1b b1 Transfected human foreskin fibroblasts (7)

Collagen I a2b1 Platelets (73, 74)Collagen Ia KB, REF52, Balbc/3T3 fibroblasts (24, 76)Collagen IVa KB cells (24)

1 a3b1 KB cells (24)ICAM-1 aLb2 T cells (80)

1b b3 Transfected human foreskin fibroblasts (7)Fibrinogen aIIbb3 Platelets (73, 74)

1 aIIbb3 Transfected CHO cells (26)Vitronectin aVb3 Transfected (b3) CS-1 cells (75)

1 aVb3 Bovine pulmonary artery endothelial cells (77)1b b5 Transfected human foreskin fibroblasts (7)

Vitronectin aVb5 Transfected (b5) CS-1 cells (75)Vitronectina REF52, KB, bovine pulmonary artery endothelial cells (24, 76, 77)Laminina REF 52 and KB cells (24, 77)

1 a4b7 B cells (29)

aThe exact integrin receptor/s engaged by the ligand that induce FAK phosphorylation was not specific in the reference.bTo crosslink the integrin, antibodies were directed against the interleukin-2 (IL-2) extracellular domain of the chimeric IL-2-integrin molecule.

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1070 BioEssays 21.12

ligation during platelet aggregation.(21,22) Furthermore, whena hybrid molecule that possesses the carboxy terminus ofFAK and the amino terminus of PYK-2 is expressed in cells inwhich PYK-2 is not normally localised to focal contacts, thenthe hybrid molecule localises to these structures and isphosphorylated.(23) These data suggest that FAK and othermembers of this family of kinases need to localize in focalcontacts in order to be tyrosine phosphorylated.

Integrin clustering at focal contacts inducestyrosine phosphorylation of FAKThe use of antibodies to artificially induce clustering ofspecific integrins has demonstrated that cross-linking ofdifferent members of the b1, b3, b5, and b7 induces tyrosinephosphorylation of FAK (Table 1). More recently, the use ofmembrane-permeable dimerizers to cluster transfected inte-grin constructs that encode tandem repeats of binding pro-teins intracellularly has demonstrated that clustering of aIIb3integrin is sufficient to stimulate fibrinogen-dependent FAKtyrosine phosphorylation.(30) The conclusion drawn from thesestudies is that integrin clustering is necessary, and oftensufficient, to induce tyrosine phosphorylation of FAK. It isimportant to remark also that, when cells are plated onintegrin specific ligands (Table 1), integrin receptor clusteringalso takes place that may lead to FAK tyrosine phosphoryla-tion. Indeed, unbound integrins are freely diffusive in themembrane plane, but occupancy of the integrin by its ligandcauses clustering of these receptors.(31) Since integrin cluster-ing normally occurs at focal contacts in cultured cells(3) andFAK co-localises with clustered integrins, even when theligand-binding site of the integrin is not occupied,(25) then thismay explain the high correlation between the presence ofFAK in focal contacts and its tyrosine phosphorylation atthese sites.(3) Furthermore, this interpretation that considersthat FAK should be present in focal contacts to be phosphory-lated, although it lacks a direct role in focal contact formation,it is consistent with evidence obtained from the analysis ofFAK knockout cells as well as for experimental approachesthat indicate that FAK is not necessary for focal adhesionformation.(32)

Although integrin signalling has been studied most exten-sively, it is also known that FAK tyrosine phosphorylation canbe induced by engagement of other adhesion receptors,including immunoglobulin,(33) selectin,(34) and CD44(35) adhe-sion receptors. Interestingly, artificial cross-linking of some ofthese non-integrin adhesion membrane receptors also in-duces FAK phosphorylation,(33) suggesting that clusteringmay be a general mechanism of signalling by surface recep-tors.

Although it is clear that clustering of integrin and otheradhesion receptors induces FAK phosphorylation, the mech-anism of this effect is not completely understood. Probably,the simplest mechanism to explain this process is offered by

the transphosphorylation model that has been proposed tounderstand the activation of receptor tyrosine kinases.(36) Inthe case of integrin receptors, this model should take intoconsideration the observed requirement of the b integrinsubunit in the phosphorylation of FAK. The model suggeststhat the aggregation of FAK molecules, which are eitherdirectly associated with the b integrin cytoplasmic do-main(10,13) or indirectly associated with the b integrin throughother intermediate molecules,(23) would favour the transphos-phorylation and activation of FAK in focal contacts.(4) A similarmechanism of FAK activation could operate in the case ofother non-integrin adhesion receptors.(10)

One thousand and one stimuli induce tyrosinephosphorylation of FAKBesides adhesion receptors, other surface receptors, notdirectly involved in adhesive phenomena, also induce tyro-sine phosphorylation of FAK. These include neuropep-tide,(37–42) catecholamine,(43) tyrosine kinase,(44–46) chemo-kines(47) and other cytokines,(48) immunoglobulin,(49–51) bioac-tive lipid,(41,52,53) neurotransmitter,(54) and cannabinoid recep-tors.(55) Other stimuli that induce phosphorylation of FAKinclude treatment of the cells with phorbol esters,(38,46,56)

neurotoxins,(57) bioactive lipids acting intracellularly,58) subjec-tion of cells to fluid shear stress,(59.60) neuronal depolariza-tion,(54) and glucose stimulation.(61) Cell transfection withpp60src, the protein encoded by the viral oncogene v-src alsoinduces FAK phosphorylation.(62) This is a special case,however, because FAK is a good substrate for Src familymembers and is phosphorylated directly by pp60src.(9–12) Aquestion that immediately arises when examining the hetero-geneity of stimuli that induce phosphorylation of FAK is whatdo these stimuli have in common? A possible answer to thisquestion might be obtained from the study of the effect of thesmall GTP-binding protein Rho.

Enter Rho: a regulator of focal contact formationThe GTP-binding protein Rho is active when bound to GTP.The intrinsic GTPase activity of Rho, however, hydrolyses thebound GTP rendering Rho in the inactive, Rho-GDP bound,form. C3 exotransferase of Clostridium botulinum, which ADPribosylates and inactivates Rho, has proved useful in study-ing the function of Rho. Furthermore, endogenous Rho is alsoinhibited by expressing the dominant negative [N19]Rho, amutated form that results in a conformation of Rho in whichbinding to GDP is favoured. Conversely, [V14]Rho, a mutatedform with decreased GTPase activity, has been employed tostudy the effect of activated Rho.

Studies carried out mainly in Swiss 3T3 cells, which uponstarvation lose stress fibres and focal contacts, show thatmicroinjection of active [V14]Rho reforms focal contacts.(63) Incontrast, the use of C3 exotransferase or the overexpressionof the dominant negative [N19]Rho blocks integrin complex

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BioEssays 21.12 1071

assembly and focal contact formation.(63,64) These resultsdemonstrate that Rho is a major regulator of focal adhesions.Interestingly, in this and other systems, the addition ofdifferent growth factors, including the neuropeptides bombe-sin and endothelin,(37) cholecystokinin,(39) neuromedin B,(40)

and thrombin;(42,65) bioactive lipids, such as lysophosphatidicacid (LPA),(52) sphingosylphosphorylcholine (SPC),(53) andsphingosine 1-phosphate,(58) and bacterial neurotoxins(57)

also cause reformation of focal contacts and tyrosine phos-phorylation of FAK. Furthermore, the use of the C3 exoen-zyme, to block the effects of these and other stimuli on bothfocal contact formation and FAK phosphorylation, demon-strate that these two effects are dependent on Rho activ-ity.(37,39,40,42,52,53,57,58) Since, as pointed out above, FAK is notnecessary for focal contact formation,(32) it is likely that Rhofirst stimulates focal contact formation before FAK is phos-phorylated.(6)

The adhesion receptor connection:a hypothesis to explain FAK phosphorylationfrom different receptorsImportantly, one of the effects of activated Rho is thestimulation of integrin clustering at focal contacts.(66) Since,as discussed previously, integrin clustering induces FAKphosphorylation, this suggests that stimuli that activate Rho,either stimulating surface receptors (like neuropeptide recep-tors) or acting intracellularly (like bacterial toxins), inducephosphorylation of FAK by inducing integrin clustering.Conversely, the blocking of growth factor and otherstimuli(37,39,40,42,52,53,57,58) or integrin-mediated(67) phosphoryla-tion of FAK, by inhibition of Rho activity, could be explainedby considering that inactive Rho cannot induce integrinclustering.

This model for integrins and Rho can be extended to otheradhesion receptors and other molecular intermediates. Infact, it has been shown that Rho can induce clustering notonly of integrins, but of other adhesion receptors, such asselectins.(81) I hypothesise that different growth factors mayuse either Rho or other molecular intermediates to induceFAK phosphorylation. I suggest that, ultimately, however,these putative intermediates should necessarily promoteadhesion receptor clustering to stimulate phosphorylation ofFAK. I postulate that, in unstimulated cells, adhesion recep-tors are diffuse or unorganised on the cell surface, but thateither upon the direct ligation of adhesion receptors by theirligands, or upon the exposure of cells to different stimuli thatinduce FAK phosphorylation, a subsequent clustering ofintegrins or other adhesion receptors would take place thatwould be responsible for the induction of FAK phosphoryla-tion (Fig. 1). The exact nature of the clustered receptor,integrin receptor or other adhesion receptor would depend onthe cell type and the cellular context, such as the specificECM ligand that the cell encounters at any particular moment.

Several candidates can be considered with regard to thepossible molecular intermediates that may induce adhesionreceptor clustering. It has been suggested that Rho caninduce myosin light chain phosphorylation, which results inthe promotion of interaction of myosin heads with actinfilaments, generating contractility and bundling of actin fila-ments.(79) At the membrane, the bundled actin filamentswould aggregate the integrins to which they are attached.(79)

Therefore, at least in the case of integrins, the myosin-actinsystem could be a potential intermediate that could induceclustering and FAK phosphorylation.(79) In the case of otheradhesion receptors which are different to integrins, Rho couldmediate clustering through a mechanism that does notinvolve myosin light chain phosphorylation.(81) Another mol-ecule which may have a functional role in inducing integrinclustering is PKC. Phorbol dibutyrate (PDB) and other phor-bol esters, as well as other growth factors that activate PKC,are able to stimulate tyrosine phosphorylation of FAK in anumber of cell types.(38,46,54,56) Furthermore, in the case ofChinese hamster ovary cells plated on fibronectin, activationof PKC precedes FAK phosphorylation.(56) Since PKC mayinduce integrin clustering,(68) it is possible that a potentialmechanism by which PKC may induce FAK phosphorylationis the promotion of integrin clustering.

The model outlined in Figure 1 takes account of the factthat adhesion receptor clustering may be induced in twodifferent ways, either directly by binding of the ligands to thereceptor or indirectly by stimulation with other stimuli. In thismodel, it is assumed that the contribution to FAK phosphory-lation from the direct engagement of the ligand or from theindirect clustering induced by growth factors or other stimulialso depends on the cell type. In this regard, it is postulatedthat, in some cases, additional clustering induced by growthfactor receptors, may further enhance the phosphorylation ofFAK stimulated by the direct clustering induced by adhesionreceptor ligands. One such example might be the additionalphosphorylation of FAK that is observed in T cells plated onthe b1 ligand fibronectin, following engagement of the T cellreceptor.(50) In other cases, however, it is also possible that,following ligand binding, adhesion receptors are clustered insuch a way that FAK reaches a level of phosphorylation thatcannot be increased further by other stimuli. One possibleexample of this might be the effect of growth factors on FAKphosphorylation in adherent and starved rat aortic smoothmuscle cells. These cells showed near maximum levels ofFAK phosphorylation, which does not increase further upontreatment with different growth factors.(23)

The model that I propose predicts that disturbing adhesionreceptor clustering should reduce the increase in FAK phos-phorylation that is induced by adhesion receptor engagementor by growth factor stimulation. It is possible that this ef-fect might explain the inhibitory effect of cytochalasin Dpre-treatment(27,29,38–42,46,73) or the maintaining of normal cells

Hypothesis


in suspension(38,41,49,69) on growth factor or integrin mediatedstimulation of FAK. By disrupting pre-formed integrin cluster-ing in focal contacts (cytochalasin D) or preventing focalcontact formation (cell suspension), both treatments maysimilarly inhibit FAK phosphorylation.

This model may help to explain the link observed betweengrowth factor and integrin signalling pathways. In this regard,muscarinic receptor mediated phosphorylation of FAK isprevented by treating the cells with soluble peptides contain-ing the Arginyl-glycyl-aspartic acid (RGD) motif, a sequencethat inhibits the binding of the a5b1 or avb3 integrins to theirligands.(70) It is possible that, in this case, the RGD containingpeptide blocks engagement and subsequent clustering ofintegrins and FAK phosphorylation. Blocking ligand occu-pancy of the avb3 integrin also inhibits insulin-like growthfactor 1 signalling in vascular smooth muscle cells.(71) Further-more, antibodies against a1b1 and a2b1 integrins selectivelyinhibited angiogenesis induced by the cytokine, vascularendothelial growth factor.(72) Interestingly, avb3, a1b1, a2b1,I-IGI, and VEGF can induce FAK phosphorylation(45,46)

(Table 1). The possibility that both integrin and growth factorsconverge in inducing FAK phosphorylation by promotingintegrin clustering deserves to be examined.

ConclusionsReceptor clustering seems to be an important mechanism bywhich cells are able to transmit signals (see Hubbard et al.(36)

and references cited therein). It has been suggested that theactivation of tyrosine kinases observed upon oligomerizationof a variety of receptors depends on the proximity of kinasemolecules associated with the cytoplasmic domain of thereceptor.(36) Receptor oligomerization would be a convenientway for starting signals from the cell surface that could laterbe modulated by downstream molecules. FAK could be amolecular candidate to transmit these signals from adhesionreceptors.

I suggest that adhesion receptor clustering may be acommon mechanism by which distinct factors may modulatetyrosine phosphorylation of FAK. The hypothesis that I pre-sent in this essay can be tested by using specific antibodies toanalyse the effect of different stimuli that induce FAK phosphor-ylation on the formation of clusters of adhesion receptors onthe membrane. Further possible experimental approachesinclude a combination of reconstitution and transfectionexperiments in which integrin and other adhesion receptorsare co-transfected with other non-adhesion receptors in asuitable cell type in order to study the effect of a variety of

Figure 1. A model to illustrate how growth factor receptors and other stimuli may induce tyrosine phosphorylation of FAK.Neuropeptide receptors, other receptors and neurotoxins, acting through the small GTPase Rho or other putative molecules (X), caninduce adhesion receptor clustering and FAK phosphorylation. Neuropeptide, bioactive lipids and neurotoxin induced FAKphosphorylation can be blocked either by treating the cells with the C3 exoenzyme, that inhibits Rho, or by directly inhibiting integrinclustering with cytochalasin D (Cyt D) or preventing attachment to the substrate by keeping the cells in suspension. PDB treatment,antibody crosslinking or ligand binding can also induce adhesion receptor clustering. For the sake of simplicity, it is assumed thatadhesion receptors are in active conformation (that is, they are able to bind ligand).

Hypothesis


factors on integrins or other adhesion receptor aggregationand FAK phosphorylation.

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Hypothesis


why do so many stimuli induce tyrosine phosphorylation of fak?

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