Cell adhesion to the extracellular matrix triggers a rapid and dynamic assembly of multiprotein complexes that is driven by integrins. The protein complexes serve as a platform for coupling integrins to the actin polymerization and assembly machinery. The earliest identified multiprotein complexes, referred to as “focal complexes” contain integrin(s), talin, paxillin, α-actinin, and low levels of vinculin and FAK (Zaidel-Bar et al., 2003
). The focal complexes are also rich in tyrosine phosphorylated proteins, indicating that kinases are among the first proteins to be recruited and/or activated at these sites. In platelets, the αIIb
-integrin receptor constitutively interacts with Src kinases; the kinases are activated as a consequence of αIIb
-activation and ligation (Kralisz and Cierniewski, 1998
; Obergfell et al., 2002
; Arias-Salgado et al., 2003
). Platelets pretreated in vitro with an inhibitor of Src kinases, PP2, and murine platelets harvested from Src family kinase-deficient mice, failed to spread on fibrinogen, the primary integrin αIIb
extracellular matrix ligand (Obergfell et al., 2002
). Similarly, triple Src-, Yes-, and Fyn-kinase–deficient fibroblasts exhibited impaired cell migration and spreading on fibronectin (Klinghoffer et al., 1999
; Cary et al., 2002
). These findings established that Src kinases are key, early components, in the signal transduction pathway(s) from integrins to the cytoskeleton. The tyrosine kinase Syk and its downstream effector substrates Vav1, Vav3, and SLAP-130 were implicated as one signal transduction pathway linking αIIb
and Src kinases to the cytoskeleton (Obergfell et al., 2002
). Our results indicating that vinculin is tyrosine phosphorylated by Src kinases in platelets, and in reconstituted COS-7 cells, open the door to the possibility that Src kinases also may use vinculin as a vehicle to relay integrin-dependent signals to the actin cytoskeleton.
The data reported in this study demonstrate that endogenous vinculin is phosphorylated on tyrosine residue 1065 in spread platelets. The phosphorylation was not detected in unstimulated platelets or in platelets treated with the inhibitor of Src kinases, PP2. The phosphorylation of vinculin on tyrosine residue 1065 was reconstituted in vanadate-treated cells cotransfected with wild-type vinculin and a constitutively active c-Src kinase. Tyrosine residue 1065 was also phosphorylated by c-Src in vitro, thus establishing that this residue is a bona fide Src kinases substrate. Using the reconstituted COS-7 system, we identified a second phosphorylation site on residue 100. Unlike residue 1065, residue 100 was not phosphorylated by c-Src in vitro, raising the possibility that the phosphorylation of vinculin may be regulated by two, or more, distinct kinases. Importantly, we also found that although the single point mutant proteins (Y100F and Y1065F) were as effective as wild vinculin in rescuing the spreading defect of vinculin –/– cells on fibronectin, the double mutant protein (Y100F/Y1065F) was significantly less effective than wild-type vinculin, or the single point mutants. Furthermore, a second double mutant protein carrying negative charges in place of the tyrosine residues in position 100 and 1065 (Y100E/Y1065E) was significantly more effective than wild-type vinculin in rescuing the spreading defect of vinculin –/– cells on fibronectin. It is therefore possible that the activity of vinculin is optimal only when both sites are phosphorylated.
Biochemical and crystal structure data demonstrated that vinculin exists in closed and open conformations. Vinculin is held in an inactive, closed conformation by intramolecular interactions between its head and tail domains (Johnson and Craig, 1994
; Bakolitsa et al
; Borgon et al., 2004
; Izard et al., 2004
). The actin binding site in vinculin is masked when vinculin is in its closed conformation (Johnson and Craig, 1995
). Binding of acidic phospholipids to the vinculin tail domain affects head–tail interactions and converts vinculin to an active, ligand-binding competent form (Gilmore and Burridge, 1996
; Weekes et al., 1996
). Crystal structures recently resolved by Izard et al
) further revealed that binding of talin derived peptides to the vinculin head domain induces a marked conformation change in the vinculin head domain, resulting in tail displacement. Binding of α-actinin to vinculin similarly affected vinculin's head–tail interaction (Izard et al., 2004
). These observations established that the conversion of vinculin from a closed to an open conformation is regulated by several, alternative mechanisms. Based on the observation that the phosphorylation of vinculin on tyrosine residue 1065 reduced head–tail interaction in vitro, we speculated that the phosphorylation of vinculin by Src kinases also could positively regulate the activation state of vinculin. In at least one analogous situation, the valosin-containing protein (VCP) was found to be phosphorylated on tyrosine residue 805, the penultimate residue in the protein (Egerton et al., 1992
). The phosphorylation of VCP on residue 805 disrupted intramolecular head–tail interactions, which in turn exposed a nucleartargeting sequence in the VCP head domain (Madeo et al., 1998
). Similarly, the phosphorylation of ezrin on a threonine residue caused conformational changes that unmasked both membrane and actin binding sites in the protein (Fievet et al., 2004
). Here, we show that phosphorylated vinculin did not cosediment with polymerized actin, suggesting that the actin binding site in vinculin remained masked despite the phosphorylation. It thus seems that the primary function of the phosphorylation of vinculin on serine residues 1033 and 1045 (Ziegler et al., 2002
) or tyrosine residues 100 and 1065 is not to modulate the interaction between vinculin and actin. The change(s) in conformation brought about by the phosphorylation might impact the interaction of vinculin with other proteins, and/or determines how long vinculin is in an open conformation. The recent study by Subauste et al
) suggesting that the vinculin tail domain could modulate the interaction between paxillin and FAK highlights one mechanism by which a change in the tail conformation may affect cellular responses.
Vinculin is a well-established constituent of focal adhesion plaques. Recent studies revealed that the localization of some focal adhesion components is regulated by their state of phosphorylation. Interestingly, FAK and α-actinin, two of the proteins thought to directly interact with integrins (Otey et al., 1990
; Schaller et al., 1995
), were excluded from focal adhesion plaques when phosphorylated on tyrosine residues (Katz et al., 2003
; von Wichert et al., 2003
). These observations raised the possibility that the density of proteins such as FAK and α-actinin within the plaque is regulated. We have no evidence at the present time that the localization of vinculin to focal adhesion plaques is similarly affected by its state of phosphorylation; in fact, two lines of evidence argue against this possibility. First, we found that the vinculin double mutant Y100F/Y1065F and Y100E/Y1065E (unpublished data) localized to focal adhesion plaques. In addition, Volberg et al.
) reported that vinculin localized to focal adhesion plaques in Src, Yes, and Fyn triple null cells. These findings, however, do not exclude the possibility that the phosphorylation of vinculin by Src kinases affects the dynamics of its recruitment and/or residency time within the early focal complexes, where it may play an important role in regulating the assembly of actin filaments, possibly through an Arp 2/3-dependent mechanism (DeMali et al., 2002
Ziegler et al
) proposed that the phosphorylation of vinculin by protein kinase C may regulate the incorporation of vinculin into nascent cell adhesion complexes. In fact, it is possible that both Src and protein kinase C enzyme families regulate the dynamics of vinculin assembly into plaques. Indeed, treatment of platelets with PMA, a protein kinase C activator, triggered robust phosphorylation of vinculin, whereas treatment of the platelets with bisindolylmaleimide, an inhibitor of protein kinase C (Toullec et al., 1991
), inhibited platelet spreading on fibrinogen as well as the phosphorylation of vinculin on tyrosine 1065 (Zhang, Lin, and Haimovich, unpublished data). It is also possible that as shown in T cells, protein kinase C regulates the activity of a Src kinase family member(s) (Niu et al., 2003
). Further studies are required to determine whether platelet vinculin is phosphorylated on serine residues 1033 and 1045 and whether the phosphorylation on these sites is a prerequisite for the phosphorylation on tyrosine residue 1065.
The crystal structure of chicken vinculin tail domain (residues 879-1066), the human vinculin head (residues 1–258), and tail (residues 879-1066) domain complex, and that of intact vinculin were resolved previously (Bakolitsa et al
; Borgon et al., 2004
; Izard et al., 2004
). In the intact vinculin, residue 100 is fully exposed, whereas residue 1065 is occluded by a stretch of amino acids derived from the proline-rich region (Bakolitsa et al., 2004
). The fact that residue 1065 is buried explains why intact vinculin was not phosphorylated by c-Src in vitro (Zhang, Lin, and Haimovich, unpublished data) and is consistent with the possibility that the phosphorylation of vinculin by c-Src requires a priming input. The atomic structure of the vinculin tail domain revealed a bundle of five helices connected by short loops and packed in an antiparallel orientation with the N- and C-terminal ends emerging from the same side of the bundle (Bakolitsa et al., 1999
). Extending from the last helix is a stretch of 21 amino acids, referred to as the C-terminal arm (1045–1066). Bakoltisa et al.
(1999) identified three potential regions within the C-terminal arm: a flexible loop (1047–1052), a β clamp (1053–1061), and a hydrophobic hairpin (1062–1066). A mutant protein lacking residues 1052–1066 failed to cosediment with acidic phospholipid vesicles at a physiological pH (Bakolitsa et al., 1999
). This observation, the hydrophobic nature of the vinculin hairpin tail, and the possibility that tryptophans may orient proteins toward membranes, or perhaps play a role in the bilayer insertion process (Wallace and Janes, 1999
), led Bakolitsa et al.
) to propose that the last five amino acid residues in vinculin, Thr-ProTrp-Tyr-Gln (TPWYQ), are inserted into membranes. Whether this observation holds true or not for the intact protein is a question that needs to be addressed in more detail because Johnson et al
) have shown that native vinculin does not spontaneously associate with acidic phospholipid vesicles under physiological conditions. The phosphorylation of vinculin on residue 1065 may generate a transient conformation that is more favorable for membrane recognition than the unphosphorylated tail, particularly if, as our data suggest, this phosphorylation affects head–tail interactions. One intriguing possibility is that the phosphorylation may help unmask residues 916–970 shown to interact with, and insert into, acidic phospholipids (Johnson et al., 1998
). On the other hand, it is also possible that the phosphorylation creates a deliberate obstacle for the membrane insertion step and thus serves to regulate the membrane binding activity of vinculin, and/or the targeting of vinculin to specific sites, where it may help facilitate the assembly of actin filaments. As a follow-up to this notion, it is also possible to envision that the recruitment of vinculin to specific sites is regulated by a phosphatase that can rapidly dephosphorylate vinculin thus rendering the phosphorylation a transient event. This may explain why the phosphorylation of vinculin in COS-7 cells is not detected unless the cells are pretreated with vanadate. Such a model would also imply that the activation state of vinculin is tightly regulated by Src kinases and a counteracting phosphatase(s).
α-Actinin (Izaguirre et al., 1999
) and as shown here, vinculin, undergo robust tyrosine phosphorylation in spread platelets. The significance of these tyrosine phosphorylation events is clearly not limited to platelets (von Wichert et al., 2003
) but rather is easier to detect in platelets than in other types. Why is that the case? It seems that platelet spreading is an “all-out” process. Because platelet spreading is an irreversible process, platelets can afford to unleash robust tyrosine phosphorylation of proteins that are only transiently or sparsely phosphorylated in other cell types thus limiting their detection. Considering the large number of proteins that are phosphorylated on tyrosine in spread platelets this system should prove to be informative in efforts to dissect events regulating focal adhesion assembly.