Expression of the capacity of integrins to mediate cellular adhesion has several distinct facets: (a) the conformational state of integrins, affecting the affinity of ligand binding; (b) ligand-induced integrin clustering, enhancing the strength of attachment and promoting interactions between kinases and their substrates important to signal transduction; and (c) signaling itself, activating a cascade of events leading to organization of the cytoskeleton and cell spreading (Schwartz et al., 1995
; Burridge and Chrzanowska-Wodnicka, 1996
; Parsons, 1996
). Numerous in vitro studies and recent evaluations of mice with targeted deletions in nonreceptor tyrosine kinases implicate FAK and Src family kinases as key mediators of integrin signaling (Furata et al., 1995; Ilic et al., 1997
; Meng and Lowell, 1998
). Thus, the molecular events leading to assembly and activation of these kinases surrounding integrins are important determinants of integrin function. This conclusion has focused much attention on nonintegrin membrane proteins which might regulate this process and our data identify one such protein as caveolin. While caveolin does not affect integrin expression or the intrinsic capacity of β1 integrins to bind fibronectin (Figs. and ), caveolin is required for the normal assembly of adhesion plaques which develop in response to ligand engagement and integrin clustering. In the absence of sufficient β1 integrin–associated caveolin, there is loss of integrin-associated Src kinase activity, little or no FAK activation after ligand binding, and impaired association of integrins with structural proteins such as tyrosine-phosphorylated cortactin important to β1 integrin– dependent adhesion (Fig. ). As a result, the characteristic accumulation of enzymes and structural proteins which comprise integrin-dependent adhesion sites fails to develop (Fig. ).
Our results confirm and extend the recent findings of Wary and colleagues (1998) that caveolin expression is required for the association of Fyn kinase with β1 integrins and ligand-dependent Shc phosphorylation. These investigators began with cells (Fisher rat thyroid cells) expressing little or no caveolin or α5 integrin and transfected both proteins. They found that physical association of the Src family kinase Fyn with α5/β1 required concurrent coexpression of caveolin-1. Fyn but not Src was required for α5/β1-dependent Shc phosphorylation. Beginning with cells having functional β1 integrins and expressing caveolin-1, we suppressed caveolin expression and found β1 integrin association with several Src family kinases to be completely dependent on caveolin. In both cases, deficient β1 integrin–associated Src family kinase activity led to correspondingly marked changes in integrin function. Together, these findings indicate caveolin is a general regulator of β1 integrin function.
How does caveolin promote integrin signaling? Although our understanding is incomplete, we favor a model (Fig. ) in which caveolin functions primarily to sequester Src family kinases in an inactive configuration at sites proximate to integrins, promoting their presentation to integrins and activation during ligand-induced integrin clustering. Caveolin has been shown previously to interact predominantly with the inactive form of Src and to reduce Src activity when overexpressed in 293 cells, perhaps by sequestering Src kinases from activating phosphatases and substrates (Li et al., 1996b
; Rodgers and Rose, 1996
). Consistent with this model, caveolin-deficient 293 cells were found to have two- to fourfold higher levels of basal c-Src kinase activity (not shown) and to exhibit striking hyperphosphorylation of a major Src substrate, cortactin (Fig. ). If one function of caveolin is to suppress basal Src kinase activity, the model in Fig. also offers a possible molecular explanation of how caveolin contributes to Src family kinase activation as a consequence of integrin clustering. Caveolin interactions with Src family kinases are thought to occur through the same membrane proximal sites on caveolin as those involved in formation of caveolin homo-oligomers (Okamoto et al., 1998
). Because coclustering of caveolin with integrins (Fig. ) could be expected to favor caveolin homo-oligomer formation, integrin clustering is likely to modify the physical interaction of caveolin with Src kinases. This may lead to outright release of these kinases from caveolin (as shown in Fig. ) and/or an altered, more accessible position of these kinases in the cluster favoring their activation through contact with substrate or through a phosphatase. In either case, these studies directly implicate caveolin function in the membrane proximate events of ligand-induced, β1 integrin–dependent tyrosine kinase activation. As caveolin levels are reportedly low in a number of transformed cells (Koleske et al., 1995
; Okamoto et al., 1998
), our data raise the possibility that low caveolin levels contribute to the defective fibronectin adhesion and matrix assembly seen in many cancer cells and thought to be important to their metastatic potential (Hynes, 1992
; Schwartz, 1997
Figure 7 Model for regulation of β1 integrin signaling by caveolin. The model indicates that caveolin binds and maintains Src family kinases in an inactive configuration (dark shade) proximate to a fraction of β1 integrins (complex is encircled). (more ...)
A further implication of the studies reported here and the recent studies of Wary and colleagues (1998) is that Fyn and Src may have overlapping but distinct roles in β1 integrin signaling: Fyn being primarily required for Shc phosphorylation and activation of an MAPK-dependent growth pathway and Src being primarily required for FAK and cytoskeletal phosphorylation and assembly of focal adhesion sites. This concept is supported by results of prior studies examining mice deficient in the negative regulator of Src family kinase activity, Csk (Thomas et al., 1995
). Fibroblasts from Csk− embryos exhibit hyperphosphorylation of adhesion sites and striking hyperphosphorylation of cortactin, reminiscent of that seen in caveolin-deficient cells (Fig. ). The hyperphosphorylation of adhesion sites and cortactin was largely corrected by crossing the Csk− mice with Src− mice but not by crossing with Fyn− mice, again implicating Src as primarily being involved in regulation of integrin-dependent adhesion. Clearly this is an oversimplification since we also observe Yes as well as Fyn and Src in β1 integrin/caveolin complexes and even kinase-inactive Src can promote focal contact assembly in Src− cells (Kaplan et al., 1995
). Still, these recent studies support the possibility that different members of the src
gene family have favored roles in integrin signaling even in cells expressing multiple family members.
The structural basis for the association between caveolin and β1 integrins is uncertain. First, most if not all cellular caveolin is reportedly found in purified caveolae (Lisanti et al., 1994
; Schnitzer et al., 1995
). Although integrins have not been observed in purified caveolae, when we subject 293 cells to mechanical lysis, sonicate, and centrifuge the postnuclear supernatant through a sucrose gradient, caveolin in 293 cells appears at the 5/35% sucrose interface, typical for that reported for caveolae preparations in 293 and other cells (Song et al., 1996
; Ikezu et al., 1998
). Almost all of the β1 integrins appear in the fractions containing caveolin (Yang, X., unpublished observation). These findings are consistent with the scenario that β1 integrin/ caveolin/Src family kinase complexes (encircled in Fig. ) exist as signaling units in caveolae and separate from caveolae in response to ligand-induced clustering and cytoskeletal reorganization. However, the exact composition of caveolae remains controversial and appears strongly dependent on the method of purification (Lisanti et al., 1994
; Schnitzer et al., 1995
; Mineo et al., 1996
; Stan et al., 1998
). An alternative possibility is that some fraction of caveolin traffics outside caveolae, as has been argued by Wary and colleagues (1998). This will be an important issue for future experiments. In either case, the fraction of β1 integrins which colocalize and cocluster with caveolin (Fig. ) appears to be critical to β1 integrin signaling.
Whether β1 integrins directly bind caveolin is also uncertain. Wary and colleagues (1998) reported that the α5 transmembrane domain was required for coprecipitation of α5/β1 and caveolin. However, no direct binding has been demonstrated. Most if not all other proteins that are reported to bind caveolin bind sequences of caveolin that are membrane proximal but in the cytoplasmic compartment (Okamoto et al., 1998
). Indeed, in SMC we observed a peptide comprised of a membrane proximal region of caveolin (amino acids 82–101) blocking coprecipitation of caveolin with β1 integrins (Fig. ). Thus, it is uncertain whether β1 integrins and caveolin directly bind or associate indirectly through a common affinity for certain membrane lipids or a third protein such as uPAR which may directly interact both with integrins and cholesterol-rich lipid domains enriched in caveolin. The physical basis for the association of β1 integrins and caveolin also requires further investigation.
The marked inhibition of β1 integrin function by uPAR-binding peptides in human SMC (Fig. ) is remarkable. Although we could detect uPAR in SMC by immunostaining and could cocluster uPAR along with β1 integrins, no uPAR was detected in the β1 immunoprecipitations of SMC. This suggests the association of uPAR with β1 integrins and caveolin in these cells is relatively weak. There also appears to be a stoichiometric excess of β1 integrins compared with uPAR in SMC. Thus, it is surprising that the coprecipitation of most caveolin- and integrin-associated Src kinases with β1 integrins would be blocked by peptide 25, raising the possibility of an in vitro artifact. This is an unlikely explanation for the findings because the uPAR-binding peptide (peptide 25), but not two control peptides (peptide 36 and a scrambled version of peptide 25), also had clear biochemical and functional effects on intact SMC. Prior treatment of SMC with the active peptide blocked Src kinase association with β1 integrins and inhibited both signaling and migration of the cells on fibronectin (Fig. ). These results indicate that, although not intrinsically required for the function of integrin/caveolin (Fig. ), the presence of uPAR organizes caveolin and its associated signaling molecules in an interdependent manner such that integrins, caveolin, and uPAR form a unit promoting integrin function. Perhaps this is possible because only a fraction of β1 integrins associates with caveolin (Fig. ) and uPAR associates mainly with activated integrins (Wei et al., 1996
). As indicated in Fig. , integrin activation initiated by ligand-induced integrin clustering may promote formation of these signaling complexes. Similar mechanisms may account for observations that uPAR is required for normal function of the β2 integrins, CD11/ CD18 (Mac-1; Simon et al., 1996
; Sitrin et al., 1996
; May et al., 1998
), and for migration on vitronectin mediated by αvβ5 (Yebra et al., 1996
), suggesting a promoting effect of uPAR on integrin function may be a widespread phenomenon.