The major finding of this study is that thrombin and angII stimulate bundling of actin filaments (stress fibers) and focal adhesion assembly in VSMCs that is mediated, in part, by c-Src. As discussed later here, changes in VSMC stress fibers are likely to be important in VSMC functions such as contraction, migration, and proliferation. Thus, understanding the mechanisms by which physiological stimuli promote cytoskeletal rearrangement in VSMCs may provide insight into pathological processes such as hypertension, atherosclerosis, and restenosis. Although focal adhesions have been recognized as dense plaques in VSMCs by electron microscopy, the effects of growth factors on cytoskeletal reorganization in VSMCs have not been well studied. The current paradigm for such cytoskeletal rearrangement is that focal adhesions are formed by activated myosin that bundles F-actin (31
). Myosin is activated by Rho kinase/ROCK/ROK, which are effectors of the small G protein Rho. These kinases regulate myosin indirectly by phosphorylating the p130 binding subunit of myosin phosphatase, thereby suppressing the phosphatase activity, and directly by phosphorylating myosin light chain (33
). In VSMCs, therefore, stress fiber formation and muscle contraction share common molecular mechanisms. Of great interest, a recent study by Uehata et al
) showed that a specific inhibitor of ROCK had a potent vasorelaxing effect in isolated vessels and lowered blood pressure when administered in vivo
to hypertensive rats. Because this inhibitor blocks actin bundling in cultured cells, it appears possible that vasoconstrictors such as angII and thrombin work, in part, by activating ROCK and thereby stimulating actin bundling and focal adhesion assembly.
In this study, we focused on tyrosine phosphorylation of focal adhesion proteins and the role of c-Src in VSMC cytoskeletal reorganization. Both thrombin and angII stimulated tyrosine phosphorylation of Cas, paxillin, and tensin. Our data indicate that c-Src is the major tyrosine kinase activated by angII and thrombin that phosphorylates these proteins in VSMCs. Cas and paxillin are thought to be important molecules in cytoskeletal reorganization by serving as linker proteins. Tensin cross-links actin filaments, possesses a barbed-end capping activity, and therefore is thought to be involved in actin assembly/disassembly (19
). Cas appears particularly important, as it binds to Src via its SH3 domain and to other signaling molecules such as Crk, Nck, FAK, and PTP-PEST via SH2-binding motifs. Cas is tyrosine-phosphorylated and localizes to focal adhesions upon integrin-mediated cell adhesion. This study shows that both thrombin and angII stimulate association of Cas with Crk. In v-Crk–transformed cells, total cellular tyrosine phosphorylation is markedly increased, and it is thought that Crk potentiates the kinase activity of Src (36
). Thus, the association between Cas and Crk induced by thrombin and angII may regulate c-Src kinase activity. Recently, Klemke et al.
) showed that Cas–Crk association serves as a molecular switch for induction of cell migration. Tyrosine phosphorylation of Cas and tensin by thrombin and angII almost completely depended on c-Src, indicating the importance of c-Src in regulating focal adhesions in VSMCs. In contrast, tyrosine phosphorylation of paxillin was only 50% dependent on c-Src, suggesting that paxillin may also be phosphorylated by other kinases, such as Fyn or Pyk2. FAK is unlikely to be the paxillin kinase in VSMCs because we could not detect tyrosine phosphorylation of FAK in response to thrombin or angII (data not shown).
One of the major findings of this study is that F-actin was poorly bundled in src–/–
VSMCs both in the unstimulated state and after thrombin stimulation. In contrast, expression of KI-Src failed to inhibit actin filament bundling. These findings suggest that c-Src may be required for actin filament bundling by serving as a scaffold molecule via its SH2 and SH3 domains, rather than as a tyrosine kinase. In contrast, the kinase activity of c-Src is critical for cell spreading, in which actin turnover is very dynamic. Thus, we propose that c-Src kinase activity and the tyrosine phosphorylation of focal adhesion proteins by c-Src are involved in actin turnover (38
). This hypothesis is supported by our findings that Cas association with Crk induced by thrombin and angII also depends on Src kinase activity. Recently, it has been shown that Cas plays an important role in cell migration, which also requires dynamic actin turnover, especially in cooperation with Crk (37
). Additional support is provided by Richardson et al
), who showed that cell spreading was inhibited by overexpression of FRNK (FAK-related nonkinase) and rescued by overexpression of WT c-Src, but not KI c-Src. However, Kaplan et al
) showed that the defect in cell spreading of src–/–
fibroblasts could be restored by Src mutants that lack kinase activity. Thus, the role of c-Src in integrin and cytoskeletal reorganization remains controversial. It is possible that the compensation by other Src family kinases may explain the varying results. In fact, the phenomena exhibited in src–/–
VSMCs and VSMCs expressing KI-Src became less obvious with serial passage, which correlated with compensatory expression of other Src family kinases (Ishida, T., et al
., unpublished data). Introduction of several Src mutants, such as c-Src that lack kinase domain, SH2 domain, and SH3 domain, into src–/–
VSMCs would be very informative to determine which domain is critical for the regulation of actin bundling, cell spreading, and cell migration.
Data from v-Src–transformed cells and Csk-deficient (csk–/–
) cells have suggested that Src disrupts actin filaments, in contrast to the results of this study (20
). In fact, we did not observe defects in actin filaments in VSMCs overexpressing WT c-Src (Ishida, T., et al.
, unpublished data). There are, however, critical differences between c-Src–overexpressing cells and v-Src–transformed or csk–/–
cells. First, in v-Src–transformed cells and cells derived from csk–/–
mice, Src kinase activity is constitutively (and dramatically) increased without negative regulation. Second, the SH2 domain of v-Src can interact promiscuously with tyrosine residues of many proteins in these cells, whereas in WT c-Src, the SH2 domain of c-Src is intramolecularly bound to phosphorylated Tyr527 when it is inactive. In addition, v-Src is predominantly distributed in focal adhesions, whereas c-Src localizes in the cytoplasm when it is inactive (15
Our findings that Cas is translocated from the cytoplasm to focal adhesions after stimulation by thrombin and angII suggest a role for Cas in c-Src function, such as targeting Src to focal adhesions. Cas is localized in the cytoplasm in serum-starved, unstimulated VSMCs as well as in fibroblasts (42
). As already discussed here, in unstimulated cells, c-Src is primarily cytosolic and translocates to focal adhesions upon stim ulation (15
). In contrast, paxillin and tensin are already present in focal adhesions because of their localization sequences (18
). Thus, we propose that Cas is phosphorylated by c-Src in the cytoplasm immediately upon stimulation of VSMCs by angII and thrombin and is then recruited to focal adhesions with c-Src, which then phosphorylates paxillin and tensin.
Rearrangement of actin filaments and focal adhesions plays an essential role in cell growth, survival, locomotion, and shape. For example, cell-cycle progression depends on cell spreading and adhesion, whereas apoptosis is initiated after cells are detached (11
). In cardiovascular diseases, such as hypertension, atherosclerosis, and restenosis after angioplasty, vascular remodeling clearly requires rearrangement of VSMC cytoskeleton and focal adhesions (9
). Both thrombin and angII are thought to have important roles in remodeling by virtue of their effects on VSMC migration and growth (1
). The receptors for both thrombin and angII are highly expressed in atherosclerotic plaque and in restenotic lesions after balloon arterial injury (5
). In summary, rearrangement of VSMC focal adhesions and actin filaments in response to thrombin and angII may play an important role in vascular remodeling as well as in vascular tone.