In response to agonist stimulation, the αIIbβ3 integrin on platelets is converted to an active conformation that binds fibrinogen and mediates platelet aggregation. This process contributes to both normal hemostasis and thrombosis. Activation of αIIbβ3 is believed to occur in part via engagement of the β3 cytoplasmic tail with talin; however, the role of the αIIb tail and its potential binding partners in regulating αIIbβ3 activation is less clear. We report that calcium and integrin binding protein 1 (CIB1), which interacts directly with the αIIb tail, is an endogenous inhibitor of αIIbβ3 activation; overexpression of CIB1 in megakaryocytes blocks agonist-induced αIIbβ3 activation, whereas reduction of endogenous CIB1 via RNA interference enhances activation. CIB1 appears to inhibit integrin activation by competing with talin for binding to αIIbβ3, thus providing a model for tightly controlled regulation of αIIbβ3 activation.

p21-activated kinases (PAKs) regulate many cellular processes, including cytoskeletal rearrangement and cell migration. In this study, we report a direct and specific interaction of PAK1 with a 22-kD Ca 2+ -binding protein, CIB1, which results in PAK1 activation both in vitro and in vivo. CIB1 binds to PAK1 within discrete regions surrounding the inhibitory switch domain in a calcium-dependent manner, providing a potential mechanism of CIB1-induced PAK1 activation. CIB1 overexpression significantly decreases cell migration on fibronectin as a result of a PAK1-and LIM kinase–dependent increase in cofilin phosphorylation. Conversely, the RNA interference–mediated depletion of CIB1 increases cell migration and reduces normal adhesion-induced PAK1 activation and cofilin phosphorylation. Together, these results demonstrate that endogenous CIB1 is required for regulated adhesion-induced PAK1 activation and preferentially induces a PAK1-dependent pathway that can negatively regulate cell migration. These results point to CIB1 as a key regulator of PAK1 activation and signaling.

Specificity and modulation of integrin function have important consequences for cellular responses to the extracellular matrix, including differentiation and transformation. The Ras-related GTPase, R-Ras, modulates integrin affinity, but little is known of the signaling pathways and biological functions downstream of R-Ras. Here we show that stable expression of activated R-Ras or the closely related TC21 (R-Ras 2) induced integrin-mediated migration and invasion of breast epithelial cells through collagen and disrupted differentiation into tubule structures, whereas dominant negative R-Ras had opposite effects. These results imply novel roles for R-Ras and TC21 in promoting a transformed phenotype and in the basal migration and polarization of these cells. Importantly, R-Ras induced an increase in cellular adhesion and migration on collagen but not fibronectin, suggesting that R-Ras signals to specific integrins. This was further supported by experiments in which R-Ras enhanced the migration of cells expressing integrin chimeras containing the α2, but not the α5, cytoplasmic domain. In addition, a transdominant inhibition previously noted only between integrin β cytoplasmic domains was observed for the α2 cytoplasmic domain; α2β1-mediated migration was inhibited by the expression of excess α2 but not α5 cytoplasmic domain-containing chimeras, suggesting the existence of limiting factors that bind the integrin α subunit. Using pharmacological inhibitors, we found that R-Ras induced migration on collagen through a combination of phosphatidylinositol 3-kinase and protein kinase C, but not MAPK, which is distinct from the other Ras family members, Rac, Cdc42, and N- and K-Ras. Thus, R-Ras communicates with specific integrin α cytoplasmic domains through a unique combination of signaling pathways to promote cell migration and invasion.