CIN85 Is a New Protein Phosphatase 2Ac Interacting Protein That Regulates α_IIbβ₃ Adhesion to Fibrinogen

Abstract 95

Platelet activation at the site of injury is dependent on signal transduction events that are mediated by protein kinases and protein phosphatases. Reversible tyrosine and/or serine/threonine phosphorylation dependent assembly of effector (cytoskeletal, signaling and adaptor) proteins are critical for propagating signaling downstream of platelet receptors. Several studies have indicated a key role for protein kinases and their effectors in regulating the functions of integrin α_IIbβ₃. In contrast, much less is known about the contribution of serine/threonine phosphatases in integrin function, and the identities of effectors regulated by phosphatases are unknown. In this context, we have previously noticed that depletion of the catalytic subunit of protein phosphatase 2A (PP2Ac) enhanced Src activation and augmented α_IIbβ₃ adhesiveness to immobilized fibrinogen. Since protein-protein interactions form the foundation of cell signaling networks, we sought to identify the potential effectors of PP2Ac. We employed yeast two-hybrid interaction studies with the full length PP2Ac fused to GAL4 binding domain as bait and screened human bone marrow library. A novel interaction of PP2Ac with a protein called CIN85 was identified. Although CIN85 associates with several proteins, an interaction with PP2Ac has not been reported in any cell types. CIN85 (Cbl-interacting protein of 85 kDa) also known as Ruk or SETA is an adaptor protein with three SH3 domains, followed by a proline rich region, a serine rich region and a coiled-coil region. CIN85 participates in vesicle mediated transport and cytoskeleton remodeling. Co-immunoprecipitation (co-IP) experiments validated the interaction of the HA tagged PP2Ac with FLAG tagged CIN85 in 293 cells expressing PP2Ac-HA and CIN85-FLAG. Purified PP2Ac bound to recombinant CIN85-GST protein but not to GST protein, indicating that the in vitro interaction of PP2Ac with CIN85 was direct. Transfection and co-IP experiments with several FLAG tagged truncation mutants of CIN85 in 293 cells revealed that the interaction of PP2Ac with CIN85 was mediated by the proline rich region of CIN85. These studies established a direct interaction of PP2Ac with CIN85. Importantly, the interaction of purified PP2Ac with recombinant CIN85 decreased PP2Ac activity, suggesting that this complex has signaling consequence in vitro. We explored and showed for the first time that CIN85 is expressed in platelets. More importantly, PP2Ac co-immunoprecipitated with CIN85 in human platelets and in 293 α_IIbβ₃ cells suspended over BSA substrate. Interestingly, adhesion of platelets and 293 α_IIbβ₃ cells to immobilized fibrinogen induced dissociation of this complex. These studies suggest that the dissociation of PP2Ac-CIN85 complex following integrin stimulation enables CIN85 to propagate outside-in signals by efficiently engaging with other downstream effectors. Consistent with this notion, siRNA mediated depletion of CIN85 significantly (p<0.001) decreased adhesion of 293 α_IIbβ₃ cells to immobilized fibrinogen. These studies reveal that platelet activation events involve the coupling of the integrin α_IIbβ₃ adhesion initiated signaling with the phosphatase effector CIN85.