Distinct Roles for the α, β and γ Isoforms of the Catalytic Subunit of Protein Phosphatase 1 in Outside-in Integrin α_IIb^β₃ Signaling.

Abstract 154

Outside-in integrin α_IIb^γ₃ signaling mediated by kinases and phosphatases regulate platelet adhesion and thrombus formation. The catalytic subunit of protein phosphatase 1 (PP1c), expressed as α, β and γ isoforms regulates a variety of cellular functions. We have previously demonstrated that fibrinogen-α_IIb^β₃ engagement during outside-in signaling dissociated integrin α_IIb*^β₃ anchored PP1c, which was followed by PP1c activation and dephosphorylation of myosin light chain (MLC). However, it is unknown whether any functions regulated by outside-in integrin α_IIb^β₃ signaling are modulated by PP1c. To overcome the lack of specificity of pharmacological agents towards PP1c, we have pursued a genetic approach to inhibit PP1c in 293 cells overexpressing α_IIb^β₃. In this study, we demonstrated that PP1cα interacts with α_IIb^β₃ in platelets and 293 cells by co-immunoprecipitation and GST pull down assays. Knockdown of PP1cα by short interference RNA resulted in a 50-60% enhanced adhesion (p<0.003) of 293 cells to immobilized fibrinogen. Conversely, overexpression of PP1cα inhibited (p<0.002) α_IIb^β₃ adhesiveness to fibrinogen. Furthermore, the enhanced adhesiveness of PP1cα depleted 293 cells was also observed with von Willebrand factor, indicating that the differential adhesion due to the lack of PP1cα was not ligand specific. Signaling pathways implicated in the reorganization of cellular cytoskeleton, like extracellular-regulated kinase (ERK1/2) and AKT were activated in PP1cα depleted 293 cells. Such observations imply that PP1cα, knockdown of PP1cγ, which also associates with α_IIb^β₃, did not affect the adhesion to fibrinogen. Indeed, platelets from the PP1cγ null and wild type mice showed comparable adhesion to fibrinogen. Unexpectedly, depletion of PP1cβ isoform in 293 cells significantly (p<0.01) inhibited the ability of these cells to adhere to fibrinogen. Since MLC is a specific substrate of PP1cβ, it is likely that alterations in the MLC phosphorylation may underlie the loss of adhesiveness in PP1cβ depleted cells. Finally, depletion of all the PP1c isoforms also significantly (p<0.001) inhibited the adhesion of 293 cells to fibrinogen. These data illustrate that each isoform of PP1c contributes distinctly to the adhesive phenotype of integrin α_IIb^β₃ and implies that the control of outside-in integrin α_IIb^β₃ signaling response might be a composite of all PP1c isoforms. Perhaps, each isoforms have specific substrates that contribute to the process of adhesion, such that alterations in the phosphorylation of these substrates may underlie the distinct functional roles for PP1c.