Protein Phosphatase 2A (PP2A) Associates with Integrin α_IIbβ₃ and Regulates Adhesion to Fibrinogen.

We have previously demonstrated that protein phosphatase 1 (PP1) associates constitutively with the integrin α_IIb subunit and regulates myosin light chain phosphorylation. In this study, we considered whether other member of the serine/threonine (Ser/Thr) phosphatase family namely, protein phosphatase 2A (PP2A) associates with the integrin α_IIbβ₃. Co-immunoprecipitation assays using lysates from resting human platelets revealed the presence of a catalytic subunit of PP2A (PP2Ac) in the α_IIb immunoprecipitates, and in a reciprocal experiment, α_IIb was detected in the PP2Ac immunoprecipitates. In contrast, another platelet abundant Ser/Thr phosphatase, protein phosphatase 2C (PP2C) was not detected in the α_IIb immunoprecipitates. Furthermore, the association of PP2Ac with integrin α_IIbβ₃ was also observed in 293 cells overexpressing α_IIbβ₃. These results indicate a constitutive and specific interaction of PP2Ac with the integrin α_IIbβ₃. Polystyrene beads coated with purified PP2Ac but not BSA supported the binding of purified integrin α_IIbβ₃ in a dose dependent manner, suggesting that the interaction of PP2Ac with α_IIbβ₃ was direct. Furthermore, purified PP2Ac as well as PP2Ac in lysates obtained from the resting platelets bound specifically to a biotinylated α_IIb cytoplasmic peptide encompassing residues 985–995 but not to a scrambled peptide, suggesting that the integrin α_IIb is sufficient to mediate the interaction of PP2Ac in vitro. The association of PP2Ac with the platelet integrin α_IIbβ₃ was not altered during platelet adhesion to fibrinogen (α_IIbβ₃ outside-in signaling) or during thrombin or ADP stimulation (inside-out signaling to α_IIbβ₃). In contrast, we have previously shown that integrin-bound PP1 dissociated from the α_IIbβ₃ complex upon platelet adhesion and thrombin-induced platelet activation. The association of PP2Ac with the integrin α_IIbβ₃ correlates well with the dephosphorylation of a PP2A substrate, extracellular-signal regulated kinase 2 (ERK2) during thrombin-induced platelet aggregation that we and others have previously demonstrated. More importantly, ERK2 dephosphorylation was not observed in platelets from Glanzmann thrombasthenic patients or in normal platelets pretreated with RGDS or integrilin, suggesting a critical role for integrin α_IIbβ₃ in the dephosphorylation of ERK2. To ascertain a physiological relevance for the PP2A-α_IIbβ₃ association, we used short interfering RNAs (siRNAs) to knock down the expression of PP2Ac in the 293/α_IIbβ₃ cells. Knock down was maximal (~55–70%) and specific to PP2Ac because PP1 and actin protein levels were not different between the control and PP2Ac siRNA treated cells. Consistent with the reduction in the PP2Ac protein level in the PP2Ac knock down cells there was ~70% reduction in the PP2Ac phosphatase activity, and a concomitant increased basal ERK2 phosphorylation. PP2Ac knock down significantly (P≤0.006) increased the adhesion of 293/α_IIbβ₃ cells to fibrinogen. The adhesion was α_IIbβ₃ specific because it could be abolished with an α_IIbβ₃ function blocking antibody (10E5). These findings supports a mechanism whereby the integrin associated Ser/Thr phosphatases might regulate α_IIbβ₃ adhesive functions via dephosphorylation of key cytoskeletal proteins.