Abstract
We have previously demonstrated that protein phosphatase 1 (PP1) associates constitutively with the integrin αIIb 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 αIIbβ3. Co-immunoprecipitation assays using lysates from resting human platelets revealed the presence of a catalytic subunit of PP2A (PP2Ac) in the αIIb immunoprecipitates, and in a reciprocal experiment, αIIb was detected in the PP2Ac immunoprecipitates. In contrast, another platelet abundant Ser/Thr phosphatase, protein phosphatase 2C (PP2C) was not detected in the αIIb immunoprecipitates. Furthermore, the association of PP2Ac with integrin αIIbβ3 was also observed in 293 cells overexpressing αIIbβ3. These results indicate a constitutive and specific interaction of PP2Ac with the integrin αIIbβ3. Polystyrene beads coated with purified PP2Ac but not BSA supported the binding of purified integrin αIIbβ3 in a dose dependent manner, suggesting that the interaction of PP2Ac with αIIbβ3 was direct. Furthermore, purified PP2Ac as well as PP2Ac in lysates obtained from the resting platelets bound specifically to a biotinylated αIIb cytoplasmic peptide encompassing residues 985–995 but not to a scrambled peptide, suggesting that the integrin αIIb is sufficient to mediate the interaction of PP2Ac in vitro. The association of PP2Ac with the platelet integrin αIIbβ3 was not altered during platelet adhesion to fibrinogen (αIIbβ3 outside-in signaling) or during thrombin or ADP stimulation (inside-out signaling to αIIbβ3). In contrast, we have previously shown that integrin-bound PP1 dissociated from the αIIbβ3 complex upon platelet adhesion and thrombin-induced platelet activation. The association of PP2Ac with the integrin αIIbβ3 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 αIIbβ3 in the dephosphorylation of ERK2. To ascertain a physiological relevance for the PP2A-αIIbβ3 association, we used short interfering RNAs (siRNAs) to knock down the expression of PP2Ac in the 293/αIIbβ3 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/αIIbβ3 cells to fibrinogen. The adhesion was αIIbβ3 specific because it could be abolished with an αIIbβ3 function blocking antibody (10E5). These findings supports a mechanism whereby the integrin associated Ser/Thr phosphatases might regulate αIIbβ3 adhesive functions via dephosphorylation of key cytoskeletal proteins.
Protein Phosphatase 2A and Cdc7 Kinase Regulate the DNA Unwinding Element-binding Protein in Replication Initiation