Cross Talk Between Serine/Threonine Protein Phosphatase 2A and Tyrosine Kinase Src Regulates αIIbβ3 Function

Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase that regulates a variety of cellular functions. In the context of the platelets, we have previously shown that a pool of the catalytic subunit of PP2A (PP2Ac) associates constitutively with the resting αIIbβ3 and negatively regulates αIIbβ3 signaling. However, the mechanism by which PP2Ac controls αIIbβ3 adhesive function is incompletely understood. In this study, we demonstrated that PP2Ac expressed as a GST fusion protein interacts with the tyrosine kinase Src. Activation of Src is essential to initiate αIIbβ3 outside-in signaling. Small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells overexpressing αIIbβ3 (293-αIIbβ3) and murine megakaryocytes, resulted in the activation of Src, as evidenced by the dephosphorylation of Src Tyr-529 and phosphorylation of Src Tyr-418. In contrast to PP2Acα, knockdown of the catalytic subunit of protein phosphatase 1 (PP1cα) did not activate Src, indicating that the regulation of Src activity by PP2Ac is specific. Dephosphorylation of Src Tyr-529 was not observed in PP2Aca depleted 293 cells treated with sulfanamido-benzbromarone compound, a selective protein tyrosine phosphatase 1 (PTP-1) inhibitor. These results suggest that inhibition of PP2Ac may activate a tyrosine phosphatase, capable of dephosphorylating Src Tyr-529. Activation of Src in PP2Ac depleted 293-αIIbβ3 cells had functional consequences for integrin αIIbβ3. PP2Ac depleted 293-αIIbβ3 cells exhibited ~2 fold increased adhesion to immobilized fibrinogen. Inhibition of Src kinase with a pharmacological agent PP2 but not by PP3 an inactive analogue of PP2, abolished the increased adhesiveness of PP2Acα–depleted 293 cells to fibrinogen. Finally, the increased activation of extracellular signal-regulated kinase (ERK1/2) in PP2Acα-depleted cells that we previously demonstrated was also blocked by Src inhibitor PP2 but not by PP3. These results indicate that both Src and ERK1/2 are activated in response to PP2Ac inhibition, with activation of Src being upstream of ERK1/2. These studies illustrate that inhibition of PP2Ac promotes αIIbβ3 adhesiveness by activating Src, and imply that the control of αIIbβ3 adhesive function can be further fine-tuned by a cross talk between the serine/threonine phosphatase PP2A and the tyrosine kinase Src.