Cross-talk between Serine/Threonine Protein Phosphatase 2A and Protein Tyrosine Phosphatase 1B Regulates Src Activation and Adhesion of Integrin αIIbβ3 to Fibrinogen

Subhashree Pradhan,Nawaf Alrehani,Vimal Patel,Tanvir Khatlani,K Vinod Vijayan

doi:10.1074/jbc.m109.085167

Subhashree Pradhan, Nawaf Alrehani + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.085167

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Abstract

Integrin α(IIb)β(3) signaling mediated by kinases and phosphatases participate in hemostasis and thrombosis, in part, by supporting stable platelet adhesion. Our previous studies indicate that the genetic manipulation of PP2Acα (α isoform of the catalytic subunit of protein phosphatase 2A) negatively regulate the adhesion of human embryonal kidney 293 cells expressing α(IIb)β(3) to fibrinogen. Here, we demonstrated that small interference RNA (siRNA) mediated knockdown of PP2Acα in 293 α(IIb)β(3) cells led to the dephosphorylation of Src Tyr-529, phosphorylation of Src Tyr-418 and an increased Src kinase activity. Conversely, overexpression of PP2Acα decreased the basal Src activity. Pharmacological inhibition of PP2Ac in human platelets or PP2Acα knockdown in primary murine megakaryocytes resulted in Src activation. PP2Acα-depleted 293 α(IIb)β(3) cells did not alter the serine (Ser) phosphorylation of Src but enhanced the Ser-50 phosphorylation of protein tyrosine phosphatase 1B (PTP-1B) with a concomitant increase in the PTP-1B activity. Src activation in the PP2Acα-depleted 293 α(IIb)β(3) cells was abolished by siRNA mediated knockdown of PTP-1B. Pharmacological inhibition of Src or knockdown of Src, PTP-1B blocked the enhanced activation of extracellular signal-regulated kinase (ERK1/2) and the increased adhesiveness of PP2Acα-depleted 293 α(IIb)β(3) cells to fibrinogen, respectively. Thus, inactivation of PP2Acα promotes hyperphosphorylation of PTP-1B Ser-50, elevates PTP-1B activity, which dephosphorylates Src Tyr-529 to activate Src and its downstream ERK1/2 signaling pathways that regulate α(IIb)β(3) adhesion. Moreover, these studies extend the notion that a cross-talk between Ser/Thr and Tyr phosphatases can fine-tune α(IIb)β(3) outside-in signaling.

Highlights

To ascertain whether the Src activation was specific to PP2A holoenzyme contains the catalytic subunit C (PP2Ac)␣ inhibition, we examined Src Tyr-529 and Src Tyr-418 phosphorylation in 293 ␣IIb␤3 cells that were depleted for the catalytic subunits of ␣, ␤, and ␥ isoforms of PP1 (PP1c)
Because Src activation in the PP2Ac␣-depleted cells is dependent on protein tyrosine phosphatase 1B (PTP-1B), we examined the role of PTP-1B in the adhesion of PP2Ac␣-depleted cells
Densitometric quantification of phospho-Src Tyr-529/total Src in 293 cells treated with small interference RNA (siRNA) described in panel A. n ϭ 3. *, p ϭ 0.02

Summary

Introduction

Spreading events [1]. Signaling molecules, including the tyrosine kinase c-Src [2], protein tyrosine phosphatase 1 B (PTP-1B)3 [3], and the catalytic subunit of protein phosphatase 2A (PP2Ac) [4] either associate constitutively or are recruited to the ␣IIb␤3 complex in response to integrin engagement. PTP-1B Activity Assay—Lysate obtained from the control and PP2Ac␣ siRNA-treated 293 ␣IIb␤3 cells were immunoprecipitated with anti-PTP-1B antibody or mouse IgG. Src was immunoprecipitated from the control and PP2Ac␣ siRNA-treated 293 ␣IIb␤3 cells (Fig. 2D) and Src kinase activity analyzed as previously described [14].

Results

Conclusion