The Serine/Threonine Protein Phosphatase 2A (PP2A) Regulates Syk Activity in Human Platelets.

Stephanie Makhoul,Kerstin Jurk,Elena Kumm,Pengyu Zhang,Ulrich Walter

doi:10.3390/ijms21238939

Abstract

Distinct membrane receptors activate platelets by Src-family-kinase (SFK)-, immunoreceptor-tyrosine-based-activation-motif (ITAM)-dependent stimulation of spleen tyrosine kinase (Syk). Recently, we reported that platelet activation via glycoprotein (GP) VI or GPIbα stimulated the well-established Syk tyrosine (Y)-phosphorylation, but also stoichiometric, transient protein kinase C (PKC)-mediated Syk serine(S)297 phosphorylation in the regulatory interdomain-B, suggesting possible feedback inhibition. The transient nature of Syk S297 phosphorylation indicated the presence of an unknown Syk pS297 protein phosphatase. In this study, we hypothesize that the S-protein phosphatase 2A (PP2A) is responsible for Syk pS297 dephosphorylation, thereby affecting Syk Y-phosphorylation and activity in human washed platelets. Using immunoblotting, we show that specific inhibition of PP2A by okadaic acid (OA) alone leads to stoichiometric Syk S297 phosphorylation, as analyzed by Zn2+-Phos-tag gels, without affecting Syk Y-phosphorylation. Pharmacological inhibition of Syk by PRT060318 or PKC by GF109203X only minimally reduced OA-induced Syk S297 phosphorylation. PP2A inhibition by OA preceding GPVI-mediated platelet activation induced by convulxin extended Syk S297 phosphorylation but inhibited Syk Y-phosphorylation. Our data demonstrate a novel biochemical and functional link between the S-protein phosphatase PP2A and the Y-protein kinase Syk in human platelets, and suggest that PP2A represents a potential enhancer of GPVI-mediated Syk activity caused by Syk pS297 dephosphorylation.

Highlights

Human platelets are small anucleate blood cells, which are essential components of the hemostasis system and widely recognized as circulating sentinels of the vessel wall [1,2,3]
We observed that activation of both GPIbα or GPVI caused a strong but transient stimulation of spleen tyrosine kinase (Syk) S297 phosphorylation, suggesting activity of both a very active Syk S297 protein kinase and protein phosphatase [33]
In order to characterize this protein phosphatase further, we studied the effect of phosphatase 2A (PP2A) and protein phosphatase 1 (PP1) on the regulation of Syk phosphorylation using conditions recently established in our laboratory for human platelets [34]

Summary

Introduction

Human platelets are small anucleate blood cells, which are essential components of the hemostasis system and widely recognized as circulating sentinels of the vessel wall [1,2,3]. Metabolic, inflammatory, and infectious vascular injuries result in newly exposed or altered vessel wall proteins, such as collagen, von Willebrand factor (vWF), fibrin, and podoplanin, which recruit platelets via distinct adhesion receptors/membrane glycoproteins (GP), such as GPVI, GPIb-V-IX, C-type lectin-2 (CLEC-2), and integrin αIIbβ. Metabolic, inflammatory, and infectious vascular injuries result in newly exposed or altered vessel wall proteins, such as collagen, von Willebrand factor (vWF), fibrin, and podoplanin, which recruit platelets via distinct adhesion receptors/membrane glycoproteins (GP), such as GPVI, GPIb-V-IX, C-type lectin-2 (CLEC-2), and integrin αIIbβ3 This initial adhesion leads to platelet activation with multiple responses, including cytoskeletal remodeling, integrin αIIbβ, activation, degranulation, synthesis/release of thromboxane A2 (TxA2), and surface exposure of anionic phospholipids. Global proteomic analyses of both human and murine platelets established that these anucleate cells have, similar to most other cells, a repertoire of hundreds of protein kinases/phosphatases [16,17,18]

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