Dissociation of SHP-1 from spinophilin during platelet activation exposes an inhibitory binding site for protein phosphatase-1 (PP1).

Peisong Ma,Lawrence F Brass,Darci C Foote,Andrew J Sinnamon,Kathleen Freson

doi:10.1371/journal.pone.0119496

Peisong Ma, Lawrence F Brass + Show 3 more

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https://doi.org/10.1371/journal.pone.0119496

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Journal: PloS one	Publication Date: Mar 18, 2015
Citations: 18	License type: CC BY 4.0

Affiliation: University of Pennsylvania

Abstract

We have recently shown that a critical regulatory node in the platelet signaling network lies immediately downstream of platelet receptors for thrombin and TxA2. This node is comprised of a scaffold protein (spinophilin, SPL), a protein tyrosine phosphatase (SHP-1), and either of the two members of the Regulators of G protein Signaling family predominantly expressed in platelets (RGS10 or RGS18). The SPL/RGS/SHP-1 complex is present in resting platelets, dissociating when thrombin or TxA2, but not ADP or collagen, activate SHP-1 and release RGS10 and RGS18 to dampen signaling. Here we demonstrate an additional regulatory role for spinophilin, showing that dissociation of SHP-1 from spinophilin is followed by an increase in the binding of spinophilin to PP1, a serine/threonine phosphatase whose binding site maps to a region close to the SHP-1 binding site. The increase in PP1 binding to spinophilin is limited to platelet agonists that cause dissociation of the complex and is selective for the α and γ isoforms of PP1. Studies in cell culture show that SHP-1 and PP1 can compete for binding to spinophilin and that binding inhibits PP1 activity since over-expression of wild type spinophilin, but not spinophilin with a disabled PP1 binding site, causes an increase in the phosphorylation of myosin light chain, a well-characterized PP1 substrate. Collectively, these results indicate that in addition to regulating RGS protein availability in resting platelets, spinophilin can serve as a time-dependent, agonist- and isoform-selective regulator of PP1, inhibiting its activity when decay of the SPL/RGS/SHP-1 complex releases SHP-1 from spinophilin, exposing a binding site for PP1.

Highlights

With the exception of collagen, most platelet agonists work through G protein coupled receptors, invoking signaling events that lead to platelet aggregation and thrombus formation [1]
That a key difference between the Chinese hamster ovary (CHO) cell and platelet studies is that 1) CHO cells do not normally express SHP-1 [2] and 2) it was not necessary to stimulate the CHO cells with thrombin or SFLLRN in order to see an association of PP1α and PP1γ with spinophilin (Fig. 2B), an observation that proved useful in subsequent studies
Our previous studies show that in resting platelets spinophilin is phosphorylated on tyrosines Y398 and Y483, forming a trimolecular complex with SHP-1 plus either RGS10 or RGS18

Summary

Introduction

With the exception of collagen, most platelet agonists work through G protein coupled receptors, invoking signaling events that lead to platelet aggregation and thrombus formation [1]. We showed that in resting platelets RGS10 and RGS18 are bound to a scaffold protein, spinophilin (SPL or neurabinII), forming a complex in which spinophilin is phosphorylated on tyrosines 398 and 483 [2]. Activation of SHP-1 leads to dephosphorylation of spinophilin and dissociation of the SPL/RGS/SHP-1 complex, releasing RGS10 and RGS18, which can dampen signaling that otherwise favors platelet activation [10, 11]. Of relevance for the present studies, dissociation of the SPL/RGS/SHP-1 complex is agonist-selective, occurring in response to thrombin and thromboxane A2 (TxA2) mimetics, but not in response to ADP or collagen, neither of which signals potently via Gq [1]

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