A newly identified complex of spinophilin and the tyrosine phosphatase, SHP-1, modulates platelet activation by regulating G protein–dependent signaling

Abstract

Platelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury . Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/ RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/ RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI 2 ). Thus, we propose that the role of the SPL/RGS/ SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI 2 . After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs. IntroductionPlatelet responses to most agonists are mediated by G proteincoupled receptors, giving rise to the intracellular events that trigger platelet aggregation and granule exocytosis. 1 It has been known for some time that signaling by G proteins in platelets is subject to regulation by extrinsic factors arising from endothelial cells, especially nitric oxide and prostacyclin (PGI 2 ). 2 However, intrinsic modulators of platelet activation also exist, including members of the RGS (regulator of G protein signaling) family, 3 proteins that suppress G protein signaling by accelerating the hydrolysis of GTP bound to active G ␣ . 4,5 In contrast to nitric oxide and PGI 2 , RGS proteins are thought to have their effect once activation has begun; hence, the gain of function that we observed when an RGSinsensitive variant of G i2␣ was introduced into platelets. 3 This inhibitory role for RGS proteins produces a potential conundrum: although preventing unwarranted platelet activation is desirable, preventing the rapid onset of the hemostatic response to injury is not. We have, therefore, sought the means by which the onset of signal suppression by RGS proteins can be delayed, allowing signaling to begin. That search brought us to spinophilin (SPL or neurabin-II), a 130-kDa scaffold protein originally identified in screens for brain proteins that can bind to the serine/ threonine phosphatase, PP1, 6 and F-actin, 7 and subsequently found to associate with other proteins as well, 8 including a limited set of G protein-coupled receptors and RGS proteins. [9][10][11][12] Prior evidence suggests that one reg...

Cyclic nucleotide‐dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

“…The well established antagonism between activators and endothelial inhibitors in the regulation of Ca 2+ levels has recently been confirmed in a systematic study comparing effects of ADP, thrombin, convulxin and U46619 in combination with activators of cAMP and cGMP pathways 61. The Gq GAP RGS18 contributes to differential control of Ca 2+ levels by platelet activators and inhibitors 7, 35. PKA also inhibits IP 3 ‐induced Ca 2+ ‐release through phosphorylation of the IP 3 ‐receptor and the IP 3 ‐receptor‐associated G‐kinase substrate (IRAG, MRVI1) 1.…”

“…In resting platelets, RGS18 interacts with the scaffold protein spinophilin (neurabin‐2, PPP1R9B), the tyrosine phosphatase SHP‐1, and the phospho‐serine/threonine binding adapter protein 14‐3‐3 (Figure 5, RGS18 complex in transition i). Platelet activation by thrombin and TxA 2 induces a dissociation of RGS18 and SHP‐1 from spinophilin35 and increases binding of 14‐3‐3γ to RGS18 7. 14‐3‐3 bound RGS18 is less active leading to enhanced Gq signaling and Ca 2+ ‐release (Figure 5, RGS18 complex inactive).…”

“…Thrombin and TxA 2 induce SHP‐1 Y536 phosphorylation leading to phosphatase activation and de‐phosphorylation of tyrosines on spinophilin thus contributing to dissolution of the spinophilin/SHP‐1/RGS18 complex 37. The role of SHP‐1 phosphatase activity in inducing complex dissociation is supported by pharmacological studies using a nonselective SHP‐1/SHP‐2 inhibitor (NSC‐87877) 35. Thrombin, TxA 2 , and ADP induce phosphorylation of RGS18 on S49 resulting in enhanced 14‐3‐3 binding to RGS18 38.…”

“…Interestingly, spinophilin knockout platelets exhibit reduced Ca 2+ ‐release and aggregation which could, at least in part, be due to dysregulated RGS18 function 35. Absence of spinophilin also results in reduced cAMP synthesis in response to PGI 2 , highlighting multiple functions of this scaffold protein in platelets.…”

“…Certain kinases are able to activate phosphatases, as shown for thrombin‐induced phosphorylation and activation of SHP‐1 leading to de‐phosphorylation of spinophilin,35 and for PKA‐mediated activation of PP1 leading to detachment of 14‐3‐3 from RGS18 38. Although tyrosine phosphatases are important during platelet activation,86 their role in cyclic nucleotide‐mediated platelet inhibition remains unclear.…”