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  • br sGC cGMP PKG pathway in

    2022-01-13


    sGC/cGMP/PKG pathway in regulation of platelet small GTPases All classical Rho family members of small GTPases including RhoA, Rac, Cdc42, some atypical Rho GTPases like RhoB, RhoF and RhoG, and also Ras family members N-Ras, K-Ras, Rap1A, Rap1B, Rap2A and Rap2B, and the Ras-related GTPases of the ADP-ribosylation family like Arf6, are expressed in platelets [57,121]. Generally, the activity of small GTPases is controlled by SC 144 nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs facilitate the dissociation of GDP and rebinding of GTP that activates the GTPases, whereas GAPs stimulate GTP hydrolysis and inactivate GTPases. The cGMP/PKG pathway is involved in inhibition of small GTPases by direct phosphorylation or by phosphorylation of specific GEFs and GAPs. Phosphorylation of RhoA by PKG at S188 inhibited its activity [122] [123]. In platelets, phosphorylation of RhoA at the same site by PKG prevented its association with Rho kinase (ROCK), resulting in activation of phosphatase, decrease of MLC phosphorylation, and inhibition of platelet shape change [124]. A similar response has been described for PKA-mediated phosphorylation of RhoA in platelets [125]. In human TXA2-stimulated platelets, activation of RhoA is inhibited by PKG and PKA [126]. Two other classical Rho family GTPases, Rac and Cdc42, are strongly involved in platelet activation, filopodia and lamellipodia formation, and stimulate p21-activated kinases (PAK) [121]. Rac and Cdc42 themselves appear not to be direct PKG substrates, whereas PKG phosphorylates PAK1 at S21 site in endothelial and HeLa cells, stimulating PAK/VASP interaction and cell polarization [127]. There is no evidence in the literature that similar events occur in platelets as well, although all three proteins, Rac1, PAK (mainly isoforms 2 and 4) and VASP are expressed in platelets [57]. PKG activation inhibited Rac1 activity by reducing Rac1-GTP levels in platelets [126], indicating that Rac1-specific GEFs and/or GAPs could be direct PKG substrates. Indeed, we recently reported that Rac1-specific GEF ARHGEF6, and Rac1-specific GAP ArhGAP17, are direct PKA and PKG substrates [128]. Using several different approaches, including analysis of platelet proteomic/phosphoproteomic data, Phos-tag, Western blotting, and point mutation studies, we demonstrated that ArhGEF6 is phosphorylated at S684, and ArhGAP17 at S702. ArhGEF6 formed a SC 144 stable complex with G protein-coupled receptor kinase-interactor 1 (GIT1), and its phosphorylation facilitated binding of 14-3-3 proteins to this complex. In other cell types, binding of 14-3-3 to the ArhGEF6/GIT1 complex correlated with decreased Rac1 activity [129]. Phosphorylation of ArhGAP17 at S702 leads to dissociation of Cdc42-interacting protein 4 (CIP4) from the CIP4/ArhGAP17 stable complex, stimulating ArhGAP17 function. PKG-dependent inhibition of Rac1 activity in platelets is a complex process which is mediated by phosphorylation of multiple substrates including specific GEFs, GAPs, and probably PAK1. Another small GTPase, Rap1, plays a significant role in platelet integrin αIIbβ3 activation and aggregation. Rap1B is the major isoform of Rap GTPases expressed in platelets [57,130]. Rap1B is rapidly activated by almost all platelet agonists [[131], [132], [133], [134], [135]], and Rap1B knockout mice display impaired platelet activation and aggregation [136]. Activation of the cGMP/PKG pathway strongly inhibits Rap1B activity [137]. Regulation of Rap1B activity by PKG in platelets is complex and may involve phosphorylation of Rap1B itself, as well as the major Rap1 GEF, calcium and diacylglycerol-regulated guanine nucleotide exchange factor 1 (CalDAG-GEFI, or RasGRP2) [138], and the Rap1-specific GAP Rap1GAP2 [139]. Rap1B can be phosphorylated by PKG at S179 [140,141], however the phosphorylation kinetics is much slower than inhibition of Rap1B by PKG [137], and this phosphorylation has no direct effect on Rap1B activity [140]. Phosphorylation of Rap1GAP2 at S7 by PKG leads to detachment of 14-3-3 protein from Rap1GAP2 which stimulates GAP activity, thereby inhibiting Rap1B [139,142]. CalDAG-GEFI is an important regulator of Rap1b and platelet activation. Similar to Rap1B KO mice, CalDAG-GEFI KO mice exhibit increased bleeding time [143], impaired platelet aggregation and protection from arterial thrombosis [144]. Mutation of CalDAG-GEFI gene in humans reduces Rap1 activation and causes severe bleeding [145]. CalDAG-GEFI contains four putative PKG/PKA phosphorylation sites (serines 116, 117, 147, 587) which are conserved among known mammalian CalDAG-GEFI proteins. In intact human platelets, the major site is S587, as identified by a phosphoproteomic approach. Phosphorylation of CalDAG-GEF1 at this site by PKA correlated directly with inhibition of Rap1B activity [138,146]. Importantly, in intact human platelets, S587 was strongly phosphorylated by PKA and only weakly by PKG, indicating that inhibition of Rap1B by these two kinases could involve different mechanisms [138]. Ongoing phosphoproteomic studies indicate that other GEFs like RapGEF2 might be contributing to NO-mediated regulation of Rap1.