Ste20-Related Kinase SLK Phosphorylates Ser188 of RhoA to Induce Vasodilation in Response to Angiotensin II Type 2 Receptor Activation

Abstract

Abstract-The small G protein Rho signaling pathways are recognized as major regulators of cardiovascular functions, and activation of Rho proteins appears to be a common component for the pathogenesis of hypertension and vascular proliferative disorders. Recent evidence suggests that modulation of Rho protein signaling by phosphorylation of Rho proteins provides an additional simple mechanism for coordinating Rho protein functions. Phosphorylation of RhoA by cAMP-or cGMP-activated kinase on Ser188 induces cytosolic sequestration of RhoA through increased interaction with guanine dissociation inhibitor, thereby resulting in inhibition of RhoA-dependent functions. Here we show that stimulation of angiotensin II (Ang II) type 2 receptor (AT 2 R) in vascular smooth muscle cells induces Ser188 phosphorylation of RhoA independently of cAMP-or cGMP-activated kinase. We identify the Ser/Thr kinase Ste20-related kinase SLK as a new kinase phosphorylating RhoA on Ser188. Activation of the signaling cascade involving Src homology 2 domain-containing protein-tyrosine phosphatase 1, casein kinase II and SLK is responsible for RhoA phosphorylation and inhibition of RhoA-mediated arterial contraction induced by AT 2 R activation. These results thus identify the molecular mechanism linking AT 2 R to RhoA inhibition and vasodilation. (Circ Res. 2008;102:1265-1274.)Key Words: Rho Ⅲ signal transduction Ⅲ phosphorylation Ⅲ angiotensin II Ⅲ vascular smooth muscle R hoA is a member of the Rho protein family that has been identified as an essential regulator of vascular smooth muscle cell functions. Through the activation of its target Rho kinase, RhoA is the major regulator of the tonic component of vascular smooth muscle cell contraction and plays a critical role in the control of vascular smooth muscle differentiation, proliferation, and migration. 1 Subsequent studies have demonstrated the participation of the RhoA/Rho kinase signaling pathway in several vascular pathologies, including hypertension, coronary artery spasm, effort angina, atherosclerosis, and restenosis. 1,2 Indeed, although basal RhoA activity is required for homeostatic functions in physiological conditions, its sustained overactivation has pathological consequences in the vascular system, particularly in vascular smooth muscle cells. Activation of RhoA-dependent pathways is involved in excessive contraction, and thereby increases blood pressure but also in excessive cell growth and migration that participate in pathological cardiovascular remodeling. 1 RhoA acts as a molecular switch. In the inactive GDPbound form, RhoA is locked in the cytosol by guanine dissociation inhibitors (GDIs). In the active GTP-bound form released from GDI, RhoA translocates to plasma membrane where it interacts with effectors to transduce the signal downstream. GTPase-activating proteins then turn off activation. In addition to this regulation, we and others have demonstrated that phosphorylation/dephosphorylation cycle also controls RhoA activity. 3 Cyclic GMP-dependent protein kinase...

Ste20-like kinase SLK, at the crossroads

“…41,42 In a study of vasodilation induced by angiotensin II type 2 receptor (AT2R) activation, it was reported that AT2R signals through SLK which in turn phosphorylates Ser188 of RhoA, thus inhibiting its activity, preventing vascular smooth muscle cell contraction. 43 This interpretation supports a mechanism by which SLK mediates cytoskeletal reorganization and stress fiber breakdown through a Rho/Rac pathway.…”

“…60 FA turnover during cell migration induces the formation of a functional FAK/ src complex initiated by auto-phosphorylation of FAK at tyrosine residue 397 (pY397), stable focal adhesion assembly and FA turnover and migration. [41][42][43][57][58][59][60] Similarly, nocodazole treatment of fibroblasts results in microtubule depolymerization and FA stabilization as evidenced by high levels of phospho-FAK-Y397. 61 Nocodazole wash-out and microtubule regrowth is accompanied by cyclical changes in the levels of FAK pY397.…”

Angiotensin II regulates the LARG/RhoA/MYPT1 axis in rat vascular smooth muscle in vitro

“…The rings were incubated in culture medium containing either an siRNA against LARG (10 nmol/L) or scrambled siRNA (10 nmol/L) for 48 h at 37 °C [20,21] . The siRNA was introduced using the siPORT TM NeoFX TM Transfection Agent.…”

A novel role for the Ste20 kinase SLK in adhesion signaling and cell migration

“…[12][13][14] Recently, SLK was identified as a member of a new signaling pathway that induce vasodilatation in response to angiotensin II type 2 receptor activation. 15 It was reported that SLK negatively regulates RhoA-dependent functions by phosphorylation of RhoA at Ser188. 15 These findings suggest that SLK represents a novel relaxation signal involved in cytoskeletal remodeling and cell migration.…”

“…15 It was reported that SLK negatively regulates RhoA-dependent functions by phosphorylation of RhoA at Ser188. 15 These findings suggest that SLK represents a novel relaxation signal involved in cytoskeletal remodeling and cell migration.…”