Serine Phosphorylation Negatively Regulates RhoA in Vivo

Abstract

Previous work indicates that RhoA phosphorylation on Ser188 by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMPdependent kinase phosphorylation site. RhoA phosphorylation or phosphomimetic substitution did not affect Rho guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity in vitro but promoted binding to the Rho guanine-dissociation inhibitor as measured by exchange factor competition assays. The in vitro similarities between RhoA phosphomimetic proteins and phosphorylated RhoA allowed us to study function of phosphorylated RhoA in vivo. RhoA phosphomimetic proteins display depressed GTP loading when transiently expressed in NIH 3T3 cells. Stable-expressing RhoA and RhoA(S188A) clones spread significantly slower than mock-transfected or RhoA(S188E) clones. RhoA(S188A) clones were protected from the morphological effects of a cAMP agonist, whereas phosphomimetic clones exhibit stress fiber disassembly similar to control cells. Together, these data provide in vivo evidence that addition of a charged group to Ser 188 upon phosphorylation negatively regulates RhoA activity and indicates that this occurs through enhanced Rho guanine-dissociation inhibitor interaction rather than direct perturbation of guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity.

Pituicyte Stellation is Prevented by RhoA-or Cdc42-Dependent Actin Polymerization

“…In fact, our results are not surprising considering the following points. Ser phosphorylation of RhoA is normally related to its inhibition by a guaninenucleotide dissociation inhibitor (GDI; Lang et al 1996;Ellerbroek et al 2003). In this case, GTP-bound RhoA is not amenable to pull-down analysis (Ellerbroek et al 2003), which is inconsistent with our previous pull-down data (Rosso et al 2002a).…”

“…In this case, GTP-bound RhoA is not amenable to pull-down analysis (Ellerbroek et al 2003), which is inconsistent with our previous pull-down data (Rosso et al 2002a). RhoA inhibition by a GTPase-activating protein (GAP) in turn can be measured by pull-down (Ellerbroek et al 2003). Furthermore, as RhoA-G14V, which is protected from GAP-mediated GTP hydrolysis, is an efficient stellation inhibitor in our hands, we believe that a GAP-related mechanism is more likely to mediate RhoA inhibition by adenosine.…”

“…Relevant to adenosine-induced pituicyte stellation, protein kinase A phosphorylation of a Ser residue has been suggested as one mechanism for RhoA inhibition (Lang et al 1996;Ellerbroek et al 2003). This indeed might represent an intriguing link between metabotropic receptor stimulation and monomeric GTPase inhibition.…”

“…4, lower right panel), consistent with our previous finding that adenosine induces stellation via RhoA inhibition. In order to determine whether the mechanism of inhibition involves serine phosphorylation of RhoA, as was shown in other cell types (Lang et al 1996;Ellerbroek et al 2003), we transfected pituicytes with two types of mutants: RhoA-S188A, a phosphorylation-resistant mutant, or RhoA-S188D, a phosphomimetic mutant. The former had no effect on spread pituicytes nor did it prevent adenosine from inducing stellation, whereas the latter mutant by itself did not induce stellation (Fig.…”

PKA-induced dimerization of the RhoGAP DLC1 promotes its inhibition of tumorigenesis and metastasis

“…Furthermore, PKA has been shown to hinder multiple components of Rho signalling, which are crucial to cytoskeletal dynamics and cell movement 36,37 . PKA directly phosphorylates RhoA at Ser188 and inhibits its function by enhancing the interaction between RhoA and the Rho guanine-dissociation inhibitor 38 . In addition, PKA also negatively regulates the upstream activator of RhoA, and activated PKA associates and phosphorylates AKAP-Lbc at Ser1656.…”