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 transfera… Show more
“…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).…”
Section: Discussioncontrasting
confidence: 56%
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
Section: Effects Of Monomeric Gtpase Expression On Pituicyte Morphologymentioning
Our aim was to shed light on different steps leading from metabotropic receptor activation to changes in cell shape, such as those that characterize the morphological plasticity of neurohypophysial astrocytes (pituicytes). Using explant cultures of adult rat pituicytes, we have previously established that adenosine A1 receptor activation induces stellation via inhibition of RhoA monomeric GTPase and subsequent disruption of actin stress fibers. Here, we rule out RhoA phosphorylation as a mechanism for that inhibition. Rather, our results are more consistent with involvement of a GTPase-activating protein (GAP). siRNA and pull-down experiments suggest that a step downstream of RhoA might involve Cdc42, another GTPase of the Rho family. However, RhoA activation, e.g., in the presence of serum, induces stress fibers, whereas direct Cdc42 activation appears to confine actin within a submembrane-i.e., cortical-network, which also prevents stellation. Therefore, we propose that RhoA may activate Cdc42 in parallel with an effector, such as p160Rho-kinase, that induces and maintains actin stress fibers in a dominant fashion. Racl is not involved in the stellation process per se but appears to induce a dendritogenic effect. Ultimately, it may be stated that pituicyte stellation is inducible upon mere actin depolymerization, and preventable upon actin organization, be it in the form of stress fibers or in a cortical configuration.
“…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).…”
Section: Discussioncontrasting
confidence: 56%
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
Section: Effects Of Monomeric Gtpase Expression On Pituicyte Morphologymentioning
Our aim was to shed light on different steps leading from metabotropic receptor activation to changes in cell shape, such as those that characterize the morphological plasticity of neurohypophysial astrocytes (pituicytes). Using explant cultures of adult rat pituicytes, we have previously established that adenosine A1 receptor activation induces stellation via inhibition of RhoA monomeric GTPase and subsequent disruption of actin stress fibers. Here, we rule out RhoA phosphorylation as a mechanism for that inhibition. Rather, our results are more consistent with involvement of a GTPase-activating protein (GAP). siRNA and pull-down experiments suggest that a step downstream of RhoA might involve Cdc42, another GTPase of the Rho family. However, RhoA activation, e.g., in the presence of serum, induces stress fibers, whereas direct Cdc42 activation appears to confine actin within a submembrane-i.e., cortical-network, which also prevents stellation. Therefore, we propose that RhoA may activate Cdc42 in parallel with an effector, such as p160Rho-kinase, that induces and maintains actin stress fibers in a dominant fashion. Racl is not involved in the stellation process per se but appears to induce a dendritogenic effect. Ultimately, it may be stated that pituicyte stellation is inducible upon mere actin depolymerization, and preventable upon actin organization, be it in the form of stress fibers or in a cortical configuration.
“…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.…”
Deleted in Liver Cancer 1 (DLC1) is a tumour suppressor that encodes a RhoGTPase-activating protein (RhoGAP) and is frequently inactivated in many human cancers. The RhoGAP activity of DLC1 against Rho signalling is well documented and is strongly associated with the tumour suppressor functions of DLC1. However, the mechanism by which the RhoGAP activity of DLC1 is regulated remains obscure. Here, we report that phosphorylation of DLC1 at Ser549 by cyclic AMP-dependent protein kinase A contributes to enhanced RhoGAP activity and promotes the activation of DLC1, which suppresses hepatoma cell growth, motility and metastasis in both in vitro and in vivo models. Intriguingly, we found that Ser549 phosphorylation induces the dimerization of DLC1 and that inducible dimerization of DLC1 can rescue the tumour suppressive and RhoGAP activities of DLC1 containing a Ser549 deletion. Our study establishes a novel regulatory mechanism for DLC1 RhoGAP activity via dimerization induced by protein kinase A signalling.
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