Serine phosphorylation differentially affects RhoA binding to effectors: implications to NGF-induced neurite outgrowth

Activation of RhoA prevents NGF-induced outgrowth and causes retraction of neurites in neuronal cells, including PC12 cells. Despite its inhibitory effect on neurite outgrowth, NGF activates GTP loading of and effector binding to RhoA, setting up an apparent contradiction. According to the molecular switch hypothesis of GTPase function GTP-loading of RhoA should be sufficient to activate its effectors uniformly. However, when monitoring NGF-induced binding of GTP-RhoA to multiple targets, we noted differential interactions with its effectors. We found that NGF elicits a protein kinase A-mediated phosphorylation of RhoA on serine(188), which renders it unable to bind to Rho-associated kinase (ROK), whereas it retains the ability to interact with other RhoA targets including rhotekin, mDia-1 and PKN. We show in vitro and in vivo that phosphorylation of serine(188) represents an additional switch, capable of directing signals among effector pathways. In the context of PC12 cell differentiation, NGF-induced phosphorylation of RhoA on serine(188) prevents it from interacting with ROK, which would otherwise block neurite outgrowth. Transfection of RhoA(S188A) mutant into PC12 cells prevents NGF-induced neurite outgrowth, just like constitutively activated RhoA(14V) does, indicating the requirement of this phosphorylation site. Replacement of serine(188) with the phosphomimetic glutamate residue in RhoA(V14/S188E) selectively impairs interaction with ROK and when transfected into PC12 cells restores NGF-induced neurite outgrowth. Therefore, phosphorylation of serine(188) may serve as a novel secondary switch of RhoA capable of overriding GTP-binding-elicited effector activation to a subset of targets such as ROK, which interact with the C-terminus of RhoA.     

Models of the cooperative mechanism for Rho effector recognition: implications for RhoA-mediated effector activation

Activated GTPases of the Rho family regulate a spectrum of functionally diverse downstream effectors, initiating a network of signal transduction pathways by interaction and activation of effector proteins. Although effectors are defined as proteins that selectively bind the GTP-bound state of the small GTPases, there have been also several indications for a nucleotide-independent binding mode. By characterizing the molecular mechanism of RhoA interaction with its effectors, we have determined the equilibrium dissociation constants of several Rho-binding domains of three different effector proteins (Rhotekin, ROCKI/ROK beta/p160ROCK, PRK1/PKNalpha where ROK is RhoA-binding kinase) for both RhoA.GDP and RhoA.GTP using fluorescence spectroscopy. In addition, we have identified two novel Rho-interacting domains in ROCKI, which bind RhoA with high affinity but not Cdc42 or Rac1. Our results, together with recent structural data, support the notion of multiple effector-binding sites in RhoA and strongly indicate a cooperative binding mechanism for PRK1 and ROCKI that may be the molecular basis of Rho-mediated effector activation.     

Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Galpha12/13.

A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. We found that Tec and Bmx activate serum response factor (SRF), in synergy with constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where thrombin's effect is mediated by G12/13 proteins. Moreover, expression of Galpha12 and Galpha13 stimulates autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3 did not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed.     

Activation of RhoA by Lysophosphatidic Acid and Gα12/13 Subunits in Neuronal Cells: Induction of Neurite Retraction

Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein-coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Galpha12 and Galpha13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity.     

Socius, a novel binding partner of Galpha12/13, promotes the Galpha12-induced RhoA activation

Heterotrimeric G proteins act as a molecular switch that conveys signals from G protein-coupled receptors in the cell membrane to intracellular downstream effectors. The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), have many cellular functions through their specific downstream effectors. On the other hand, regulatory systems of the activity of Galpha(12) and Galpha(13) have not been fully clear. Here, we show that Socius, a previously identified Rho family small GTPase Rnd1 interacting protein, binds directly to Galpha(12) and Galpha(13) through its NH(2)-terminal region. Socius increased the amounts of GTP-bound active form of Galpha(12) in 293T cells. Furthermore, Socius promotes the Galpha(12)-induced RhoA activation in 293T cells. These results demonstrate that Socius is a novel activator of the Galpha(12) family.     

cGMP-dependent protein kinase phosphorylates and inactivates RhoA

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.     

RhoA inhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway

The Rho family of small GTPases has been shown to be involved in the regulation of neuronal morphology, and Rac and Rho exert antagonistic actions in neurite formation. In this study, we have examined the cross-talk between Rac and Rho in relation to the nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. NGF induced a rapid activation of Rac1 and suppression of RhoA activity. Constitutively active RhoA, RhoA(V14), or constitutively active Galpha(12)-induced endogenous RhoA activation inhibited the NGF-induced Rac1 activation without any effect on the NGF-induced extracellular signal-regulated kinase activation. Moreover, Y-27632, an inhibitor of Rho-associated kinase, completely abolished the RhoA-induced down-regulation of the NGF-induced Rac1 activation. We also revealed that NGF induced a rapid recruitment of Rac1 to the cell surface protrusion sites and formed filamentous actin-rich protrusions. Activation of RhoA and Rho-associated kinase formed a thick ringlike structure of cortical actin filaments at the cell periphery and then inhibited the NGF-induced recruitment of Rac1 to protrusions. These results indicate that RhoA down-regulates the NGF- induced Rac1 activation through Rho-associated kinase, inhibiting the neurite formation.     

Physiological roles of Rho and Rho effectors in mammals

Rho GTPase is a master regulator controlling cytoskeleton in multiple contexts such as cell migration, adhesion and cytokinesis. Of several Rho GTPases in mammals, the best characterized is the Rho subfamily including ubiquitously expressed RhoA and its homologs RhoB and RhoC. Upon binding GTP, Rho exerts its functions through downstream Rho effectors, such as ROCK, mDia, Citron, PKN, Rhophilin and Rhotekin. Until recently, our knowledge about functions of Rho and Rho effectors came mostly from in vitro studies utilizing cultured cells, and their physiological roles in vivo were largely unknown. However, gene-targeting studies of Rho and its effectors have now unraveled their tissue- and cell-specific roles and provide deeper insight into the physiological function of Rho signaling in vivo. In this article, we briefly describe previous studies of the function of Rho and its effectors in vitro and then review and discuss recent studies on knockout mice of Rho and its effectors.     

Galpha(12) and galpha(13) inhibit Ca(2+)-dependent exocytosis through Rho/Rho-associated kinase-dependent pathway

The release of neurotransmitters is known to be regulated by activation of heterotrimeric G protein-coupled receptors, although precise mechanisms have not yet been elucidated. To assess the role of the G(12) family of heterotrimeric G proteins in the regulation of neurotransmitter release, we established PC12 cell lines that expressed constitutively active Galpha(12) or Galpha(13) using an isopropyl-beta-D-thiogalactoside-inducible expression system. In the cells, expression of constitutively active Galpha(12) or Galpha(13) inhibited the high K(+)-evoked [(3)H]dopamine release without any effect on the high K(+)-induced increase in intracellular Ca(2+) concentration. A Ca(2+) ionophore ionomycin-induced [(3)H]dopamine release was also inhibited by the expression of active Galpha(12) or Galpha(13). These inhibitory effects of Galpha(12) and Galpha(13) on [(3)H]dopamine release were mimicked by the expression of constitutively active RhoA. In addition, Y-27632, and inhibitor of Rho-associated kinase, a downstream Rho effector, completely abolished the inhibition of [(3)H]dopamine release by Galpha(12), Galpha(13), and RhoA. These results indicate that Ca(2+)-dependent exocytosis is regulated by Galpha(12) and Galpha(13) through a Rho/Rho-associated kinase-dependent pathway.