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/cGMP-dependent 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 Ser188 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.
Highlights
The intracellular protein RhoA, a member of the Ras superfamily of low molecular weight G proteins, regulates cell cycle progression, gene expression, focal adhesion assembly/disassembly, and the acto-myosin generated contraction and tension events of cell motility, matrix remodeling, and cytokinesis (1)
These data provide in vivo evidence that addition of a charged group to Ser188 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
Constitutively active RhoA containing an S188A mutation is more effective in blocking actin dissolution promoted by dibutyryl cAMP (23) or 8-Bromo-cGMP (24), and constitutively active RhoA require a S188A mutation to promote stress fibers in cells co-transfected with constitutively active PKG (14)
Summary
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 Ser188 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. Administration of forskolin, an activator of adenylate cyclase, or dibutyryl cAMP to cells stimulates morphological changes that are strikingly similar to those observed upon introduction of the Rho-specific inhibitor C3-transferase (13) From this initial observation, cAMP- and cGMP-dependent kinase (PKA and PKG) were demonstrated to phosphorylate RhoA on Ser188 (13, 14), thereby joining RhoA with Rap1a and Rap1b as small GTPases regulated by carboxyl PKA phosphorylation (15, 16). Manganello et al (26) demonstrated recently that PKA phosphorylates G␣13 subunit to promote ␥ subunit uncoupling, thereby effectively shutting down receptor activation cysteine-rich domain; GST, glutathione S-transferase; mant, N-methylanthraniloyl; RBD, Rho-binding domain of Rhotekin; RhoGDI, Rho guanine-dissociation inhibitor; ggRhoA, geranylgeranylated RhoA; PKA, cAMP-dependent kinase; PKG, cGMP-dependent kinase
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