Serine phosphorylation negatively regulates RhoA in vivo.
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.
Ellerbroek
Shawn M
SM
Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599,USA. hawkeye@med.unc.edu
Wennerberg
Krister
K
Burridge
Keith
K
D016475:3T3 Cells; D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D051379:Mice; D016296:Mutagenesis; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins; D012694:Serine / Q000378:metabolism*; D014162:Transfection; D020741:rho GTP-Binding Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; C489577:Rhog protein, mouse; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D005576:Colforsin; D012694:Serine; D006160:Guanosine Triphosphate; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D020558:GTP Phosphohydrolases; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein