cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKalpha.
Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). We demonstrate that RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKalpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKalpha. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKalpha may be involved in this antagonism.
Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609.
J M
JM
Dong
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T
Leung
E
E
Manser
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Lim
D002460:Cell Line; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008845:Microinjections; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D005576:Colforsin; D012694:Serine; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D013917:Thrombin; D020558:GTP Phosphohydrolases