AKAPs competing peptide HT31 disrupts the inhibitory effect of PKA on RhoA activity.
A-kinase can inhibit RhoA activation through phosphorylating ser188 of RhoA. AKAP is a novel protein that can target PKA to different subcellular compartment. Evidence has been presented that PKA anchorage by AKAP is important for the kinase to exert its function. This study analyzed the role of PKA anchorage in PKA-induced antagonism against RhoA activity and function. The cells transfected with pcDNA HT31wt/mut were treated with LPA and/or CPT-cAMP. The amount of GTP-RhoA and phosphorylation of RhoA was detected by Western blotting with specific antibodies. The formation of stress fiber was visualized under fluorescent microscope. The gene expression activity was analyzed by luciferase reporter gene assay. The motility and the anchorage-independent growth assays were carried out with stably transfected cells expressing the AKAP inhibitory peptide HT31. The results showed that HT31 not only blocked the PKA-induced phosphorylation of RhoA but also prevented the PKA-induced inhibition on RhoA activation. The disruption of PKA anchorage abolished its inhibition on the LPA-induced expression of reporter gene SRE-luciferase. The ability of PKA to antagonize the LPA-induced stress fiber formation was partly impaired upon the disruption of the PKA anchorage. The control of PKA on migration and the proliferation excited by LPA disappeared in stably transfected cells highly expressing HT31. The results revealed that PKA anchorage was necessary for the kinase to exert its inhibitory effect on RhoA activation and RhoA-dependent biological activities.
Wang
Ying
Y
School of Medicine, Jiangu University, Jiangu Province 212001, P.R. China.
Chen
Yongchang
Y
Chen
Min
M
Xu
Wenrong
W
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D004791:Enzyme Inhibitors; C400224:Ht 31 protein, synthetic; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C100494:RTKN protein, human; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein