Phosphorylation of RGS13 by the cyclic AMP-dependent protein kinase inhibits RGS13 degradation.
Regulators of G-protein signaling (RGS) proteins are scaffolds that control diverse signaling pathways by modulating signalosome formation and by accelerating the GTPase activity of heterotrimeric G proteins. Although expression of many RGS proteins is relatively low in quiescent cells, transcriptional and post-translational responses to environmental cues regulate both their abundance and activity. We found previously that RGS13, one of the smallest RGS proteins in the family, inhibited cyclic AMP-dependent protein kinase (PKA)-induced gene expression through interactions with the transcription factor cAMP-response element-binding (CREB) protein. Here, we show that PKA activation also leads to increased steady-state RGS13 expression through RGS13 phosphorylation, which inhibits RGS13 protein degradation. RGS13 turnover was significantly reduced in cells stimulated with cAMP, which was reversed by expression of the PKA-specific inhibitory peptide PKI. RGS13 phosphorylation was diminished by mutation of an N-terminal Thr residue (T41) identified as a phosphorylation site by mass spectrometry. Mutation of Thr41 in RGS13 to Ala (T41A) reduced steady-state RGS13 levels and its ability to inhibit M2 muscarinic receptor-mediated Erk phosphorylation compared with wild-type RGS13 by attenuating the protective effect of cAMP on RGS13 degradation. RGS13 underwent ubiquitylation, indicating that it is a likely target of the proteasome. These studies are the first to demonstrate post-translational mechanisms controlling the expression of RGS13. Stabilization of RGS13 through PKA-mediated phosphorylation could enhance RGS13 functions, providing negative feedback regulation that promotes cellular desensitization.
Molecular Signal Transduction Section, Laboratory of Allergic Diseases, NIAID/NIH, 10 Center Drive, Room 11N242, Bethesda, MD 20892, USA.
Zhihui
Z
Xie
Zhao
Z
Yang
Kirk M
KM
Druey
D019943:Amino Acid Substitution; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043585:Receptor, Muscarinic M2 / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination
D020710:RGS Proteins; C456322:RGS13 protein, human; D043585:Receptor, Muscarinic M2; D025801:Ubiquitin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D046988:Proteasome Endopeptidase Complex