Molecular Signal Transduction Section, Laboratory of Allergic Diseases, NIAID/NIH, 10 Center Drive, Room 11N242, Bethesda, MD 20892, USA.
J Mol Cell Biol. 2010 Dec;2(6):357-65. doi: 10.1093/jmcb/mjq031. Epub 2010 Oct 25.
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.
G 蛋白信号调节蛋白（RGS）是一种支架蛋白，通过调节信号小体的形成和异源三聚体 G 蛋白的 GTP 酶活性，控制多种信号通路。虽然许多 RGS 蛋白在静止细胞中的表达相对较低，但对环境信号的转录和翻译后反应调节它们的丰度和活性。我们之前发现，RGS 蛋白家族中最小的之一 RGS13 通过与转录因子 cAMP 反应元件结合蛋白（CREB）相互作用，抑制环腺苷酸依赖性蛋白激酶（PKA）诱导的基因表达。在这里，我们表明 PKA 激活还通过 RGS13 磷酸化导致稳态 RGS13 表达增加，从而抑制 RGS13 蛋白降解。在 cAMP 刺激的细胞中，RGS13 周转率显著降低，这可以通过表达 PKA 特异性抑制肽 PKI 逆转。通过质谱鉴定的一个 N 端 Thr 残基（T41）的磷酸化突变，RGS13 磷酸化减少。RGS13 中的 Thr41 突变为 Ala（T41A）与野生型 RGS13 相比，降低了稳态 RGS13 水平及其抑制 M2 毒蕈碱受体介导的 Erk 磷酸化的能力，减弱了 cAMP 对 RGS13 降解的保护作用。RGS13 发生泛素化，表明它是蛋白酶体的一个可能靶标。这些研究首次证明了调节 RGS13 表达的翻译后机制。通过 PKA 介导的磷酸化稳定 RGS13 可以增强 RGS13 的功能，提供促进细胞脱敏的负反馈调节。
