Moroi Kayoko, Nishiyama Mariko, Kawabata Shin-ichirou, Ichiba Hideaki, Yajima Takehiko, Kimura Sadao
Department of Biochemistry and Molecular Pharmacology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.
Life Sci. 2007 Jun 13;81(1):40-50. doi: 10.1016/j.lfs.2007.04.022. Epub 2007 May 1.
RGS5 is a member of regulators of G protein signaling (RGS) proteins that attenuate heterotrimeric G protein signaling by functioning as GTPase-activating proteins (GAPs). We investigated phosphorylation of RGS5 and the resulting change of its function. In 293T cells, transiently expressed RGS5 was phosphorylated by endogenous protein kinases in the basal state. The phosphorylation was enhanced by phorbol 12-myristate 13-acetate (PMA) and endothelin-1 (ET-1), and suppressed by protein kinase C (PKC) inhibitors, H7, calphostin C and staurosporine. These results suggest involvement of PKC in phosphorylation of RGS5. In in vitro experiments, PKC phosphorylated recombinant RGS5 protein at serine residues. RGS5 protein phosphorylated by PKC showed much lower binding capacity for and GAP activity toward Galpha subunits than did the unphosphorylated RGS5. In cells expressing RGS5, the inhibitory effect of RGS5 on ET-1-induced Ca(2+) responses was enhanced by staurosporine. Mass spectrometric analysis of the phosphorylated RGS5 revealed that Ser166 was one of the predominant phosphorylation sites. Substitution of Ser166 by aspartic acid abolished the binding capacity to Galpha subunits and the GAP activity, and markedly reduced the inhibitory effect on ET-1-induced Ca(2+) responses. These results indicate that phosphorylation at Ser166 of RGS5 by PKC causes loss of the function of RGS5 in G protein signaling. Since this serine residue is conserved in RGS domains of many RGS proteins, the phosphorylation at Ser166 by PKC might act as a molecular switch and have functional significance.
RGS5是G蛋白信号调节因子（RGS）家族的成员之一，它作为GTP酶激活蛋白（GAP），减弱异源三聚体G蛋白信号。我们研究了RGS5的磷酸化及其功能变化。在293T细胞中，瞬时表达的RGS5在基础状态下被内源性蛋白激酶磷酸化。佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）和内皮素-1（ET-1）可增强这种磷酸化，而蛋白激酶C（PKC）抑制剂H7、钙泊三醇C和星形孢菌素则可抑制这种磷酸化。这些结果表明PKC参与了RGS5的磷酸化。在体外实验中，PKC使重组RGS5蛋白的丝氨酸残基磷酸化。与未磷酸化的RGS5相比，被PKC磷酸化的RGS5蛋白对Gα亚基的结合能力和GAP活性要低得多。在表达RGS5的细胞中，星形孢菌素增强了RGS5对ET-1诱导的Ca(2+)反应的抑制作用。对磷酸化RGS5的质谱分析表明，Ser166是主要的磷酸化位点之一。将Ser166替换为天冬氨酸消除了对Gα亚基的结合能力和GAP活性，并显著降低了对ET-1诱导的Ca(2+)反应的抑制作用。这些结果表明，PKC介导的RGS5的Ser166磷酸化导致RGS5在G蛋白信号传导中功能丧失。由于该丝氨酸残基在许多RGS蛋白的RGS结构域中保守，PKC介导的Ser166磷酸化可能作为一种分子开关并具有功能意义。
