Yue C, Dodge K L, Weber G, Sanborn B M
Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School, Houston, Texas 77225, USA.
J Biol Chem. 1998 Jul 17;273(29):18023-7. doi: 10.1074/jbc.273.29.18023.
The mechanism by which protein kinase A (PKA) inhibits Galphaq -stimulated phospholipase C activity of the beta subclass (PLCbeta ) is unknown. We present evidence that phosphorylation of PLCbeta3 by PKA results in inhibition of Galphaq -stimulated PLCbeta3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCbeta3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Galphaq with PLCbeta3 or PLCbeta (Ser1105--> Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Galphaq, PLCbeta3, or PLCbeta3 (Ser1105 --> Ala mutant alone. Whereas Galpha1-stimulated PLCbeta3, activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, Galphaq-stimulated PLCbeta3 (Ser1105 --> Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Galpha1-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCbeta3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCbeta3 inhibits Galphaq-stimulated PLCbeta3 activity. This can at least partially explain the inhibitory effect of PKA on Galphaq-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.
蛋白激酶A（PKA）抑制Gαq刺激的β亚类磷脂酶C（PLCβ）活性的机制尚不清楚。我们提供的证据表明，PKA对PLCβ3的磷酸化导致Gαq刺激的PLCβ3活性受到抑制，并且我们确定了磷酸化位点。对体外磷酸化的PLCβ3进行二维磷酸氨基酸分析，发现一个磷酸丝氨酸作为假定的PKA位点，肽图谱分析产生了一个磷酸肽。通过反相高压液相色谱分离的磷酸肽直接测序和定点诱变，确定该残基为Ser1105。在COSM6细胞中过表达Gαq与PLCβ3或PLCβ（Ser1105→Ala）突变体，与单独表达Gαq、PLCβ3或PLCβ3（Ser1105→Ala）突变体相比，[3H]磷脂酰肌醇1,4,5-三磷酸的形成增加了5倍。虽然过表达PKA催化亚基可使Gα1刺激的PLCβ3活性受到58-71%的抑制，但Gαq刺激的PLCβ3（Ser1105→Ala）突变体活性不受影响。此外，在仅表达PLCβ3的RBL-2H3细胞中，用8-[4-氯苯硫基]-cAMP预处理细胞可完全抑制由推测的Gα1偶联的M1毒蕈碱受体和催产素受体刺激的磷脂酰肌醇转换。这些数据表明，PKA对PLCβ3假定G盒中Ser1105的直接磷酸化抑制了Gαq刺激的PLCβ3活性。这至少可以部分解释PKA对在多种细胞和组织中观察到的Gαq刺激的磷脂酰肌醇转换的抑制作用。
