Yue C, Ku C Y, Liu M, Simon M I, Sanborn B M
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77225, USA.
J Biol Chem. 2000 Sep 29;275(39):30220-5. doi: 10.1074/jbc.M004276200.
Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLCbeta) pathway. In turn, phosphorylation of PLCbeta by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca(2+) release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Galpha(q)-coupled (oxytocin and M1 muscarinic) and Galpha(i)-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCbeta(3). Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLCbeta(3) was stimulated directly by Galpha(q) or Gbetagamma in overexpression assays. PKC phosphorylated PLCbeta(3) at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCbeta(3) and site-directed mutagenesis identified Ser(1105) as the predominant phosphorylation site. Ser(1105) is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023-18027). Similar to PKA, the inhibition by PKC of Galpha(q)-stimulated PLCbeta(3) activity was completely abolished by mutation of Ser(1105) to Ala. In contrast, mutation of Ser(1105) or Ser(26), another putative phosphorylation target, to Ala had no effect on inhibition of Gbetagamma-stimulated PLCbeta(3) activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Galpha(q)-stimulated PLCbeta(3) as a result of phosphorylation of Ser(1105). Moreover, PKC and PKA both inhibit Gbetagamma-stimulated activity by mechanisms that do not involve Ser(1105).
蛋白激酶C（PKC）的激活可由受体 - G蛋白 - 磷脂酶C（PLCβ）途径的刺激引发。反过来，PKC对PLCβ的磷酸化可能在受体介导的磷脂酰肌醇（PI）周转和细胞内Ca(2+)释放的调节中发挥作用。佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯对内源性PKC的激活在表达PLCβ(3)的PHM1、HeLa、COSM6和RBL - 2H3细胞中，使Gα(q)偶联（催产素和M1毒蕈碱）以及Gα(i)偶联（甲酰 - 甲硫氨酸 - 亮氨酸 - 苯丙氨酸）受体刺激的PI周转抑制了50 - 100%。用百里酚毒素激活传统的PKC同样抑制了催产素或甲酰 - 甲硫氨酸 - 亮氨酸 - 苯丙氨酸受体刺激的PI周转。在过表达实验中，当PLCβ(3)直接由Gα(q)或Gβγ刺激时，也观察到了PKC的抑制作用。PKC在体内和体外都在相同的主要位点磷酸化PLCβ(3)。对体外磷酸化的重组PLCβ(3)进行肽段测序和定点诱变确定Ser(1105)为主要磷酸化位点。Ser(1105)也可被蛋白激酶A（PKA；Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023 - 18027）磷酸化。与PKA类似，将Ser(1105)突变为丙氨酸可完全消除PKC对Gα(q)刺激的PLCβ(3)活性的抑制作用。相反，将Ser(1105)或另一个假定的磷酸化靶点Ser(26)突变为丙氨酸，对PKC或PKA抑制Gβγ刺激的PLCβ(3)活性没有影响。这些数据表明，PKC和PKA的作用方式相似，即它们通过Ser(1105)的磷酸化抑制Gα(q)刺激的PLCβ(3)。此外，PKC和PKA都通过不涉及Ser(1105)的机制抑制Gβγ刺激的活性。
