Fleming I N, Elliott C M, Buchanan F G, Downes C P, Exton J H
Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.
J Biol Chem. 1999 Apr 30;274(18):12753-8. doi: 10.1074/jbc.274.18.12753.
A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro.
已经鉴定出许多鸟嘌呤核苷酸交换因子，它们通过促进GTP与这些蛋白质的结合来激活Rho家族GTP酶。我们最近证明，溶血磷脂酸和其他几种激动剂可刺激瑞士3T3成纤维细胞中Rac1特异性交换因子Tiam1的磷酸化，并且蛋白激酶C参与Tiam1的磷酸化（Fleming，I.N.，Elliott，C.M.，Collard，J.G.和Exton，J.H.（1997）J.Biol.Chem.272，33105 - 33110）。我们现在通过操纵细胞内[Ca2 +]并使用蛋白激酶抑制剂表明，蛋白激酶Cα和Ca2 + /钙调蛋白依赖性蛋白激酶II在体内均参与Tiam1的磷酸化。此外，我们表明Ca2 + /钙调蛋白依赖性蛋白激酶II在体外使Tiam1磷酸化，在SDS - 聚丙烯酰胺凝胶电泳上产生电泳迁移率减慢。值得注意的是，Ca2 + /钙调蛋白依赖性蛋白激酶II而非蛋白激酶C对Tiam1的磷酸化使其对Rac1的核苷酸交换活性提高了约2倍。此外，Tiam1在体外优先被蛋白磷酸酶1去磷酸化，用这种磷酸酶处理消除了Ca2 + /钙调蛋白依赖性蛋白激酶II对Tiam1的激活。这些数据表明，蛋白激酶Cα和Ca2 + /钙调蛋白依赖性蛋白激酶II在体内使Tiam1磷酸化，并且后者激酶在体外调节该交换因子的活性中起关键作用。
