Benner G E, Dennis P B, Masaracchia R A
Department of Biological Sciences, University of North Texas, Denton 76203-5018, USA.
J Biol Chem. 1995 Sep 8;270(36):21121-8. doi: 10.1074/jbc.270.36.21121.
The S6/H4 kinase purified from human placenta catalyzes phosphorylation of the S6 ribosomal protein, histone H4, and myelin basic protein. In vitro activation of the p60 S6/H4 kinase requires removal of an autoinhibitory domain by mild trypsin digestion and autophosphorylation of the catalytic domain (p40 S6/H4 kinase). The two autophosphorylation/autoactivation sites contain the sequences SSMVGTPY (site 1) and SVIDPVPAPVGDSHVDGAAK (site 2). These sequences identify S6H4 kinase as the rac-activated PAK65 (Martin, G. A., Bollag, G., McCormick, F. and Abo, A. (1995) EMBO J. 14, 1971-1978). Site 1 phosphorylation is most rapid, but activation does not occur until site 2 is autophosphorylated. The site 1 phosphorylation occurs by an intramolecular mechanism whereas site 2 autophosphorylation occurs by an intermolecular mechanism. A model is proposed in which phosphorylation of sites 1 and 2 occurs sequentially. The model proposes that trypsin treatment of the inactive holoenzyme removes an inhibitory rac-binding domain which blocks MgATP access to the catalytic site. The pseudosubstrate domain at site 1 is autophosphorylated and subsequent bimolecular autophosphorylation at site 2 fully opens the catalytic site. Phosphorylation by a regulatory protein kinase may occur at site 2 in vivo.
从人胎盘中纯化得到的S6/H4激酶可催化S6核糖体蛋白、组蛋白H4和髓鞘碱性蛋白的磷酸化。p60 S6/H4激酶的体外激活需要通过温和的胰蛋白酶消化去除自抑制结构域，并使催化结构域（p40 S6/H4激酶）进行自磷酸化。两个自磷酸化/自激活位点包含序列SSMVGTPY（位点1）和SVIDPVPAPVGDSHVDGAAK（位点2）。这些序列将S6H4激酶鉴定为rac激活的PAK65（Martin, G. A., Bollag, G., McCormick, F.和Abo, A. (1995) EMBO J. 14, 1971 - 1978）。位点1的磷酸化最快，但直到位点2自磷酸化后才会发生激活。位点1的磷酸化通过分子内机制发生，而位点2的自磷酸化通过分子间机制发生。提出了一个模型，其中位点1和位点2的磷酸化依次发生。该模型提出，对无活性全酶进行胰蛋白酶处理可去除抑制性的rac结合结构域，该结构域会阻止MgATP进入催化位点。位点1的假底物结构域进行自磷酸化，随后位点2的双分子自磷酸化会完全打开催化位点。在体内，调节蛋白激酶的磷酸化可能发生在位点2。
