Yamamoto H, Arakane F, Ono T, Tashima K, Okumura E, Yamada K, Hisanaga S, Fukunaga K, Kishimoto T, Miyamoto E
Department of Pharmacology, Kumamoto University School of Medicine, Japan.
J Neurochem. 1995 Aug;65(2):802-9. doi: 10.1046/j.1471-4159.1995.65020802.x.
We identified two major substrates for the proline-directed protein kinases--cdc2 kinase and tau protein kinase II (TPKII)--in the cytosol fraction from rat brains. The molecular masses of the proteins were 80 and 46 kDa. Because the 80-kDa protein was phosphorylated by protein kinase C and was heat stable, we examined the possibility that the protein might be myristoylated alanine-rich C kinase substrate (MARCKS). On the basis of a comparison between the properties of the 80-kDa protein and purified MARCKS, we concluded that the 80-kDa protein is indeed MARCKS. The amounts of phosphate incorporated into MARCKS by protein kinase C, cdc2 kinase, and TPKII were 1.7, 1.4, and 0.6 mol/mol of the protein, respectively. Two-dimensional tryptic peptide mapping indicated that phosphorylation sites by protein kinase C and proline-directed protein kinases completely differed. Only the seryl residue was phosphorylated by protein kinase C, whereas both seryl and threonyl residues were phosphorylated by cdc2 kinase and TPKII. Phosphorylation of MARCKS by protein kinase C inhibited the binding to calmodulin, whereas phosphorylation by cdc2 kinase and TPKII significantly increased the binding to calmodulin. The holoenzyme of protein phosphatase 2A dephosphorylated MARCKS that had been phosphorylated by protein kinase C, cdc2 kinase, or TPKII, whereas calcineurin was unable to dephosphorylate it. These results suggest that cdc2 kinase and TPKII regulate the functions of MARCKS in different ways from protein kinase C.
我们在大鼠脑的胞质溶胶组分中鉴定出脯氨酸定向蛋白激酶的两种主要底物——细胞分裂周期蛋白2激酶（cdc2激酶）和tau蛋白激酶II（TPKII）。这些蛋白质的分子量分别为80 kDa和46 kDa。由于80 kDa的蛋白质可被蛋白激酶C磷酸化且热稳定，我们研究了该蛋白质可能是肉豆蔻酰化富含丙氨酸的C激酶底物（MARCKS）的可能性。基于对80 kDa蛋白质与纯化的MARCKS特性的比较，我们得出结论，80 kDa的蛋白质确实是MARCKS。蛋白激酶C、cdc2激酶和TPKII掺入到MARCKS中的磷量分别为1.7、1.4和0.6摩尔/摩尔蛋白质。二维胰蛋白酶肽图谱表明，蛋白激酶C和脯氨酸定向蛋白激酶的磷酸化位点完全不同。蛋白激酶C仅使丝氨酸残基磷酸化，而cdc2激酶和TPKII则使丝氨酸和苏氨酸残基都磷酸化。蛋白激酶C对MARCKS的磷酸化抑制了其与钙调蛋白的结合，而cdc2激酶和TPKII的磷酸化则显著增加了其与钙调蛋白的结合。蛋白磷酸酶2A的全酶可使已被蛋白激酶C、cdc2激酶或TPKII磷酸化的MARCKS去磷酸化，而钙调神经磷酸酶则无法使其去磷酸化。这些结果表明，cdc2激酶和TPKII以与蛋白激酶C不同的方式调节MARCKS的功能。
