Tada Yukiyo, Nimura Takaki, Sueyoshi Noriyuki, Ishida Atsuhiko, Shigeri Yasushi, Kameshita Isamu
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kagawa 761-0795, Japan.
Arch Biochem Biophys. 2006 Aug 15;452(2):174-85. doi: 10.1016/j.abb.2006.06.005. Epub 2006 Jun 21.
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.
钙（2+）/钙调蛋白依赖性蛋白激酶磷酸酶（CaMKP）是丝氨酸/苏氨酸蛋白磷酸酶家族的成员，其催化结构域与蛋白磷酸酶2Cα（PP2Cα）具有29%的序列同一性。为了研究CaMKP的功能结构域，利用在大肠杆菌中表达的各种重组CaMKP进行了突变分析。对N端缺失突变体的分析表明，CaMKP的N端区域在酶催化活性结构的形成中起重要作用，并且在多阳离子刺激中起关键作用。一种嵌合突变体，即CaMKP的N端结构域与PP2Cα的催化结构域的融合体，表现出与CaMKP相似但与PP2Cα不同的底物特异性，这表明CaMKP的N端区域对其独特的底物特异性至关重要。PP2C家族催化结构域中高度保守的氨基酸残基精氨酸-162、天冬氨酸-194、组氨酸-196和天冬氨酸-400的点突变导致磷酸酶活性显著丧失，表明这些氨基酸残基可能在CaMKP的催化活性中起重要作用。虽然C端截短突变体CaMKP（1-412）保留了磷酸酶活性，但发现在37℃孵育时其稳定性远低于野生型CaMKP，这表明CaMKP的C端区域对维持催化活性构象很重要。结果表明，CaMKP的N端和C端序列对CaMKP的调节和稳定性至关重要。
