Alexander K A, Cimler B M, Meier K E, Storm D R
J Biol Chem. 1987 May 5;262(13):6108-13.
P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.
P - 57是一种具有新型钙调蛋白结合特性的神经特异性钙调蛋白结合蛋白。与存在Ca2 + 时相比，P - 57在无游离Ca2 + 情况下对钙调蛋白 - 琼脂糖具有更高的亲和力（安德烈亚森，T.J.，吕特杰，C.W.，海德曼，W.和斯托姆，D.R.（1983年）《生物化学》22卷，4615 - 4618页；西姆勒，B.M.，安德烈亚森，T.J.，安德烈亚森，K.I.和斯托姆，D.R.（1985年）《生物化学杂志》260卷，10784 - 10788页）。在本研究中，测定了低离子强度和高离子强度条件下P - 57与免疫纯化的5 - [[（碘乙酰氨基）乙基] - 氨基] - 1 - 萘磺酸标记的钙调蛋白（AEDANS - CaM）的解离常数。在未添加KCl的情况下，无论是否存在过量Ca2 + 螯合剂，P - 57×AEDANS - CaM复合物的解离常数分别为2.3×10（ - 7）±6×10（ - 8）M和1.0×10（ - 6）±3×10（ - （ - 7）M。将KCl添加至150 mM会使不依赖Ca2 + 和依赖Ca2 + 的解离常数分别增加至3.4×10（ - 6）±9×10（ - 7）M和3.0×10（ - 6）±9×10（ - 7）M。在低Ca2 + 条件下，测定了P - 57与AEDANS - CaM的结合与KCl浓度的关系。考虑到脑中P - 57的含量及其对钙调蛋白的亲和力，得出结论：在未受刺激的细胞中，大部分或全部钙调蛋白会与P - 57形成复合物。P - 57被Ca2 + - 磷脂依赖性蛋白激酶（蛋白激酶C）磷酸化，磷酸：蛋白质摩尔比为1.3。磷酸氨基酸分析表明在丝氨酸残基处发生了磷酸化。钙调蛋白降低了蛋白激酶C对P - 57的磷酸化速率，且磷酸化阻止了P - 57与钙调蛋白 - 琼脂糖的结合。P - 57未被cAMP依赖性蛋白激酶的催化亚基磷酸化。有人提出，P - 57在细胞内特定部位结合并定位钙调蛋白，并且游离钙调蛋白会因蛋白激酶C对P - 57的磷酸化和/或细胞内游离Ca2 + 的增加而在局部释放。这种调节机制似乎是脑特有的，它将有助于缩短Ca2 + - 钙调蛋白调节过程的响应时间。
