Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src
Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src
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DOI：
10.1016/j.jmb.2004.02.041
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发表时间：
2004-04-16
影响因子：
5.6
通讯作者：
Matsushima, N
中科院分区：
文献类型：
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作者：
Hayashi, N;Nakagawa, C;Matsushima, N
pp 60（v-src）酪氨酸蛋白激酶可能与钙离子结合的钙调素相互作用（1）pp 60（v-src）的N端与CAP-23/NAP-22一样被豆蔻酰化，而CAP-23/ NAP-22是一种豆蔻酰化的神经元特异性蛋白，其豆蔻酰基团是与Ca ~（2+）/CaM相互作用所必需的;（2）赖氨酸残基是豆蔻酰化依赖性相互作用所必需的，丝氨酸残基被认为通过位于pp 60（v-src）的N-末端区域的磷酸化来调节相互作用。为了验证这种可能性，我们研究了pp 60（v-src）和Ca 2 +/CaM之间的直接相互作用，使用豆蔻酰化肽对应于pp 60（v-src）的N-末端区域。结合试验表明，只有豆蔻酰化的肽与Ca 2 +/CaM结合，而非豆蔻酰化的肽不能与Ca 2 +/CaM结合。结合动力学分析显示两个独立反应，解离常数（K-D）分别为2.07 x 10（-9）M（K-D1）和3.93 x 10（-6）M（K-D2）。两个丝氨酸残基附近的豆蔻酰部分的肽（Ser 2，Ser 11）被磷酸化的蛋白激酶C在体外，和磷酸化大大降低了相互作用。NMR实验表明，两个豆蔻酰化肽分子结合在Ca 2 +/CaM分子的疏水裂缝周围。小角X-射线散射分析表明，肽-Ca ~（2+）/CaM复合物的尺寸比已知的Ca ~（2+）/CaM-靶分子复合物的尺寸小2-3埃。这些结果清楚地表明pp 60（v-src）与Ca ~（2+）/CaM之间的直接相互作用以不同于已知的Ca ~（2+）/CaM（靶分子）相互作用的新方式进行。(C)2004爱思唯尔有限公司保留所有权利。
pp60(v-src) tyrosine protein kinase was suggested to interact with Ca2+-bound calmodulin (Ca2+/CaM) through the N-terminal region based on its structural similarities to CAP-23/NAP-22, a myristoylated neuron-specific protein, whose myristoyl group is essential for interaction with Ca2+/CaM; (1) the N terminus of pp60(v-src) is myristoylated like CAP-23/ NAP-22; (2) both lysine residues are required for the myristoylation-dependent interaction and serine residues that are thought to regulate the interaction through the phosphorylations located in the N-terminal region of pp60(v-src). To verify this possibility, we investigated the direct interaction between pp60(v-src) and Ca2+/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60(v-src). The binding assay indicated that only the myristoylated peptide binds to Ca2+/CaM, and the non-myristoylated peptide is not able to bind to Ca2+/CaM. Analyses of the binding kinetics revealed two independent reactions with the dissociation constants (K-D) of 2.07 x 10(-9) M (K-D1) and 3.93 x 10(-6) M (K-D2), respectively. Two serine residues near the myristoyl moiety of the peptide (Ser2, Ser11) were phosphorylated by protein kinase C in vitro, and the phosphorylation drastically reduced the interaction. NMR experiments indicated that two molecules of the myristoylated peptide were bound around the hydrophobic clefts of a Ca2+/CaM molecule. The small-angle X-ray scattering analyses showed that the size of the peptide-Ca2+/CaM complex is 2-3 Angstrom smaller than that of the known Ca2+/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60(v-src) and Ca2+/CaM in a novel manner different from that of known Ca2+/CaM, the target molecules, interactions. (C) 2004 Elsevier Ltd. All rights reserved.