Identification of major Ca2+/calmodulin-dependent protein kinase phosphatase-binding proteins in brain:: biochemical analysis of the interaction
Identification of major Ca2+/calmodulin-dependent protein kinase phosphatase-binding proteins in brain:: biochemical analysis of the interaction
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DOI：
10.1016/j.abb.2004.11.022
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发表时间：
2005-03-01
影响因子：
3.9
通讯作者：
Kameshita, I
中科院分区：
文献类型：
--
作者：
Ishida, A;Tada, Y;Kameshita, I
Ca 2 +/钙调蛋白依赖性蛋白激酶磷酸酶（CaMKP）是一种特异性去磷酸化和调节多功能Ca 2 +/钙调蛋白依赖性蛋白激酶（CaMK）的蛋白磷酸酶。为了阐明CaMKP的生理意义，我们确定了甘油醛-3-磷酸脱氢酶（GAPDH）和果糖二磷酸醛缩酶作为主要的结合伴侣的CaMKP在大鼠脑的可溶性部分，使用二维远蛋白质印迹技术，结合肽质量指纹分析。我们分析了这些相互作用的亲和力。在GST下拉测定中与GAPDH相关的野生型CaMKP-谷胱甘肽S-转移酶（GST）。缺失分析表明CaMKP催化结构域的N端侧负责与GAPDH的结合。此外，抗CaMKP抗体在大鼠脑提取物中共免疫沉淀GAPDH。GAPDH在体外被CaMKI或CaMKIV磷酸化;然而，当CaMKP共存时，磷酸化显著减弱。在这些条件下，CaMKP显着去磷酸化CaMKI和CaMKIV，这已经被CaMK激酶磷酸化，而它没有去磷酸化先前磷酸化的GAPDH。结果表明，CaMKP通过去磷酸化和失活负责GAPDH磷酸化的CaMK来调节CaMKP-GAPDH复合物中GAPDH的磷酸化水平。(C)2004爱思唯尔公司All rights reserved.
Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP-glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP was responsible for the binding to GAPDH. Further, anti-CaMKP antibody coimmunoprecipitated GAPDH in a rat brain extract. GAPDH was phosphorylated by CaMKI or CaMKIV in vitro; however, when CaMKP coexisted, the phosphorylation was markedly attenuated. Under these conditions, CaMKP significantly dephosphorylated CaMKI and CaMKIV, which had been phosphorylated by CaMK kinase, whereas it did not dephosphorylate the previously phosphorylated GAPDH. The results suggest that CaMKP regulates the phosphorylation level of GAPDH in the CaMKP-GAPDH complex by dephosphorylating and deactivating CaMKs that Lire responsible for the phosphorylation of GAPDH. (C) 2004 Elsevier Inc. All rights reserved.