Ishida Atsuhiko, Tada Yukiyo, Nimura Takaki, Sueyoshi Noriyuki, Katoh Tsuyoshi, Takeuchi Masayuki, Fujisawa Hitoshi, Taniguchi Takanobu, Kameshita Isamu
Department of Biochemistry, Asahikawa Medical College, Asahikawa, Japan.
Arch Biochem Biophys. 2005 Mar 1;435(1):134-46. doi: 10.1016/j.abb.2004.11.022.
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca(2+)/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 are responsible for the phosphorylation of GAPDH.
钙（2+）/钙调蛋白依赖性蛋白激酶磷酸酶（CaMKP）是一种独特的蛋白磷酸酶，它特异性地使多功能钙（2+）/钙调蛋白依赖性蛋白激酶（CaMKs）去磷酸化并对其进行调节。为了阐明CaMKP的生理意义，我们使用二维远 Western 印迹技术结合肽质量指纹图谱分析，在大鼠脑的可溶性部分中鉴定出甘油醛-3-磷酸脱氢酶（GAPDH）和果糖二磷酸醛缩酶是CaMKP的主要结合伴侣。我们分析了这些相互作用的亲和力。在GST下拉实验中，野生型CaMKP-谷胱甘肽S-转移酶（GST）与GAPDH结合。缺失分析表明，CaMKP催化结构域的N端负责与GAPDH的结合。此外，抗CaMKP抗体在大鼠脑提取物中与GAPDH进行了共免疫沉淀。GAPDH在体外被CaMKI或CaMKIV磷酸化；然而，当CaMKP共存时，磷酸化明显减弱。在这些条件下，CaMKP使已被CaMK激酶磷酸化的CaMKI和CaMKIV显著去磷酸化，而它不会使先前磷酸化的GAPDH去磷酸化。结果表明，CaMKP通过使负责GAPDH磷酸化的CaMKs去磷酸化并失活，从而调节CaMKP-GAPDH复合物中GAPDH的磷酸化水平。
