Haribabu B, Hook S S, Selbert M A, Goldstein E G, Tomhave E D, Edelman A M, Snyderman R, Means A R
Department of Pharmacology, Duke University Medical Center, Durham, NC 27710, USA.
EMBO J. 1995 Aug 1;14(15):3679-86. doi: 10.1002/j.1460-2075.1995.tb00037.x.
Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.
人钙调蛋白（CaM）依赖性蛋白激酶I（CaMKI）编码一种含370个氨基酸的蛋白质，计算所得分子量（M(r)）为41337。1.5 kb的CaMKI信使核糖核酸（mRNA）在多种不同的人体组织中表达，是位于人类3号染色体上单个基因的产物。CaMKI 1 - 306无法结合钙 - 钙调蛋白（Ca(2+)-CaM），且完全无活性，由此确定CaM结合结构域的一个必需成分位于306位残基的C末端。CaMKI 1 - 294不结合CaM，但在没有Ca(2+)-CaM的情况下完全有活性，这表明295 - 306位残基足以使CaMKI维持在自抑制状态。CaMKI在苏氨酸177（Thr177）位点被磷酸化，其活性通过CaMKI激酶以钙 - 钙调蛋白依赖性方式增强约25倍。用丙氨酸（Ala）或天冬氨酸（Asp）取代Thr177可防止CaMKI激酶的磷酸化和激活，后一种取代在没有CaMKI激酶的情况下也导致部分激活。虽然CaMKI 1 - 306对CaMKI激酶无反应，但1 - 294突变体在有和没有Ca(2+)-CaM的情况下均被CaMKI激酶磷酸化并激活，不过在有Ca(2+)-CaM时激活速率更快。这些结果表明，CaMKI中的自抑制结构域以钙 - 钙调蛋白依赖性方式控制底物与底物结合裂隙的可及性以及CaMKI激酶与Thr177的可及性。此外，CaMKI激酶直接对钙 - 钙调蛋白作出反应，活性增加。
