Regulation of gene expression mediated by Ca^<2+>/calmodulin-dependent protein kinase cascade

钙调素依赖的蛋白激酶(CaM-Ks)是一组不同的酶，参与细胞内钙离子浓度升高所介导的许多细胞反应。以往的研究表明，两种多功能的CaM-KK酶，CaM-KI和IV，通过上游CaM-KK(CaM-KK)对激活环Thr残基的磷酸化而激活，从而大大提高了催化效率。在本研究中，我们发现CaM-KKα中的Ile441是CaM-KK自身抑制所必需的，而CaM与CaM-KKα的结合方向并不是解除自身抑制的关键。首次通过核磁共振分析发现CaM与CaM-KKα结合的独特取向，也被线虫CaM-KK的X-射线结晶学证实。与CaM-KKα相比，CaM-KKβ-异构体表现出更强的钙非依赖性活性，这是由于第二调节域(第129-151位)位于催化结构域的N端。该结构域抑制CaM-KKβ的自身抑制，导致其自主活性的产生。我们还培育了携带Cre-GFP报告基因的线虫，并克隆了线虫CREB。用线虫CaM-K级联蛋白(CaM-KK/CaM-KI)诱导线虫CREB介导的基因表达。在活体虫体中，线虫CaM-Ki 1-295(成分活性突变体)过表达可诱导部分神经元表达GFP-，而CREB缺陷型虫体则未见此现象。这表明CaM-K级联调节依赖于CREB的转录激活在线虫中是保守的。

Ca^<2+>/calmodulin-dependent protein kinases (CaM-Ks) constitute a diverse group of enzymes, which are involved in many cellular responses mediated by an increase in the concentration of intracellular calcium. Previous studies have demonstrated that two multifunctionla CaM-kinases, CaM-KI and IV, are activated byphosphorylation of an activation loop Thr residue by an upstream CaM-kinase (CaM-KK) resulting in a large increase in catalytic efficiency. In this study, we have found that Ile441 in CaM-KKα is essential for the autoinhibition of CaM-KK and the binding orientation of CaM to CaM-KKα is not critical for relief of the autoinhibition. The unique binding orientation of CaM to CaM-KKα which was originally discovered using NMR analysis, was also confirmed with C.elegans CaM-KK by using X-ray crystallography. In contrast to CaM-KKα, CaM-KKβ-isoform has shown to exhibit enhanced Ca^<2+>/CaM-independent activity which is due to the second regulatory domain (residues 129-151) located in N-terminal of the catalytic domain. This domain inhibits the autoinhibition of CaM-KKβ resulting in generation of its autonomous activity. We also have generated C.elegans carrying CRE-GFP reporter gene and cloned C.elegans CREB. C.elegans CREB-mediated gene expression was induced by C.elegans CaM-K cascade (CaM-KK/CaM-KI) in transfected cells. In living worm, GFP-expression was induced by overexpression of C.elegans CaM-KI 1-295 (constitutively active mutant) in some neurons, which was not observed in CREB-deficient worm. This indicates that CaM-K cascade mediats CREB-dependent transcriptional activation is conserved in C.elegans.