Leddy J J, Murphy B J, Doucet J P, Pratt C, Tuana B S
Department of Pharmacology, University of Ottawa, Ontario, Canada.
Biochem J. 1993 Nov 1;295 ( Pt 3)(Pt 3):849-56. doi: 10.1042/bj2950849.
Activation of a calmodulin (CaM)-dependent protein kinase associated with rabbit skeletal-muscle sarcoplasmic reticulum (SR) results in the phosphorylation of polypeptides of 450, 360, 165, 105, 89, 60, 34 and 20 kDa. Radioligand-binding studies indicated that a membrane-bound 60 kDa polypeptide contained both CaM- and ATP-binding domains. Under renaturing conditions on nitrocellulose blots, the 60 kDa polypeptide of the membrane exhibited CaM-dependent autophosphorylation activity, suggesting that it was the CaM-dependent protein kinase of SR. Ca2+/CaM-independent autophosphorylation of polypeptides of 62 and 45 kDa was found to occur in the light SR, whereas the Ca2+/CaM-dependent autophosphorylation activity was enriched in the heavy SR. Both these kinase activities were absent from transverse tubules, although these membranes were enriched in CaM-binding polypeptides of 160, 100 and 80 kDa. In the absence of Ca2+, CaM bound to a 33 kDa polypeptide of the membrane. The purified ryanodine receptor was not phosphorylated by the purified CaM kinase, although it was a substrate for protein kinase C. Affinity-purified antibodies to brain CaM kinase II cross-reacted with the 60 kDa polypeptide in Western blots and immunoprecipitated the 60 kDa polypeptide, along with the 360, 105, 89, 34 and 20 kDa phosphoproteins, from Nonidet-P-40-solubilized SR membranes. Antibodies raised against the 60 kDa kinase polypeptide did not cross-react with the other phosphoproteins, suggesting that these polypeptides were distinct and unrelated. Subcellular distribution of the 60 kDa kinase indicated the specific association of the polypeptide with the junctional-face membrane of SR. The CaM-dependent incorporation of 32P into various membrane proteins was inhibited by the CaM kinase II fragment (290-309), with an IC50 value of 2 nM for the inhibition of incorporation into the 60 kDa kinase polypeptide. Recent studies [Wang and Best (1992) Nature (London) 359, 739-741] have shown that a CaM kinase activity intrinsic to the membrane can inactivate the Ca(2+)-release channel of skeletal muscle SR. Since our results demonstrate that the 60 kDa polypeptide of SR is a CaM-dependent protein kinase, we suggest that this kinase, through its associations, may be responsible for gating the Ca(2+)-release channel.
与兔骨骼肌肌浆网（SR）相关的钙调蛋白（CaM）依赖性蛋白激酶的激活会导致450、360、165、105、89、60、34和20 kDa的多肽发生磷酸化。放射性配体结合研究表明，一种膜结合的60 kDa多肽同时包含CaM和ATP结合结构域。在硝酸纤维素印迹上的复性条件下，膜的60 kDa多肽表现出CaM依赖性的自磷酸化活性，表明它是SR的CaM依赖性蛋白激酶。发现62和45 kDa的多肽在轻肌浆网中发生Ca2+/CaM非依赖性的自磷酸化，而Ca2+/CaM依赖性的自磷酸化活性在重肌浆网中富集。尽管横管富含160、100和80 kDa的CaM结合多肽，但这两种激酶活性在横管中均不存在。在没有Ca2+的情况下，CaM与膜的一种33 kDa多肽结合。纯化的兰尼碱受体不是纯化的CaM激酶的磷酸化底物，尽管它是蛋白激酶C的底物。针对脑CaM激酶II的亲和纯化抗体在蛋白质印迹中与60 kDa多肽发生交叉反应，并从Nonidet-P-40溶解的SR膜中免疫沉淀出60 kDa多肽以及360、105、89、34和20 kDa的磷蛋白。针对60 kDa激酶多肽产生的抗体不与其他磷蛋白发生交叉反应，表明这些多肽是不同且不相关的。60 kDa激酶的亚细胞分布表明该多肽与肌浆网的连接面膜有特异性关联。CaM激酶II片段（290 - 309）抑制了32P向各种膜蛋白中的CaM依赖性掺入，对掺入60 kDa激酶多肽的抑制IC50值为2 nM。最近的研究[Wang和Best（1992年）《自然》（伦敦）359，739 - 741]表明，膜固有的一种CaM激酶活性可以使骨骼肌肌浆网的Ca(2+)释放通道失活。由于我们的结果表明肌浆网的60 kDa多肽是一种CaM依赖性蛋白激酶，我们认为这种激酶通过其关联作用可能负责调控Ca(2+)释放通道。
