Characterization of calreticulin as a protein interacting with protein kinase C
Characterization of calreticulin as a protein interacting with protein kinase C
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DOI：
10.1042/0264-6021:3440469
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发表时间：
1999-12-01
影响因子：
4.1
通讯作者：
Robles-Flores, M
中科院分区：
文献类型：
--
作者：
Rendón-Huerta, E;Mendoza-Hernández, G;Robles-Flores, M
从大鼠肝细胞匀浆中的Triton不溶性组分中纯化蛋白激酶C（PKC）结合蛋白。通过N-末端氨基酸测序和其与针对C-末端KDEL（单字母代码）序列产生的抗钙网蛋白抗体的免疫反应性，鉴定该蛋白为成熟钙网蛋白链。计算的分子量为46.6 kDa，但蛋白质在SDS/PAGE中迁移为表观分子量为60和55 kDa的双峰。体外研究纯化钙网蛋白与使用的覆盖试验方法表明，它结合到激活的PKC同工酶在大鼠肝细胞中表达。磷酸化的纯化钙网蛋白与PKC同工酶特异性免疫复合物激酶测定表明，它也是一个非常好的底物，所有的PKC亚型在体外。用佛波酯或用肾上腺素（肾上腺素）加普萘洛尔处理完整细胞增加钙网蛋白磷酸化，其通过用PKC特异性抑制剂Ro 31-8220预处理细胞来阻断。钙网蛋白免疫沉淀的分析表明，从控制或处理的细胞，PKC α，PKC β，PKC θ，PKC ζ和PKC μ，但不是PKC δ或PKC β，与钙网蛋白共免疫沉淀。两者合计，我们的研究结果表明，PKC在体内与钙网蛋白相互作用，并表明它们可以在共同的信号转导途径。
A protein kinase C (PKC)-binding protein was purified to homogeneity from the Triton-insoluble fraction from rat hepatocytes homogenates. The protein was identified as the mature calreticulin chain by N-terminal amino acid sequencing and by its immunoreactivity with anti-calreticulin antibody raised against the C-terminal KDEL (single-letter code) sequence. The calculated molecular mass was 46.6 kDa but the protein migrates in SDS/PAGE as a doublet with apparent molecular masses of 60 and 55 kDa. Studies in vitro with purified calreticulin with the use of an overlay assay approach demonstrated that it binds to activated PKC isoenzymes expressed in rat hepatocytes. Phosphorylation of purified calreticulin with a PKC isoenzyme-specific immune complex kinase assay showed that it is also a very good substrate for all PKC isoforms in vitro. The treatment of intact cells with phorbol ester or with adrenaline (epinephrine) plus propranolol increased calreticulin phosphorylation, which was blocked by the pretreatment of cells with the PKC-specific inhibitor Ro 31-8220. The analysis of calreticulin immunoprecipitates from control or treated cells indicated that PKC alpha, PKC beta, PKC theta, PKC zeta and PKC mu, but not PKC delta or PKC epsilon, coimmunoprecipitated with calreticulin. Taken together, our results indicate that PKC interacts in vivo with calreticulin and suggest that they can operate in common signalling pathways.