Brady M J, Palfrey H C
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
J Biol Chem. 1993 Aug 25;268(24):17951-8.
Nerve growth factor (NGF) treatment of PC12 cells led to the rapid phosphorylation of a calmodulin-binding protein of 100 kDa (CaM-BP100) identified on blot overlays with 125I-labeled CaM. The effect was detected as a retardation in the mobility of the protein by an apparent 10 kDa on SDS gels. The mobility shift was complete within 5 min and was maintained for 24 h in the continued presence of NGF. The protein was present in both the soluble and crude particulate fractions, and the gel mobility shift occurred in both fractions. Epidermal growth factor elicited a similar response, but the mobility shift was reversed within 12 h. The gel retardation was due to phosphorylation of CaM-BP100, as it could be reversed if cytoplasmic extracts were held under dephosphorylating conditions at 37 degrees C for 10 min prior to electrophoresis; dephosphorylation was inhibited by okadaic acid but not vanadate, suggesting the participation of a Ser/Thr phosphatase. Treatment with either acid or alkaline phosphatase also reversed the mobility shift. CaM-BP100 phosphorylation was stimulated by 12-O-tetradecanoylphorbol-13-acetate in intact cells, but the effect of NGF did not involve a protein kinase C-dependent process, because it occurred in PC12 cells depleted of protein kinase C. The phosphorylation event appeared to be due to an NGF-stimulated protein kinase, as mixing extracts from NGF-treated cells with extracts from control cells in the presence of ATP and Mg2+ reconstituted the mobility shift in vitro. CaM-BP100 appears to be a minor cellular phosphoprotein, as 32P labeling of the protein could not be detected in crude cell extracts. These results suggest that receptor tyrosine kinases communicate with at least one component of the Ca2+/calmodulin-signaling pathway early in signal transduction.
用神经生长因子（NGF）处理PC12细胞，导致一种100kDa的钙调蛋白结合蛋白（CaM - BP100）快速磷酸化，该蛋白在与125I标记的钙调蛋白的印迹覆盖实验中得以鉴定。在SDS凝胶上，该效应表现为该蛋白迁移率明显减慢10kDa。迁移率变化在5分钟内完成，并在持续存在NGF的情况下维持24小时。该蛋白存在于可溶性和粗颗粒部分，凝胶迁移率变化在两个部分均发生。表皮生长因子引发类似反应，但迁移率变化在12小时内逆转。凝胶阻滞是由于CaM - BP100的磷酸化，因为如果在电泳前将细胞质提取物在37℃的去磷酸化条件下保持10分钟，迁移率变化可以逆转；去磷酸化被冈田酸抑制，但不被钒酸盐抑制，提示丝氨酸/苏氨酸磷酸酶参与其中。用酸性或碱性磷酸酶处理也能逆转迁移率变化。在完整细胞中，12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯刺激CaM - BP100磷酸化，但NGF的作用不涉及蛋白激酶C依赖的过程，因为在缺乏蛋白激酶C的PC12细胞中也会发生。磷酸化事件似乎是由于NGF刺激的蛋白激酶，因为在ATP和Mg2+存在的情况下，将NGF处理细胞的提取物与对照细胞的提取物混合，可在体外重建迁移率变化。CaM - BP100似乎是一种次要的细胞磷蛋白，因为在粗细胞提取物中无法检测到该蛋白的32P标记。这些结果表明，受体酪氨酸激酶在信号转导早期与Ca2+/钙调蛋白信号通路的至少一个组分进行通讯。
