Rosado J A, Salido G M, García L J
Department of Physiology, University of Extremadura, Cáceres, 10080-, Spain.
Arch Biochem Biophys. 2000 May 1;377(1):85-94. doi: 10.1006/abbi.2000.1761.
Tyrosine phosphorylation plays a key role in transmembrane and cytoplasmic signal transduction mechanisms stimulated by oncogenes, integrins, growth factors, neuropeptides, and bioactive lipids. Moreover, recent studies show that stimulation of odd-numbered muscarinic receptors increases the tyrosine phosphorylation of several proteins in different cellular types. The present study was aimed at examining whether activation of m3 muscarinic receptors in rat pancreatic acini evokes tyrosine phosphorylation of p125(FAK), and its substrates, p130(cas) and paxillin. Results show that stimulation of pancreatic acini with carbachol resulted in a rapid and transient increase in tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin. Tyrosine phosphorylation of these proteins occurred in a time- and concentration-dependent manner. Simultaneous blockage of both PKC activation and increases in Ca(2+) partially decreased p125(FAK), p130(cas), and paxillin tyrosine phosphorylation stimulated by carbachol. Pretreatment of pancreatic acini with Clostridium botulinum C3 transferase, which specifically inactivates p21(rho), partially inhibited carbachol-induced p125(FAK), p130(cas), and paxillin tyrosine phosphorylation. In contrast, this treatment had no effect on amylase release stimulated by carbachol. Cytochalasin D, which disrupts actin microfilaments network, completely inhibited carbachol stimulated tyrosine phosphorylation of these proteins without having significant effects in carbachol-stimulated amylase secretion. These results dissociate tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin from amylase secretion after m3 muscarinic receptors occupation in rat pancreatic acini. Taken together, these data suggest that (a) activation of m3 muscarinic receptors in rat pancreatic acini increases tyrosine phosphorylation of p125(FAK) and its substrates, p130(cas) and paxillin by diacylglycerol-activated PKC- and calcium- dependent, and independent pathways, (b) these responses require activation of p21(rho) and an intact actin cytoskeleton, and (c) p125(FAK), p130(cas), and paxillin are unlikely related to secretion in rat pancreatic acinar cells.
酪氨酸磷酸化在由癌基因、整合素、生长因子、神经肽和生物活性脂质刺激的跨膜和细胞质信号转导机制中起关键作用。此外，最近的研究表明，刺激奇数毒蕈碱受体可增加不同细胞类型中几种蛋白质的酪氨酸磷酸化。本研究旨在检测大鼠胰腺腺泡中m3毒蕈碱受体的激活是否会引起p125（黏着斑激酶）及其底物p130（接头蛋白cas）和桩蛋白的酪氨酸磷酸化。结果显示，用卡巴胆碱刺激胰腺腺泡会导致p125（黏着斑激酶）、p130（接头蛋白cas）和桩蛋白的酪氨酸磷酸化迅速且短暂增加。这些蛋白质的酪氨酸磷酸化呈时间和浓度依赖性。同时阻断蛋白激酶C（PKC）的激活和细胞内钙离子浓度（Ca(2+)）的升高，可部分降低卡巴胆碱刺激引起的p125（黏着斑激酶）、p130（接头蛋白cas）和桩蛋白的酪氨酸磷酸化。用肉毒杆菌C3转移酶预处理胰腺腺泡，该酶可特异性使p21（小G蛋白rho）失活，可部分抑制卡巴胆碱诱导的p125（黏着斑激酶）、p130（接头蛋白cas）和桩蛋白的酪氨酸磷酸化。相比之下，这种处理对卡巴胆碱刺激的淀粉酶释放没有影响。细胞松弛素D可破坏肌动蛋白微丝网络，完全抑制卡巴胆碱刺激的这些蛋白质的酪氨酸磷酸化，而对卡巴胆碱刺激的淀粉酶分泌没有显著影响。这些结果表明，在大鼠胰腺腺泡中m3毒蕈碱受体被占据后，p125（黏着斑激酶）、p130（接头蛋白cas）和桩蛋白的酪氨酸磷酸化与淀粉酶分泌无关。综上所述，这些数据表明：（a）大鼠胰腺腺泡中m3毒蕈碱受体的激活通过二酰甘油激活的PKC和钙依赖性及非依赖性途径增加p125（黏着斑激酶）及其底物p130（接头蛋白cas）和桩蛋白的酪氨酸磷酸化；（b）这些反应需要p21（小G蛋白rho）的激活和完整的肌动蛋白细胞骨架；（c）p125（黏着斑激酶）、p130（接头蛋白cas）和桩蛋白不太可能与大鼠胰腺腺泡细胞的分泌有关。
