Regulation of protein phosphorylation in hamster insulinoma cells. Identification of Ca2+-regulated cytoskeletal and cAMP-regulated cytosolic phosphoproteins by two-dimensional electrophoresis.
Regulation of protein phosphorylation in hamster insulinoma cells. Identification of Ca2+-regulated cytoskeletal and cAMP-regulated cytosolic phosphoproteins by two-dimensional electrophoresis.
复制标题
DOI：
10.1016/s0021-9258(18)33705-0
复制
发表时间：
1982-10
期刊：
影响因子：
--
通讯作者：
U. Schubart
中科院分区：
文献类型：
--
作者：
U. Schubart
用双向凝胶电泳法和放射自显影技术分析了~(32)P标记的仓鼠胰岛素瘤细胞中依赖于钙和cAMP的蛋白磷酸化。在40m~K‘的条件下孵育细胞，引起电压依赖性的钙离子内流和胰岛素释放，刺激三种电荷略有变化的60,000道尔顿蛋白的磷酸化。胰升糖素(3p~)通过增加细胞内cAMP浓度促进胰岛素的释放，刺激两个16,000道尔顿多肽的磷酸化。用双向电泳法对几种蛋白质的~(32)P含量进行了定量分析，结果表明，放射自显影鉴定的3种MR=60,000蛋白质的~(32)P掺入的刺激程度随着蛋白质酸性电荷的增加而递增，在钾去极化细胞中分别为28、36和9176。胰升糖素使这两种M，=16,000蛋白的~(32)P标记分别增加了50%和100%。细胞分离研究表明，cAMP刺激的MR=16,000两个磷酸蛋白是胞浆中的次要成分。用1%Triton X-100和0.6M Kc1提取细胞后的残留物中回收了M_1=60,000个蛋白质，似乎是细胞骨架的结构蛋白。部分蛋白水解肽图谱显示，在双向电泳胶上，Fm，=60,000个蛋白质序列相互靠近地迁移。由两种蛋白质组成，每一种都以非磷酸化形式存在，并以几种酸性稍强的磷酸化衍生物的形式出现。二维胰酶磷酸肽图谱证实了这些观察结果。在去极化诱导的Ca“内流作用下，两个M_1=60,000蛋白的负电性较强，它们的磷酸化增强。由于M_1=16,000蛋白的浓度较低，不可能进行类似的研究。酸解后，从PSO中回收磷丝氨酸和磷苏氨酸，而从16,000道尔顿磷蛋白中仅回收磷丝氨酸。40 nm K+和胰升糖素分别诱导这些蛋白的磷酸化增强，与前人研究的细胞内钙内流增加和cAMP升高相一致。这些磷酸化事件可能是钙和cAMP刺激胰岛素释放的中间步骤。
Ca2+-and CAMP-dependent protein phosphorylations were analyzed in 32P-labeled hamster insulinoma cells by two-dimensional gel electrophoresis and autoradiography. Incubation of the cells in the presence of 40 m~ K', which induces voltage-dependent Ca2+ influx and insulin release, stimulates phosphorylation of three 60,000-dalton proteins varying slightly in charge. Glucagon (3 p~) which promotes insulin release by raising the cellular concentration of CAMP stimulates phosphorylation of two 16,000-dalton peptides. Quantitative analysis of the 32P content of several proteins resolved by two-dimensional electrophoresis revealed that the degree of stimulation of 32P incorporation into the three Mr= 60,000 proteins identified by autoradiography rose progressively with increasing acidic charge of the protein and was 28, 36, and 9176, respectively, in potassium-depolarized cells. Glucagon enhanced the 32P-labeling of the two M,= 16,000 proteins by 50 and loo%, respectively. Cell fractionation studies showed that the two CAMP-stimulated Mr= 16,000 phosphoproteins are minor cytosolic components. The M,= 60,000 proteins were recovered in the residue remaining after extracting the cells with 1% Triton X-100, 0.6 M KC1 and appear to be structural proteins of the cytoskeleton. Partial proteolytic peptide maps revealed that the series ofM,= 60,000 proteins migrating in close proximity to one another on two-dimensional electrophoresis gels consists. of two proteins each occurring in unphosphorylated form and as several slightly more acidic phosphorylated derivatives. Two-dimensional tryptic phosphopeptide maps confirmed these observations. The more negatively charged of the two M,= 60,000 proteins undergoes enhanced phosphorylation in response to depolarization-induced Ca" influx. Analogous stud-ies on the M,= 16,000 proteins were not possible due to their low concentration. After acid hydrolysis, phosphoserine and phosphothreonine were recovered from Pso and only phosphoserine from the 16,000-dalton phosphoproteins. The enhanced phosphorylation of these proteins induced by 40 nm K+ and glucagon coincides, respectively, with the enhanced Ca2+ influx and rise in cellular CAMP characterized in previous studies. These phosphorylation events may be intermediate steps in the stimulation of insulin release by Ca2+ and CAMP.