Sjöholm A, Honkanen R E, Berggren P O
University of Hawaii at Manoa, Cancer Research Center of Hawaii, Honolulu 96813-2424, USA.
Endocrinology. 1995 Aug;136(8):3391-7. doi: 10.1210/endo.136.8.7628374.
Reversible protein phosphorylation is considered to be an important and versatile mechanism by which cells transduce external signals into biological responses. Cellular levels of protein phosphorylation are determined by the balanced actions of both protein kinases and protein phosphatases (PPases). Compared with protein kinases, however, serine/threonine PPases have received less attention. In the present study, the effects of known insulin secretagogues and some intracellular second messengers on the activities of serine/threonine PPases in insulin-secreting RINm5F insulinoma cells were investigated. The stimulation of intact RINm5F cells with the insulin secretagogues L-arginine, L-glutamine, KCl, or ATP elicited time-dependent changes in PPase activities with an early (1 min) decrease in type 1-like and/or type 2A-like PPase activity that gradually returned to normal levels. Addition of cAMP, cGMP, or prostaglandins E2 and F1 alpha at widely different concentrations to RINm5F cell homogenates failed to affect PPase activities. In contrast, addition of physiological concentrations of adenine nucleotides, which are known to increase upon secretory stimulation, to cell homogenates inhibited type 2A-like and, to a lesser extent, type 1-like, PPase activity (ATP > ADP > AMP > adenosine). ATP and ADP IC50 values for type 2A-like PPase were approximately 75 and 250 microM, respectively. The inhibitory effect of ATP was reproduced and of comparable magnitude when purified PPases (types 1 and 2A) were used instead of RINm5F cell homogenates. It is concluded that insulin secretagogues cause time- and concentration-dependent inhibitory effects on RINm5F cell PPase activities, which may contribute to the increase in the phosphorylation state that occurs after stimulation of insulin release. Thus, inhibition of protein dephosphorylation may be a novel regulatory mechanism controlling the stimulus-secretion coupling in insulin-producing cells.
可逆性蛋白质磷酸化被认为是一种重要且通用的机制，通过该机制细胞将外部信号转化为生物学反应。蛋白质磷酸化的细胞水平由蛋白激酶和蛋白磷酸酶（PP 酶）的平衡作用决定。然而，与蛋白激酶相比，丝氨酸/苏氨酸 PP 酶受到的关注较少。在本研究中，研究了已知的胰岛素促分泌剂和一些细胞内第二信使对胰岛素分泌型 RINm5F 胰岛素瘤细胞中丝氨酸/苏氨酸 PP 酶活性的影响。用胰岛素促分泌剂 L-精氨酸、L-谷氨酰胺、KCl 或 ATP 刺激完整的 RINm5F 细胞，会引起 PP 酶活性随时间的变化，其中 1 型样和/或 2A 型样 PP 酶活性在早期（1 分钟）降低，随后逐渐恢复到正常水平。向 RINm5F 细胞匀浆中添加浓度差异很大的 cAMP、cGMP 或前列腺素 E2 和 F1α，均未影响 PP 酶活性。相反，向细胞匀浆中添加已知在分泌刺激时会增加的生理浓度的腺嘌呤核苷酸，会抑制 2A 型样 PP 酶活性，对 1 型样 PP 酶活性的抑制作用较小（ATP > ADP > AMP > 腺苷）。2A 型样 PP 酶的 ATP 和 ADP 的 IC50 值分别约为 75 和 250 μM。当使用纯化的 PP 酶（1 型和 2A 型）代替 RINm5F 细胞匀浆时，ATP 的抑制作用得以重现且程度相当。得出的结论是，胰岛素促分泌剂对 RINm5F 细胞 PP 酶活性产生时间和浓度依赖性的抑制作用，这可能有助于胰岛素释放刺激后磷酸化状态的增加。因此，抑制蛋白质去磷酸化可能是控制胰岛素产生细胞中刺激-分泌偶联的一种新型调节机制。
