Hagiwara M, Brindle P, Harootunian A, Armstrong R, Rivier J, Vale W, Tsien R, Montminy M R
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037.
Mol Cell Biol. 1993 Aug;13(8):4852-9. doi: 10.1128/mcb.13.8.4852-4859.1993.
Cyclic AMP (cAMP) regulates a number of eukaryotic genes by mediating the protein kinase A (PKA)-dependent phosphorylation of the CREB transcription factor at Ser-133. In this study, we test the hypothesis that the stoichiometry and kinetics of CREB phosphorylation are determined by the liberation and subsequent translocation of PKA catalytic subunit (C subunit) into the nucleus. Using fluorescence imaging techniques, we observed that PKA was activated in a stimulus-dependent fashion that led to nuclear entry of C subunit over a 30-min period. The degree of CREB phosphorylation, assessed with antiserum specific for CREB phosphorylated at Ser-133, correlated with the amount of PKA liberated. The time course of phosphorylation closely paralleled the nuclear entry of the catalytic subunit. There was a linear relationship between the subsequent induction of the cAMP-responsive somatostatin gene and the degree of CREB phosphorylation, suggesting that each event--kinase activation, CREB phosphorylation, and transcriptional induction--was tightly coupled to the next. In contrast to other PKA-mediated cellular responses which are rapid and quantitative, the slow, incremental regulation of CREB activity by cAMP suggests that multifunctional kinases like PKA may coordinate cellular responses by dictating the kinetics and stoichiometry of phosphorylation for key substrates like CREB.
环磷酸腺苷（cAMP）通过介导蛋白激酶A（PKA）依赖的CREB转录因子在丝氨酸133位点的磷酸化来调节许多真核基因。在本研究中，我们检验了这样一个假设，即CREB磷酸化的化学计量和动力学由PKA催化亚基（C亚基）的释放及随后向细胞核的转位所决定。使用荧光成像技术，我们观察到PKA以刺激依赖的方式被激活，这导致C亚基在30分钟内进入细胞核。用针对丝氨酸133位点磷酸化的CREB的抗血清评估的CREB磷酸化程度与释放的PKA量相关。磷酸化的时间进程与催化亚基的核进入密切平行。随后cAMP反应性生长抑素基因的诱导与CREB磷酸化程度之间存在线性关系，这表明每个事件——激酶激活、CREB磷酸化和转录诱导——都与下一个事件紧密耦合。与其他快速且定量的PKA介导的细胞反应不同，cAMP对CREB活性的缓慢、渐进调节表明，像PKA这样的多功能激酶可能通过决定关键底物（如CREB）磷酸化的动力学和化学计量来协调细胞反应。
