Sun P, Enslen H, Myung P S, Maurer R A
Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland 97201.
Genes Dev. 1994 Nov 1;8(21):2527-39. doi: 10.1101/gad.8.21.2527.
The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser133 of CREB is essential for its activation by CaMKIV. Because both CaMKII and CaMKIV can phosphorylate CREB, we pursued further the mechanism by which CaMKII and CaMKIV differentially regulate CREB activity. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser133 only, whereas CaMKII phosphorylates CREB at Ser133 and a second site, Ser142. Transient transfection studies revealed that phosphorylation of Ser142 by CaMKII blocks the activation of CREB that would otherwise occur when Ser133 is phosphorylated. When Ser142 was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser142 to alanine enhanced the ability of Ca2+ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser142 in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca2+ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca2+ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca2+/calmodulin-dependent protein kinases.
环磷酸腺苷反应元件结合蛋白（CREB）已被证明可介导基因的转录激活，以响应环磷酸腺苷和钙内流信号转导途径。在瞬时转染研究中，利用这些激酶的全长或组成型活性形式，研究了两种多功能钙/钙调蛋白依赖性蛋白激酶CaMKIV和CaMKII的作用。结果表明，在三种不同的细胞系中，CaMKIV在激活CREB方面比CaMKII更有效。在这些研究中还发现，CREB的Ser133对其被CaMKIV激活至关重要。由于CaMKII和CaMKIV都能使CREB磷酸化，我们进一步探究了CaMKII和CaMKIV差异调节CREB活性的机制。诱变研究和磷酸肽图谱分析表明，在体外，CaMKIV仅在Ser133处使CREB磷酸化，而CaMKII在Ser133和第二个位点Ser142处使CREB磷酸化。瞬时转染研究表明，CaMKII对Ser142的磷酸化会阻断CREB的激活，否则当Ser133被磷酸化时就会发生激活。当Ser142突变为丙氨酸时，CREB被CaMKII以及CaMKIV激活。此外，Ser142突变为丙氨酸增强了钙内流激活CREB的能力，表明Ser142的磷酸化在调节CREB活性中具有生理作用。这些数据为一种新的调节CREB活性的机制提供了证据，该机制涉及转录激活域中负调控位点的磷酸化。这些研究还为环磷酸腺苷和钙信号通路在转录调控中可能的相互作用提供了新的见解。特别是，细胞内钙的变化有可能抑制或增强环磷酸腺苷刺激转录的能力，这取决于特定形式的钙/钙调蛋白依赖性蛋白激酶的存在。
