TI - Differential activation of CREB by Ca2+/calmodulin -dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity . AB - The c-AMP 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 .