MEKK1 inhibits CREB-directed transcripiton by preventing the interaction of CREB with its coactivator CBP

The cAMP response element binding protein CREB has been shown to play a pivotal role in maintaining beta-cell function. Our previous data demonstrated that in the insulin-producing pancreatic beta-cell line HIT CRE/CREB-directed transcription stimulated by membrane depolarization or by cAMP was inhibited by overexpression of the mitogen activated protein kinase kinase kinase 1 (MEKK1). In contrast, MEKK1 enhanced the transcriptional activity of the CREB coactivator CBP. In the present study it was investigated how MEKK1 inhibits stimulated CRE/CREB-directed transcription. HIT cells were transiently transfected with a luciferase reporter gene controlled by four copies of the rat somatostatin CRE and an expression vector for constitutively active MEKK1. Inhibitors for p38, JNK and ERK, putative downstream kinases of MEKK1, did not diminish the inhibitory effect of MEKK1 on stimulated CRE-directed transcription although these reagents inhibited the phosphorylation of the respective kinases as revealed by immunoblot. Phosphorylation of Ser-133 is required for CREB transactivation whereas phosphorylation of the Ser-100, 111, 121, and 142 have been shown to inhibit Ser-133-phosphorylated CREB. After cAMP and membrane depolarization MEKK1 decreased the transcriptional activity of GAL4-CREB fusion proteins, carrying Ser-100, 111, and 121 or Ser-142 to alanine mutations. Furthermore, MEKK1 did not decrease the phosphorylation of Ser-133 as revealed by immunoblot. The interaction between CREB and CBP was investigated by fluorescence resonance electron transfer (FRET). Overexpression of MEKK1 inhibited the cAMP-induced interaction between the kinase inducible domain of CREB and the CREB interaction domain of CBP. Thus, our findings show that MEKK1 inhibits stimulated CREB-directed transcription by decreasing the interaction between Ser-133-phosphorylated CREB and CBP. Given the role of CREB in beta-cell function and given the role of MEKK1 in conferring stress signals inhibition of CREB activity by MEKK1 might contribute to the development of beta-cell failure and diabetes.