CaMKIIδ-dependent Inhibition of cAMP-response Element-binding Protein Activity in Vascular Smooth Muscle

One transcription factor mediator of Ca2+-signals is cAMP response element-binding protein (CREB). CREB expression and/or activity negatively correlates with vascular smooth muscle (VSM) cell proliferation and migration. Multifunctional Ca2+/calmodulin-dependent protein kinases, including CaMKII, have been demonstrated to regulate CREB activity through both positive and negative phosphorylation events in vitro, but the function of CaMKII as a proximal regulator of CREB in intact cell systems, including VSM, is not clear. In this study, we used gain- and loss-of-function approaches to determine the function of CaMKII in regulating CREB phosphorylation, localization, and activity in VSM. Overexpression of constitutively active CaMKII specifically increased CREB phosphorylation on Ser(142) and silencing CaMKII expression by siRNA or blocking endogenous CaMKII activity with KN93 abolished thrombin- or ionomycin-induced CREB phosphorylation on Ser(142) without affecting Ser(133) phosphorylation. CREB-Ser(142) phosphorylation correlated with transient nucleocytoplasmic translocation of CREB. Thrombin-induced CREB promoter activity, CREB binding to Sik1 and Rgs2 promoters, and Sik1/Rgs2 transcription were enhanced by a kinase-negative CaMKII2 (K43A) mutant and inhibited by a constitutively active (T287D) mutant. Taken together, these studies establish negative regulation of CREB activity by endogenous CaMKII-dependent CREB-Ser(142) phosphorylation and suggest a potential mechanism for CaMKII/CREB signaling in modulating proliferation and migration in VSM cells.