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Keywords:

  • Ca2+/calmodulin-dependent protein kinase II;
  • dopamine D2 receptor promoter;
  • mitogen-activated protein kinase;
  • NB2A cell;
  • Zif268

Abstract

To investigate regulation of D2 receptor (D2R) gene expression by protein kinases, we evaluated effects of constitutively active MAPK kinase kinase (MEKK), Ca2+/calmodulin-dependent protein kinase (CaMK) II, CaMKIV and cyclic AMP-dependent protein kinase (PKA) on D2R promoter activity using luciferase reporter gene assays. A 1.5-kbp fragment containing the rat D2R promoter was cloned upstream of the reporter and transfected into D2R-expressing NB2A cells or nonexpressing NG108-15 and C6 glioma cells. MEKK and CaMKII, but not CaMKIV and PKA, increased promoter activity 4.5- and 1.5-fold, respectively, in NB2A cells. Inhibitory effects of a MEK inhibitor and lack of effect by dominant negative (DN)-JNK1 or DN-p38MAPK revealed that ERK but not JNK and p38MAPK is involved in MEKK-induced promoter activation. Deletion and mutation of the promoter revealed that the MEKK-responsive region was Sp1 site B between nucleotides −56 and −47. Overexpression of Sp1 suppressed promoter activity without affecting MEKK-induced activation. Interestingly, overexpression of Zif268 increased promoter activity through the region between nucleotides −56 and −36. Increased activity by Zif268 was additive with CaMKII-induced activation but not with activation induced by MEKK. Co-transfection with CaMKII stimulated nuclear translocation of Zif268. These results suggest that ERK and CaMKII positively regulate the D2R promoter and that Zif268 is a potential transcription factor for the CaMKII-dependent pathway.