Activation of the rat dopamine D2 receptor promoter by mitogen-activated protein kinase and Ca2+/calmodulin-dependent protein kinase II pathways.
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.
Takeuchi
Yusuke
Y
Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto, Japan. yusuket@gpo.kumamoto-u.ac.jp
Miyamoto
Eishichi
E
Fukunaga
Kohji
K
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D051766:Early Growth Response Protein 1; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology; D005799:Genes, Dominant; D017930:Genes, Reporter; D005910:Glioma / Q000378:metabolism; D017874:Immediate-Early Proteins; D048728:MAP Kinase Kinase Kinase 1; D051379:Mice; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis / Q000235:genetics*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; C486110:Egr1 protein, rat; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D017448:Receptors, Dopamine D2; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C517683:CAMK4 protein, human; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517685:Camk4 protein, mouse; C517686:Camk4 protein, rat; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; C482578:Map3k1 protein, mouse