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.
MeSH terms
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Animals
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Calcium-Calmodulin-Dependent Protein Kinase Type 4
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Calcium-Calmodulin-Dependent Protein Kinases / genetics
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
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Cell Line
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Cyclic AMP-Dependent Protein Kinases / metabolism
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DNA-Binding Proteins / metabolism
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Early Growth Response Protein 1
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Enzyme Activation / drug effects
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Enzyme Inhibitors / pharmacology
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Gene Expression Regulation / physiology
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Genes, Dominant
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Genes, Reporter
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Glioma / metabolism
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Immediate-Early Proteins*
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MAP Kinase Kinase Kinase 1*
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Mice
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Mitogen-Activated Protein Kinase 8
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Mitogen-Activated Protein Kinases / genetics
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Mitogen-Activated Protein Kinases / metabolism*
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Mutagenesis, Site-Directed
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Promoter Regions, Genetic / physiology*
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Protein Serine-Threonine Kinases / metabolism
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Rats
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Receptors, Dopamine D2 / biosynthesis
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Receptors, Dopamine D2 / genetics*
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Sequence Deletion
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Signal Transduction / drug effects
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Signal Transduction / physiology*
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Sp1 Transcription Factor / metabolism
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Transcription Factors / metabolism
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Transfection
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p38 Mitogen-Activated Protein Kinases
Substances
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DNA-Binding Proteins
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Early Growth Response Protein 1
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Egr1 protein, mouse
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Egr1 protein, rat
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Enzyme Inhibitors
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Immediate-Early Proteins
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Receptors, Dopamine D2
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Sp1 Transcription Factor
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Transcription Factors
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Protein Serine-Threonine Kinases
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Cyclic AMP-Dependent Protein Kinases
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CAMK4 protein, human
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Calcium-Calmodulin-Dependent Protein Kinase Type 4
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Calcium-Calmodulin-Dependent Protein Kinases
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Camk4 protein, mouse
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Camk4 protein, rat
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Mitogen-Activated Protein Kinase 8
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Mitogen-Activated Protein Kinases
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p38 Mitogen-Activated Protein Kinases
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MAP Kinase Kinase Kinase 1
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MAP3K1 protein, human
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Map3k1 protein, mouse