Effects of mutating different steroidogenic factor-1 protein regions on gene regulation.
The involvement of cyclic adenosine monophosphate cAMP-dependent protein kinase A (PKA) in the regulation of the steroidogenic acute regulatory protein (StAR) and the high-density lipoprotein receptor (HDL-R) genes by steroidogenic factor-1 (SF-1) and cAMP were examined. Cotransfection studies carried out in Kin 8 cells, a Y1 cell line (mouse adrenal) with a mutation in the type I PKA regulatory subunit, demonstrated that an intact PKA is required for maximal activation and that SF-1 participates in cAMP regulation of these genes. Site-directed mutational analysis was performed to examine which SF-1 regions could be involved in SF-1 transcriptional activation of the StAR and HDL-R genes. SF-1 regions protein analyzed were amino acids Thr 60, Ser 203, Ser 431, Thr 462, and the activation function-2 domain (amino acids 449-462). Plasmids encoding each of the mutated SF-1 proteins were cotransfected with the StAR and HDL-R promoter constructs into human bladder carcinoma (HTB-9) cells in the presence or absence of dibutyryl cAMP. The results of these studies suggest that although SF-1 is required for optimal promoter response to cAMP, transcriptional activation of genes by SF-1 and cAMP are promoter dependent, perhaps resulting from gene-specific interactions of this transcription factor with other regulatory proteins.
Lopez
D
D
Department of Obstetrics and Gynecology, University of South Florida, Tampa, USA.
Nackley
A C
AC
Shea-Eaton
W
W
Xue
J
J
Schimmer
B P
BP
McLean
M P
MP
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008075:Lipoproteins, HDL / Q000096:biosynthesis / Q000378:metabolism; D008156:Luciferases / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000502:physiology*; D010750:Phosphoproteins / Q000235:genetics*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D000098682:Steroidogenic Acute Regulatory Protein
D008075:Lipoproteins, HDL; D010750:Phosphoproteins; D012333:RNA, Messenger; D000098682:Steroidogenic Acute Regulatory Protein; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases