Phosphorylation of the hamster adrenal steroidogenic acute regulatory protein as analyzed by two-dimensional polyacrylamide gel electrophoreses.
Post-translational modifications such as phosphorylation and specific proteolysis affect the steroidogenic acute regulatory protein (StAR) activity. We have found that in pcDNA3.1-StAR-transfected COS-1 cells, StAR was phosphorylated on S55, S56 and S194 (Fleury et al., unpublished). In this study, we are comparing the two-dimensional gel electrophoresis (2D-PAGE) characteristics of the WT StAR with those of the S194A and S55A/S56A/S194A-StAR mutants under control and (Bu)(2)-cAMP stimulation, using an anti-StAR antibody and an anti-phospho-(Ser/Thr) PKA substrate antibody. The 2D-PAGE migration pattern of the WT StAR analyzed by immunoblotting with the anti-StAR antibody revealed many StAR species with different pI and different molecular weights. In the (Bu)(2)-cAMP-WT preparations, except for three, all these StAR species were also recognized by the anti-phospho-(Ser/Thr) PKA substrate antibody; in contrast, less phosphorylated species were found in the non-stimulated WT preparations. The two-dimensional (2D) patterns of StAR revealed by the anti-StAR and the anti-phospho-(Ser/Thr) PKA substrate antibodies were modified for the S194A mutant and further modified for the S55A/S56A/S194A mutant. Whereas many species could still be detected by the anti-StAR antibody in the triple mutant S55A/S55A/S194A, none of these could be revealed by the anti-phospho-(Ser/Thr) PKA substrate antibody. Finally we found that, in addition to phosphorylation, the formation of different StAR species was also due to the hydrolysis of the molecule at its N-terminal and to a lesser degree at its C-terminal.
LeHoux
Jean-Guy
JG
Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Que., Canada J1H 5N4. jean-guy.lehoux@usherbrooke.ca
Fleury
Alain
A
Ducharme
Lyne
L
Hales
Dale Buchanan
DB
D000302:Adrenal Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D019556:COS Cells; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional; D015151:Immunoblotting; D009154:Mutation / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D000098682:Steroidogenic Acute Regulatory Protein
D010750:Phosphoproteins; D000098682:Steroidogenic Acute Regulatory Protein; D003994:Bucladesine