Regulation of adrenocortical pregnenolone-binding protein activity by phosphorylation/dephosphorylation. Phosphatase-mediated inactivation is reversed by cytosolic kinase.
The pregnenolone-binding protein (PBP) in guinea pig adrenocortical cytosol is inactivated (converted to a nonsteroid-binding form) by incubation with calf intestinal alkaline phosphatase at pH 9. Previously bound pregnenolone does not prevent this inactivation, and dephosphorylation causes dissociation of bound ligand from the protein. Cytosolic PBP, partially purified PBP, and highly purified PBP are equally susceptible to alkaline phosphatase-mediated inactivation. No change in apparent molecular weight or immunoreactivity is evident by Western blot analysis. Loss of pregnenolone-binding capacity of cytosolic PBP (but not partially purified PBP) could be reversed by inhibiting the phosphatase, lowering the pH to approximately 7, and adding ATP to the incubation. Reactivation is absolutely and specifically dependent upon ATP, which restores binding capacity in a concentration-dependent manner. Other nucleoside triphosphates, including the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate, as well as cAMP and cGMP are ineffectual as cofactors for reactivation. These data strongly implicate a cytosolic kinase which is apparently inactivated or separated from PBP during purification. Preliminary investigations indicate that the reactivating kinase is not cAMP-dependent, but may have a requirement for calcium and/or calmodulin. The identification of phosphorylation/dephosphorylation as the regulatory mechanism for steroid binding should prove pivital in elucidating the functional role of PBP.
Driscoll
W J
WJ
Section on Adrenal Cell Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Lee
Y C
YC
Strott
C A
CA
D000302:Adrenal Cortex / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011284:Pregnenolone / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D013997:Time Factors
D002352:Carrier Proteins; D010750:Phosphoproteins; D004533:Egtazic Acid; D011284:Pregnenolone; D000242:Cyclic AMP; D011494:Protein Kinases; D000469:Alkaline Phosphatase