Protein kinase C catalyzed phosphorylation of sterol carrier protein 2.
The transport of cholesterol to the inner mitochondrial membrane, a key step in steroidogenesis, is subject to hormonal modulation that, at least in part, could be mediated by protein phosphorylation. This step is stimulated by sterol carrier protein 2 (SCP2) and Ca2+. To explore whether SCP2 itself is a potential control point for regulation by Ca2+-dependent phosphorylation we investigated whether highly purified SCP2 could serve as a substrate for major type Ca2+ and non-Ca2+-dependent protein kinases. Phosphorylation by calmodulin protein kinase II (CaM-PK II), myosin light chain kinase (MLCK), cAMP-dependent kinase (PKA) and protein kinase C (PKC) was monitored under optimal conditions for each enzyme. PKA, CaM-PK II and MLCK catalyzed the radiolabeling of histone 2A, synapsin I and myosin light chain (MLC), known substrates for these kinases, respectively, yet no phosphate transfer to SCP2 was observed. In contrast, PKC from two different sources (rat and calf brain) effectively catalyzed the phosphorylation of the highly purified SCP2. The phosphorylation of SCP2 depended on the addition of Ca2+ and phospholipids and was completely blocked by Polymyxin B, a PKC inhibitor. PKC catalyzed phosphorylation of SCP2 displayed a similar dependence on the concentration of ATP. Lineweaver Burk plots of the data indicate Km values for ATP of approximately 6 microM for the phosphorylation of SCP2. Our results, which have revealed for the first time that SCP2 is a substrate for PKC, are consistent with the possibilities that the control of steroidogenesis by tropic hormones and by PKC activation are mediated, at least in part, by the phosphorylation/dephosphorylation of SCP2.
Steinschneider
A
A
Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago 60612.
McLean
M P
MP
Billheimer
J T
JT
Azhar
S
S
Gibori
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D009994:Osmolar Concentration; D010766:Phosphorylation; D010940:Plant Proteins; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013261:Sterols
D002352:Carrier Proteins; D010940:Plant Proteins; D013261:Sterols; C072087:sterol carrier proteins; D000255:Adenosine Triphosphate; D011493:Protein Kinase C