Phosphorylation of supernatant protein factor enhances its ability to stimulate microsomal squalene monooxygenase.
Supernatant protein factor is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, a downstream enzyme in the cholesterol biosynthetic pathway. The mechanism of stimulation is poorly understood, although supernatant protein factor belongs to a family of lipid-binding proteins that includes Sec14p and alpha-tocopherol transfer protein. Because recombinant human supernatant protein factor purified from Escherichia coli exhibited a relatively weak ability to activate microsomal squalene monooxygenase, we investigated the possibility that cofactors or post-translational modifications were necessary for full activity. Addition of ATP to rat liver cytosol increased supernatant protein factor activity by more than 2-fold and could be prevented by the addition of inhibitors of protein kinases A and C. Incubation of purified recombinant supernatant protein factor with ATP and protein kinases A or C delta similarly increased activity by more than 2-fold. Addition of protein phosphatase 1 gamma, a serine/threonine phosphatase, to rat liver cytosol reduced activity by 50%, suggesting that supernatant protein factor is partially phosphorylated in vivo. To determine whether dietary cholesterol influenced the phosphorylation state, cytosols were prepared from livers of rats fed a high fat diet. Although supernatant protein factor activity was reduced by more than one-half, it could not be restored by the addition of ATP or protein kinase C delta with ATP, suggesting that dietary cholesterol reduced the expression of this protein. Supernatant protein factor thus appears to be regulated both post-translationally through phosphorylation and at the level of expression. Phosphorylation may provide a means for the rapid short term modulation of cholesterol synthesis.
Singh
Dev K
DK
Division of Pharmaceutical Sciences, College of Pharmacy and The Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536-0082, USA.
Mokashi
Vishwesh
V
Elmore
C Lee
CL
Porter
Todd D
TD
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D010105:Oxygenases / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D050603:Squalene Monooxygenase; D015534:Trans-Activators
D002352:Carrier Proteins; D008074:Lipoproteins; D011994:Recombinant Proteins; C432422:SEC14L2 protein, human; C486622:Sec14l2 protein, rat; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D010105:Oxygenases; D050603:Squalene Monooxygenase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1