Supernatant
protein factor (SPF) is a poorly characterized cytosolic
protein that stimulates
HMG-CoA reductase and
squalene monooxygenase in vitro and
cholesterol synthesis when expressed in
hepatoma cells. The activation of SPF by
protein kinases A (PKA) and Cdelta enhances its ability to stimulate these cholesterolgenic
enzymes in microsomal preparations. The present studies demonstrate that the ability of SPF to stimulate
cholesterol synthesis in cell culture is also modulated by phosphorylation. Addition of dibutyryl-cAMP, a PKA activator, to
hepatoma cells expressing SPF increased
cholesterol synthesis by 62%, whereas addition of a cell-permeable
PKA inhibitor blocked the SPF-mediated increase in
cholesterol synthesis. To confirm a role for PKA in the regulation of SPF, substitution of
alanine for serine-289 (a putative PKA recognition site) blocked the stimulation of
cholesterol synthesis by SPF. Serine-289 is located at the junction of the proposed
lipid-binding domain and the carboxyl-terminal Golgi dynamics domain, suggesting that phosphorylation may alter the interaction of these two domains. In a test of this hypothesis, deletion of the Golgi dynamics domain blocked the ability of SPF to stimulate
cholesterol synthesis, supporting a role for Golgi in SPF function; this finding was buttressed by the observation that addition of
brefeldin A, which disrupts Golgi formation, also abolished the ability of SPF to stimulate
cholesterol synthesis. The activation of SPF by PKA suggests that
cholesterol synthesis can be rapidly modulated in response to external stimuli by changes in cAMP levels, and that this regulation is dependent on an as yet undefined interaction with Golgi.