Robert M. Evans

An alteration in the phosphorylation of vimentin-type intermediate filaments is associated with mitosis in cultured mammalian cells

Analysis of cultured Chinese hamster ovary (CHO) cells has shown that vimentin exists primarily as two 57,000 dalton isoelectric variants, a nonphosphorylated form and a slightly more acidic phosphorylated form. Similar analyses of CHO cells that were treated with colcemid show the presence of at least two to three additional, more acidic, phosphorylated vimentin isoelectric variants. An increasing 32P-specific activity of these variants suggests that this alteration involves increased phosphorylation. Analysis of 32P-labeled vimentin from colcemid-treated cells indicates that the amount of the additional phosphorylated variants correlates with the accumulation of cells in mitosis. CHO cells enriched in mitotic cells without antimitotic drugs demonstrate the same alteration in the isoelectric focusing pattern of phosphorylated vimentin. When mitotic cells are replated, the amount of additional phosphorylated variants is reduced within 30 min. The data suggest that an alteration in phosphorylated vimentin is temporally related to the alteration in the organization of intermediate filaments in mitotic cells.

Cyclic AMP-dependent protein kinase-induced vimentin filament disassembly involves modification of the N-terminal domain of intermediate filament subunits

The intermediate filament protein vimentin was phosphorylated with cAMP‐dependent protein kinase under conditions that induce filament disassembly. Digestion of phosphorylated vimentin with lysine‐specific endoprotease and subsequent tryptic peptide mapping indicated that a 12 kDa N‐terminal fragment contained all the phosphorylation sites found in the intact molecule. Analysis of cyanogen bromide digests indicated that two phosphorylated peptides were produced, with the major 32P‐labeled species representing amino acid position 14–72, and a minor 32P‐labeled peptide representing amino acid positions 1–13. These results demonstrate that phosphorylation of sites within the N‐terminal head domain of vimentin are associated with phosphorylation induced filament disassembly.

Intermediate filaments and lipoprotein cholesterol

The ability of cells to utilize cholesterol derived from lipoprotein is important in plasma membrane biosynthesis, steroidogenesis and the regulation of sterol synthesis. While the endocytosis of lipoprotein-derived cholesterol has been well characterized, the subsequent events that mediate its post-lysosomal intracellular transport are not understood. Recent studies have suggested that vimentin-type intermediate filaments may have a role in cholesterol transport. The mechanism by which vimentin filaments affect this process is not known, but future studies promise to provide new insights into both the post-lysosomal transport of cholesterol and the intracellular functions of intermediate filaments.

Lipoproteins Regulate Expression of the Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenocortical Cells

The steroidogenic acute regulatory protein (StAR) is required for the movement of cholesterol from the outer to the inner mitochondrial membrane, the site of cholesterol side chain cleavage. Here we describe a novel form of regulation of StAR gene expression in steroidogenic cells. Treatment of Y-1 BS1 adrenocortical cells with either low density lipoprotein (LDL) or high density lipoprotein (HDL) increases expression of endogenous StAR mRNA and protein in a dose-dependent manner. Induction of StAR mRNA by lipoprotein requires basal cAMP-dependent protein kinase, since the inhibitor, R p -8-Br-cAMP, inhibited induction of StAR protein by LDL. Likewise, basal StAR expression or LDL induction of StAR protein was not detectable in Y-1 kin-8 cells which are deficient in cAMP-dependent protein kinase. Aminoglutethimide and ketoconazole were used to determine if side chain cleavage of lipoprotein-derived cholesterol is required for induction of StAR mRNA. Treatment with either drug alone induced StAR mRNA expression 1.5–3-fold, while induction of StAR in cells treated with either drug plus LDL, was equal to, or greater than, induction seen with either agent alone, suggesting that lipoprotein does not regulate StAR via generation of an oxysterol intermediate. Both LDL and HDL increased expression of a mouse −966 StAR promoter-reporter construct 1.5–2.5-fold, indicating that regulation occurs at the level of transcription. In contrast, neither lipoprotein was able to induce transcription from a −966 StAR promoter in which the steroidogenic factor-1 site at −135 was abolished, indicating that regulation of StAR transcription by lipoproteins requires steroidogenic factor-1. The regulation of StAR gene expression by lipoproteins may represent a positive feedback circuit which links cholesterol availability with steroidogenic output.