Protein kinase C (PKC) represents a family of serine/threonine kinases that play a central role in the regulation of cell growth, differentiation, and transformation. Posttranslational control of the PKC isoforms and their activation have been extensively studied; however, not much is known about their translational regulation. Here we report that the expression of one of the PKC isoforms, PKC, is regulated at the translational level both under normal growth conditions and during stress imposed by amino acid starvation, the latter causing a marked increase in its protein levels. The 5 untranslated region (5 UTR) of PKC is unusually long and GC rich, characteristic of many oncogenes and growth regulatory genes. We have identified two conserved upstream open reading frames (uORFs) in its 5 UTR and show their effect in suppressing the expression of PKC in MCF-7 growing cells. While the two uORFs function as repressive elements that maintain low basal levels of PKC in growing cells, they are required for its enhanced expression upon amino acid starvation. We show that the translational regulation during stress involves leaky scanning and is dependent on eIF-2␣ phosphorylation by GCN2. Our work further suggests that translational regulation could provide an additional level for controlling the expression of PKC family members, being more common than currently recognized.Studies over the last several years have focused on the activation of protein kinase C (PKC) members and their regulation by phosphorylation, membrane recruitment, and downregulation by proteolysis; however, not much is known about the regulatory mechanisms leading to PKC synthesis. Most cellular mRNAs have short 5Ј untranslated regions (UTRs
Intra-abdominal (IA) fat functionally differs from subcutaneous (SC) adipose tissue, likely contributing to its stronger association with obesity-induced morbidity and to differential response to medications. Drug-induced partial lipodystrophy, like in response to antiretroviral agents, is an extreme manifestation of the different response of different fat depots, with loss of SC but not IA. Investigating depot-specific adipocyte differences is limited by the low accessibility to IA fat and by the heterogenous cell population comprising adipose tissue. Here, we aimed at utilizing immortalized preadipocyte cell lines from IA (epididymal) or SC (inguinal) fat to investigate whether they differentially respond to the HIV protease inhibitor nelfinavir. Preadipocytes were readily amenable to adipogenesis, as evidenced by lipid accumulation, expression of adipose-specific genes, measurable lipolysis, and insulin responsiveness. Leptin secretion was higher by the SC line, consistent with known differences between IA and SC fat. As previously reported, nelfinavir inhibited adipogenesis downstream of C/EBPβ, but similarly in both cell lines. In contrast, nelfinavir's capacity to diminish insulin signaling, decrease leptin secretion, enhance basal lipolysis, and decrease expression of the lipid droplet-associated protein perilipin occurred more robustly and/or at lower nelfinavir concentrations in the SC line. This was despite similar intracellular concentrations of nelfinavir (23.8 ± 5.6 and 33.6 ± 12.2 μg/mg protein for inguinal and epididymal adipocytes, respectively, P = 0.46). The cell lines recapitulated depot-differential effects of nelfinavir observed in differentiated primary preadipocytes and with whole tissue explants. Thus, we report the use of fat depot-specific adipocyte cell lines for unraveling depot-differential responses to a drug causing partial lipodystrophy.
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