In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-zeta/lambda activity in plasma membranes and microsomes and (b) immunoreactive PKC-zeta and PKC-lambda in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-zeta and PKC-lambda were also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO(4))(3) (PIP(3)) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-zeta and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-zeta pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-zeta/lambda but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-zeta. Also, insulin in situ and PIP(3) in vitro activated and stimulated autophosphorylation of a PKC-zeta mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-zeta that lacks the regulatory (presumably PIP(3)-binding) domain; this may reflect PIP(3) effects on PDK-1 or transphosphorylation by endogenous full-length PKC-zeta. Our findings suggest that insulin activates both PKC-zeta and PKC-lambda in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP(3), PDK-1-dependent phosphorylation of activation loop sites in PKC-zeta and lambda, and subsequent autophosphorylation and/or transphosphorylation.

Lipodystrophies are a heterogeneous group of human disorders characterized by the anomalous distribution of body fat associated with insulin resistance and altered lipid metabolism. The pathogenetic mechanism of inherited lipodystrophies is not yet clear; at the molecular level they have been linked to mutations of lamin A/C, peroxisome proliferator-activated receptor (PPARγ) and other seemingly unrelated proteins. In this study, we examined lamin A/C processing in three laminopathies characterized by lipodystrophic phenotypes: Dunnigan type familial partial lipodystrophy, mandibuloacral dysplasia and atypical Werner's syndrome. We found that the lamin A precursor was specifically accumulated in lipodystrophy cells. Pre-lamin A was located at the nuclear envelope and co-localized with the adipocyte transcription factor sterol regulatory element binding protein 1 (SREBP1). Using co-immunoprecipitation experiments, we obtained the first demonstration of an in vivo interaction between SREBP1 and pre-lamin A. Binding of SREBP1 to the lamin A precursor was detected in patient fibroblasts as well as in control fibroblasts forced to accumulate pre-lamin A by farnesylation inhibitors. In contrast, SREBP1 did not interact in vivo with mature lamin A or C in cultured fibroblasts. To gain insights into the effect of pre-lamin A accumulation in adipose tissue, we inhibited lamin A precursor processing in 3T3-L1 pre-adipocytes. Our results show that pre-lamin A sequesters SREBP1 at the nuclear rim, thus decreasing the pool of active SREBP1 that normally activates PPARγ and causing impairment of pre-adipocyte differentiation. This defect can be rescued by treatment with troglitazone, a known PPARγ ligand activating the adipogenic program.

Interconnected functional strategies govern chromatin dynamics in eukaryotic cells. In this context, A and B type lamins, the nuclear intermediate filaments, act on diverse platforms involved in ti...

The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKCepsilon isoform, these are Thr(566) in the activation loop, Thr(710) in the turn motif and Ser(729) in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKCepsilon kinase activity in controlling the phosphorylation of Thr(566) and Ser(729). In NIH 3T3 fibroblasts, PKCepsilon migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr(566) and Ser(729). Cells transfected with an active PDK-1 allele also resulted in increased PKCepsilon Thr(566) and Ser(729) phosphorylation, whereas an active myristoylated PKCepsilon mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKCepsilon were not phosphorylated at Ser(729) in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr(566), an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKCepsilon in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P(3), concomitant with an increase in Ser(729) phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.