In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-/ activity in plasma membranes and microsomes and (b) immunoreactive PKC-and PKCin GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-and PKCwere 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-and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-/ but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-. Also, insulin in situ and PIP 3 in vitro activated and stimulated autophosphorylation of a PKC-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-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-. Our findings suggest that insulin activates both PKCand PKC-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-and , and subsequent autophosphorylation and/or transphosphorylation.Insulin has been reported to activate atypical forms of protein kinase C (PKC), 1 i.e. PKC-and/or PKC-, in 3T3/L1 adipocytes (1, 2), rat adipocytes (3), L6 myotubes (4), and 32D cells (5). These increases in atypical PKC enzyme activity appear to be largely dependent upon activation of phosphatidylinositol (PI) 3-kinase (1-5) and subsequent increases in D3-PO 4 polyphosphoinositides, i.e. PI 3,4,5-(PO 4 ) 3 and PI 3,4-(PO 4 ) 2 (3). Moreover, transfection studies suggest that PKC-and/or PKC-is/are required for and may be sufficient for insulin stimulation of GLUT4 translocation and subsequent glucose transport (1-4). At present, there is only limited information on the mechanism whereby D3-PO 4 polyphosphoinositides activate atypical PKCs and little or no information on the subcellular compartments in which atypical PKCs are activated or, for that matter, whether one or both atypical PKCs are activated by insulin in specific cell types. With respect to the first point, recent findings (6, 7) suggest that PI 3,4,5-(PO 4 ) 3 and PI 3,4-(PO 4 ) 2 activate, or allow access for, 3-phosphoinositide-dependent kinase-1 (PDK-1), which phosphorylates threonine 410 in the activation loop of PKC-, thereby initiating the activation of this atypical PKC. Indeed, in other studies, we have found that PDK-1 action is required for insulin-induced activation of PKC-in rat adipocytes.2 However, it is uncertain whether this requirement reflects a permissive effect of PDK-1 or whether PDK-1 mediates acute a...

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 (PPARgamma) 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 PPARgamma and causing impairment of pre-adipocyte differentiation. This defect can be rescued by treatment with troglitazone, a known PPARgamma ligand activating the adipogenic program.

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleocytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.

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 tissue homeostasis. On the nuclear side, lamins elicit large scale or fine chromatin conformational changes, affect DNA damage response factors and transcription factor shuttling. On the cytoplasmic side, bridging-molecules, the LINC complex, associate with lamins to coordinate chromatin dynamics with cytoskeleton and extra-cellular signals. Consistent with such a fine tuning, lamin mutations and/or defects in their expression or post-translational processing, as well as mutations in lamin partner genes, cause a heterogeneous group of diseases known as laminopathies. They include muscular dystrophies, cardiomyopathy, lipodystrophies, neuropathies, and progeroid syndromes. The study of chromatin dynamics under pathological conditions, which is summarized in this review, is shedding light on the complex and fascinating role of the nuclear lamina in chromatin regulation.

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.

Laminopathies are genetic diseases due to mutations or altered post-translational processing of nuclear envelope/lamina proteins. The majority of laminopathies are caused by mutations in the LMNA gene, encoding lamin A/C, but manifest as diverse pathologies including muscular dystrophy, lipodystrophy, neuropathy, and progeroid syndromes. Lamin-binding proteins implicated in laminopathies include lamin B2, nuclear envelope proteins such as emerin, MAN1, LBR, and nesprins, the nuclear matrix protein matrin 3, the lamina-associated polypeptide, LAP2alpha and the transcriptional regulator FHL1. Thus, the altered functionality of a nuclear proteins network appears to be involved in the onset of laminopathic diseases. The functional interplay among different proteins involved in this network implies signaling partners. The signaling effectors may either modify nuclear envelope proteins and their binding properties, or use nuclear envelope/lamina proteins as platforms to regulate signal transduction. In this review, both aspects of lamin-linked signaling are presented and the major pathways so far implicated in laminopathies are summarized.

Pre-lamin A undergoes subsequent steps of post-translational modification at its C-terminus, including farnesylation, methylation, and cleavage by ZMPSTE24 metalloprotease. Here, we show that accumulation of different intermediates of pre-lamin A processing in nuclei, induced by expression of mutated pre-lamin A, differentially affected chromatin organization in human fibroblasts. Unprocessed (non-farnesylated) pre-lamin A accumulated in intranuclear foci, caused the redistribution of LAP2alpha and of the heterochromatin markers HP1alpha and trimethyl-K9-histone 3, and triggered heterochromatin localization in the nuclear interior. In contrast, the farnesylated and carboxymethylated lamin A precursor accumulated at the nuclear periphery and caused loss of heterochromatin markers and Lap2alpha in enlarged nuclei. Interestingly, pre-lamin A bound both HP1alpha and LAP2alpha in vivo, but the farnesylated form showed reduced affinity for HP1alpha. Our data show a link between pre-lamin A processing and heterochromatin remodeling and have major implications for understanding molecular mechanisms of human diseases linked to mutations in lamins.

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 LAP2α 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.