Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LA⌬50/progerin). These modifications cause an abnormal association of LA⌬50/ progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G 1 is impaired in cells expressing LA⌬50/ progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.cell division ͉ nuclear lamins ͉ nuclear structure ͉ progeria ͉ protein farnesylation
Rem, Rem2, Rad, and Gem͞Kir (RGK) represent a distinct GTPase family with largely unknown physiological functions. We report here that both Rem and Rad bind directly to Ca 2؉ channel -subunits ( R em, Rem2, Rad, and Gem͞Kir (RGK) are members of a Ras-related GTPase subfamily (RGK family), with many unique characteristics that distinguish them from other members of the Ras superfamily (1-5). The common structure for all RGK proteins consists of a conserved Ras-related core domain, a series of nonconservative amino acid substitutions within regions known to be involved in guanine nucleotide binding and hydrolysis, a non-CAAX-containing C-terminal extension, and large N-terminal extensions relative to other Ras family proteins. The conservation of structural features within the RGK proteins suggests shared mechanisms of regulation and the control of common cellular signal transduction networks. However, RGK subfamily members differ from each other and from other Ras-related proteins in their putative effector (G2) domains, suggesting that they interact with distinct regulatory and effector proteins, and each exhibits distinct tissue-specific expression patterns (1-5). Thus, despite their conserved structural and biochemical properties, functional evidence to suggest a unified mechanism of action has been limited.In this study, we investigated the ability of Rem and Rad to regulate Ca 2ϩ channel activity. Our results demonstrate that, although both proteins have distinct effector interaction domains, they each bind directly to Ca V  subunits and inhibit detectable ionic current expression from the native cardiac L type, but not from T type, Ca 2ϩ channels when coexpressed in human embryonic kidney (HEK)293 cells. The Rem protein is expressed in striated muscle cells, and we demonstrate that Rem inhibits L type Ca 2ϩ channel activity in differentiated C2C12 myotubes. Deletion analysis demonstrates that the C terminus of Rem plays an important role in Ca V  subunit association and is necessary for regulation of channel activity. Taken together, these studies indicate that Rem functions as a potent regulator of L type Ca 2ϩ channel function in muscle and suggest that a conserved physiological role for the RGK GTPase gene family is the control of Ca 2ϩ channel activity via modulation of Ca V  subunit function.
Experimental ProceduresRNase Protection Assays and Cell Culture. C2C12DS and C2C12(E) mouse muscle myoblast cell lines were from Robert Krauss (Mount Sinai School of Medicine, New York) and were cultured and induced to differentiate as described (6). Primary mouse muscle cultures and MM14 cells were provided by
Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.
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