The Journal of biological chemistry
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Defining the molecular basis responsible for regulating the proliferative potential of keratinocytes has important implications for normal homeostasis and neoplasia of the skin. Under current culture conditions, neonatal foreskin-derived human keratinocytes possess a relatively short replicative lifespan. Recently it was reported that forced overexpression of the helix-loop-helix protein Id-1 was capable of immortalizing keratinocytes, secondary to activation of telomerase activity and suppression of p16/Rb-mediated growth arrest pathways. ⋯ Under these experimental conditions, Id-1 expression did not trigger induction of telomerase activity, and there was progressive shortening of the telomeres that was accompanied by elevated p16 levels and prevalence of active Rb. The ability of Id-1 to postpone, but not prevent, senescence may be related to partial inhibition of p16 expression, as the Id-1-overexpressing cultures displayed a decreased capacity for 12-O-tetradecanoylphorbol-13-acetate-mediated p16 induction. Thus, while no immortalization was observed, Id-1 could delay the onset of replicative senescence in unselected human keratinocyte populations.
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Transforming growth factor-beta (TGF-beta) is involved in multiple processes including cell growth and differentiation. In particular, TGF-beta has been implicated in the pathogenesis of fibrotic lung diseases. In this study, we examined regulation of the mitogen-activated protein kinase pathway by TGF-beta1 in primary human lung fibroblasts. ⋯ Induction of AP-1 by TGF-beta1-conditioned medium was observed at 2 h, similar to AP-1 induction in response to exogenous bFGF. Dependence of ERK/AP-1 activation on bFGF induction was demonstrated by inhibition of TGF-beta1-induced ERK/AP-1 activation when conditioned medium from TGF-beta1-treated cells was incubated with bFGF-neutralizing antibody. Together, these results demonstrate that TGF-beta1 regulates the autocrine induction of bFGF, resulting in activation of the ERK mitogen-activated protein kinase pathway and induction of AP-1 binding.
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Precise regulation of neurotransmitter release is essential for the normal function of neural networks, but the mechanisms involved are largely unclear. Using superfused synaptosomes, we have studied the readily releasable pool of synaptic vesicles, measured as the amount of release triggered by hypertonic sucrose. We show that activation of presynaptic metabotropic glutamate receptors by dihydroxyphenylglycine and stimulation of protein kinase C by phorbol esters enhance the readily releasable pool of glutamate. ⋯ NEM induced a dose-dependent increase in the readily releasable pool of neurotransmitters but by itself did not trigger release. Direct measurements of core complexes confirmed that NEM caused an increase in the levels of SNARE core complexes under these conditions. Our data suggest that in the readily releasable pool of synaptic vesicles, SNARE proteins are fully assembled into core complexes, and that SNARE complex assembly is a target of presynaptic regulation.
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The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. ⋯ Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.
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The regulation of gene expression by cell surface receptors often involves the stimulation of signaling pathways including one or more members of the MAPK superfamily of serine-threonine kinases. Upon their activation in the cytosol, MAPKs can translocate to the nucleus and affect the activity of a variety of transcription factors. Recently, it has been observed that a novel member of the MAPK superfamily, ERK5, can be potently activated by transforming G protein-coupled receptors (GPCRs) and that ERK5 participates in the regulation of c-jun expression through the activation of MEF2 transcription factors. ⋯ To investigate which heterotrimeric G proteins signal to ERK5, we used a chimeric system by which Galpha(q)- and Galpha(13)-mediated signaling pathways can be conditionally activated upon ligand stimulation. Using this system, as well as the expression of activated forms of G protein subunits, we show that the Galpha(q) and Galpha(12/13) families of heterotrimeric G proteins, but not the Galpha(i), Galpha(s), and betagamma subunits, are able to regulate ERK5. Furthermore, we provide evidence that the stimulation of ERK5 by GPCRs involves a novel signaling pathway, which is distinct from those regulated by Ras and Rho GTPases.