MyoD is a basic helix-loop-helix transcription factor involved in the activation of genes encoding skeletal muscle-specific proteins. Independent of its ability to transactivate muscle-specific genes, MyoD can also act as a cell cycle inhibitor. MyoD activity is regulated by transcriptional and posttranscriptional mechanisms. While MyoD can be found phosphorylated, the functional significance of this posttranslation modification has not been established. MyoD contains several consensus cyclin-dependent kinase (CDK) phosphorylation sites. In these studies, we examined whether a link could be established between MyoD activity and phosphorylation at putative CDK sites. Site-directed mutagenesis of potential CDK phosphorylation sites in MyoD revealed that S200 is required for MyoD hyperphosphorylation as well as the normally short half-life of the MyoD protein.Additionally, we determined that turnover of the MyoD protein requires the proteasome and Cdc34 ubiquitinconjugating enzyme activity. Results of these studies demonstrate that hyperphosphorylated MyoD is targeted for rapid degradation by the ubiquitin pathway. The targeted degradation of MyoD following CDK phosphorylation identifies a mechanism through which MyoD activity can be regulated coordinately with the cell cycle machinery (CDK2 and CDK4) and/or coordinately with the cellular transcriptional machinery (CDK7, CDK8, and CDK9).

DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. We used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Spl to its target sequence (a G+C-rich sequence known as a "GC box"). Concatemers of doublestranded 14-mers containing a GC box successfully competed with the human metallothionein iHA promoter for binding to Spl in DNase I protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Spl binding. The result was confirmed using doublestranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Spl-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in. the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Spl-DNA interactions.There is strong experimental support for the proposal that DNA methylation may be a component of a multilevel control mechanism through which the expression of certain eukaryotic genes is regulated (1). Inhibition of genomic methylation by 5-azacytidine can result in reactivation of genes on the transcriptionally inactive X chromosome (2, 3), induction of tissue-specific gene expression (4, 5), and expression of differentiated phenotypes in cultured cells (6)(7)(8). Introduction of in vitro hypermethylated sequences into recipient cells can preclude gene expression (9, 10). Despite these correlations the relationship between methylation patterns and expression of some genes remains unclear (11) and it is not known how changes in the methylation patterns of certain genes alter expression while the activities of other genes remain unaffected.Recent reports strongly suggest that cytosine methylation may be involved in the structural ordering of chromatin (12,13). Methylation of phage M13 constructs before transfection into eukaryotic cells targets the assimilated DNA into DNase I-insensitive conformations. Buschhausen et al. (13) showed that reconstitution of methylated thymidine kinase DNA with histone octamers prior to microinjection resulted in biological inactivity even in the absence of integration into the host genome. In contrast, mock-methylated constructs actively expressed thymidine kinase and the inhibitory effect of methylation could be reversed by treatment with 5-azacytidine. These observations suggest that positioning of structural nuclear proteins (histones, high-mobility group proteins, or matrix proteins) may be involved in transducing the methylation signal. What remains unclear, however, is whether methylation also alters the direct binding of specific transcription factors to defined promoter element...

Activation of transforming growth factor-beta (TGF-beta) and activin receptors leads to phosphorylation of Sma- and Mad-related protein 2 (Smad2) and Smad3, which function as transcription factors to regulate gene expression. Smad7 is a regulatory protein which is able to inhibit TGF-beta and activin signalling in a negative-feedback loop, mediated by a direct regulation by Smad3 and Smad4 via a Smad-binding element (SBE) in the Smad7 promoter. Interestingly, we found that the Smad7 promoter was also regulated by nuclear factor kappaB (NF-kappaB), a transcription factor which plays an important role in inflammation and the immune response. Expression of NF-kappaB p65 subunit was able to inhibit the Smad7 promoter activity, and this inhibition could be reversed by co-expression of IkappaB, an inhibitor of NF-kappaB. In addition, the inhibitory activity of p65 was observed in a minimal promoter that contained only the Smad7 SBE and a TATA box, without any consensus NF-kappaB binding site. This inhibitory effect appeared to be common to other TGF-beta- and activin-responsive promoters, since p65 also inhibited the forkhead-activin-signal-transducer-2-mediated activation of a Xenopus Mix.2 promoter, as well as the Smad3-mediated activation of 3TP-lux which contains PMA-responsive elements and a plasminogen-activator-inhibitor-1 promoter. Activation of endogenous NF-kappaB by tumour necrosis factor-alpha (TNF-alpha) was also able to inhibit the Smad7 promoter in human embryonic kidney 293 cells. In human hepatoma HepG2 cells, TNF-alpha was able to inhibit TGF-beta- and activin-mediated transcriptional activation. Furthermore, overexpression of the transcription co-activator p300 could abrogate the inhibitory effect of NF-kappaB on the Smad7 promoter. Taken together, these data have indicated a novel mode of crosstalk between the Smad and the NF-kappaB signalling cascades at the transcriptional level by competing for a limiting pool of transcription co-activators.

Chronic alveolar hypoxia is the major cause of pulmonary hypertension. The cellular mechanisms involved in hypoxia- induced pulmonary arterial remodeling are still poorly understood. Mitogen-activated protein kinase (MAPK) is a key enzyme in the signaling pathway leading to cellular growth and proliferation. The purpose of this investigation was to determine the roles that MAPKs, specifically Jun-N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 kinase, play in the hypoxia-induced pulmonary arterial remodeling. Rats were exposed to normobaric hypoxia (10% O(2)) for 1, 3, 7, or 14 d. Hypoxia caused significant remodeling in the pulmonary artery characterized by thickening of pulmonary arterial wall and increases in tissue mass and total RNA. JNK, ERK, and p38 kinase tyrosine phosphorylations and their activities were significantly increased by hypoxia. JNK activation peaked at Day 1 and ERK/p38 kinase activation peaked after 7 d of hypoxia. The results from immunohistochemistry show that hypoxia increased phospho-MAPK staining in both large and small intrapulmonary arteries. Hypoxia also upregulated vascular endothelial growth factor messenger RNA (mRNA) and platelet-derived growth factor receptor mRNA levels in pulmonary artery with a time course correlated to the activation of ERK and p38 kinase. The gene expressions of c-jun, c-fos, and egr-1, known as downstream effectors of MAPK, were also investigated. Hypoxia upregulated egr-1 mRNA but downregulated c-jun and c-fos mRNAs. These data suggest that hypoxia-induced activation of JNK is an early response to hypoxic stress and that activation of ERK and p38 kinase appears to be associated with hypoxia-induced pulmonary arterial remodeling.

The innate immune response is an important defense against pathogenic agents. A component of this response is the NF-B-dependent activation of genes encoding inflammatory cytokines such as interleukin-8 (IL-8) and cell adhesion molecules like E-selectin. Members of the serine/threonine innate immune kinase family of proteins have been proposed to mediate the innate immune response. One serine/threonine innate immune kinase family member, the mouse Pelle-like kinase/human interleukin-1 receptor-associated kinase (mPLK/ IRAK), has been proposed to play an obligate role in promoting IL-1-mediated inflammation. However, it is currently unknown whether mPLK/IRAK catalytic activity is required for IL-1-dependent NF-B activation. The present study demonstrates that mPLK/IRAK catalytic activity is not required for IL-1-mediated activation of an NF-B-dependent signal. Intriguingly, catalytically inactive mPLK/IRAK inhibits type 1 tumor necrosis factor (TNF) receptor-dependent NF-B activation. The pathway through which mPLK/IRAK mediates this TNF response is TRADD-and TRAF2-independent. Our data suggest that in addition to its role in IL-1 signaling, mPLK/IRAK is a component of a novel signal transduction pathway through which TNF R1 activates NF-B-dependent gene expression.

Pellino is a Drosophila protein originally isolated in a two-hybrid screen for proteins interacting with the serine/threonine kinase, pelle. Although mammalian homologs have been identified in mouse and man, the function of pellino is as yet unknown. In this study, the cloning, expression pattern, and a preliminary characterization of mouse pellino-2 is described. These studies reveal that mouse pellino-2 is expressed during embryogenesis and in a tissue-restricted manner in the adult. IL-1 induces the association of mouse pellino-2 with the mouse pelle-like kinase/IL-1R-associated kinase protein, a mammalian homolog of pelle. Ectopic pellino-2 expression did not result in NF-κB activation. However, ectopic expression of a mouse pellino-2 antisense construct inhibited IL-1 or LPS-induced activation of NF-κB-dependent IL-8 promoter activity. Our data reveal that mouse pellino-2 is a tissue-restricted component of a signaling pathway that couples the mouse pelle-like kinase/IL-1R-associated kinase protein to IL-1- or LPS-dependent signaling.

Inflammation contributes to insulin resistance in diabetes and obesity. Mouse Pelle-like kinase (mPLK, homolog of human IL-1 receptor-associated kinase (IRAK)) participates in inflammatory signaling. We evaluated IRS-1 as a novel substrate for mPLK that may contribute to linking inflammation with insulin resistance. Wildtype mPLK, but not a kinase-inactive mutant (mPLK-KD), directly phosphorylated full-length IRS-1 in vitro. This in vitro phosphorylation was increased when mPLK was immunoprecipitated from tumor necrosis factor (TNF)-␣-treated cells. In NIH-3T3 IR cells, wild-type mPLK (but not mPLK-KD) co-immunoprecipitated with IRS-1. This association was increased by treatment of cells with TNF-␣. Using mass spectrometry, we identified Ser 24 in the pleckstrin homology (PH) domain of IRS-1 as a specific phosphorylation site for mPLK. IRS-1 mutants S24D or S24E (mimicking phosphorylation at Ser 24 ) had impaired ability to associate with insulin receptors resulting in diminished tyrosine phosphorylation of IRS-1 and impaired ability of IRS-1 to bind and activate PI-3 kinase in response to insulin. IRS-1-S24D also had an impaired ability to mediate insulin-stimulated translocation of GLUT4 in rat adipose cells. Importantly, endogenous mPLK/IRAK was activated in response to TNF-␣ or interleukin 1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser 24 , we found that interleukin-1 or TNF-␣ treatment of Fao cells stimulated increased phosphorylation of endogenous IRS-1 at Ser 24 . We conclude that IRS-1 is a novel physiological substrate for mPLK. TNF-␣-regulated phosphorylation at Ser 24 in the pleckstrin homology domain of IRS-1 by mPLK/IRAK represents an additional mechanism for cross-talk between inflammatory signaling and insulin signaling that may contribute to metabolic insulin resistance.Biochemical, physiological, and epidemiological studies implicate pro-inflammatory cytokines (e.g. TNF-␣, 1 IL-1␤, and IL-6) in the development of insulin resistance and the pathophysiology of type 2 diabetes and obesity (1-8). These studies suggest an intriguing link between inflammation and metabolic dysregulation. Indeed, IB kinase ␤ (IKK␤), a critical mediator of inflammatory signaling pathways activating NF-B, has been identified as an important inhibitor of metabolic insulin signaling pathways (9 -12). Inactivation of IKK␤ signaling increases insulin sensitivity, whereas overexpression of IKK␤ or activation of IKK␤ by pro-inflammatory cytokines (e.g. TNF-␣) leads to insulin resistance (9, 12). Similarly, JNK is another inflammatory signaling molecule that may play a role in the insulin resistance of obesity (13). One potential explanation for these observations is cross-talk between inflammatory signaling and metabolic insulin signaling pathways.Metabolic actions of insulin such as enhanced glucose uptake into skeletal muscle and adipose tissue are regulated by activation of the insulin receptor tyrosine kinase and subsequent tyrosine phosphorylation o...

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