Transforming growth factor-beta1 (TGF-beta1) is a potent cytokine with pleiotropic effects, including anti-inflammatory activity. Here we show that the signaling protein Smad6 bound to Pellino-1, an adaptor protein of mammalian interleukin 1 receptor (IL-1R)-associated kinase 1 (IRAK1), and thereby promoted TGF-beta-mediated anti-inflammatory effects. Smad6-Pellino-1 interaction abrogated signaling mediated by a complex of IRAK1, Pellino-1 and adaptor protein TRAF6 that formed after stimulation by IL-1beta treatment. Blockade of IRAK1-Pellino-1-TRAF6 signaling prevented degradation of the inhibitor IkappaBalpha and subsequent nuclear translocation of transcription factor NF-kappaB and thus expression of proinflammatory genes. 'Knockdown' of endogenous Smad6 expression by RNA interference reduced anti-inflammatory activity mediated by TGF-beta1 or the TGF-beta family member BMP-4. Thus Smad6 is a critical mediator of the TGF-beta-BMP pathway that mediates anti-inflammatory activity and negatively regulates IL-1R-Toll-like receptor signals.

The kinase Mirk/dyrk1B is essential for the differentiation of C2C12 myoblasts. Mirk reinforces the G 0 /G 1 arrest state in which differentiation occurs by directly phosphorylating and stabilizing p27Kip1 and destabilizing cyclin D1. We now demonstrate that Mirk is antiapoptotic in myoblasts. Knockdown of endogenous Mirk by RNA interference activated caspase 3 and decreased myoblast survival by 75%, whereas transient overexpression of Mirk increased cell survival. Mirk exerts its anti-apoptotic effects during muscle differentiation at least in part through effects on the cell cycle inhibitor and pro-survival molecule p21Cip1 . Overexpression and RNA interference experiments demonstrated that Mirk phosphorylates p21 within its nuclear localization domain at Ser-153 causing a portion of the typically nuclear p21 to localize in the cytoplasm. Phosphomimetic GFP-p21-S153D was pancellular in both cycling C2C12 myoblasts and NIH3T3 cells. Endogenous Mirk in myotubes and overexpressed Mirk in NIH3T3 cells were able to cause the pancellular localization of wild-type GFPp21 but not the nonphosphorylatable mutant GFP-p21-S153A. Translocation to the cytoplasm enables p21 to block apoptosis through inhibitory interaction with pro-apoptotic molecules. Phosphomimetic p21-S153D was more effective than wild-type p21 in blocking the activation of caspase 3. Transient expression of p21-S153D also increased myoblast viability in colony forming assays, whereas the p21-S153A mutant had no effect. This Mirk-dependent change in p21 intracellular localization is a natural part of myoblast differentiation. Endogenous p21 localized exclusively to the nuclei of proliferating myoblasts but was also found in the cytoplasm of post-mitotic multinucleated myotubes and adult human skeletal myofibers.

Transforming growth factor-beta1 (TGF-beta1) regulates inflammation and can inhibit activation of the transcription factor NF-kappaB in certain cell types. Here we show that the TGF-beta-induced signaling protein Smad7 bound to TAB2 and TAB3, which are adaptors that link the kinase TAK1 to 'upstream' regulators in the proinflammatory tumor necrosis factor (TNF) signaling pathway. Smad7 thereby promoted TGF-beta-mediated anti-inflammatory effects. The formation of Smad7-TAB2 and Smad7-TAB3 complexes resulted in the suppression of TNF signaling through the adaptors TRAF2, TAB2 and/or TAB3, and TAK1. Furthermore, expression of a transgene encoding Smad7 in mouse skin suppressed inflammation and NF-kappaB nuclear translocation substantially and disrupted the formation of endogenous TRAF2-TAK1-TAB2 and TRAF2-TAK1-TAB3 complexes. Thus, Smad7 is a critical mediator of TGF-beta signals that block proinflammatory TNF signals.

Mirk/Dyrk1B is an arginine-directed serine/threonine protein kinase that is expressed at low levels in most normal tissues but at elevated levels in many tumor cell lines and in normal skeletal muscle. Colon carcinoma cell lines stably overexpressing Mirk proliferated in serumfree medium, but the mechanism of Mirk action is unknown. DCoHm (dimerization cofactor of hepatocyte nuclear factor 1␣ ( HNF1␣) from muscle), a novel gene of the DCoH family with 78% amino acid identity to DCoH, was identified as a Mirk-binding protein by yeast two-hybrid analysis and cloned. Mirk co-immunoprecipitated with DCoHm and bound to DCoHm in glutathione S-transferase pull-down assays. DCoH stabilizes HNF1␣ as a dimer and enhances its transcriptional activity on the ␤-fibrinogen promoter reporter, and DCoHm had similar activity. Mirk enhanced HNF1␣ transcriptional activity in a dosedependent manner, whereas two kinase-inactive Mirk mutants and a Mirk N-terminal deletion mutant did not. Mirk, DCoHm, and HNF1␣ formed a complex. Mirk bound to a specific region within the CREB-binding proteinbinding region of HNF1␣ and phosphorylated HNF1␣ at a site adjacent to the Mirk-binding region. Conversely, the HNF1␣ binding domain was located within the first five conserved kinase subdomains of Mirk. Mirk co-immunoprecipitated with the MAPK kinase MKK3, an upstream activator of p38. MKK3 enhanced Mirk kinase activity and the transcriptional activation of HNF1␣ by Mirk, suggesting that Mirk, like p38, is activated by certain environmental stress agents. The Mirk-binding protein DCoH has been shown to be selectively expressed in colon carcinomas but not in normal tissue. Mirk may function as an HNF1␣ transcriptional activator in response to an MKK3-mediated stress signal, and the selective expression of DCoH could restrict the Mirk response to carcinoma cells. Mirk1 /Dyrk1B is a serine/threonine protein kinase that is expressed at elevated levels in normal skeletal muscle and certain carcinoma cell lines and at low levels in many normal tissues (1). Colon carcinoma cell lines stably overexpressing Mirk proliferated in serum-free medium (1), but the mechanism of Mirk action that enabled this survival capacity is unknown. Mirk is a member of the Dyrk/minibrain family of dual specificity, tyrosine-regulated, arginine-directed protein kinases (2-4) and is identical to Dyrk1B (5). Mirk/Dyrk1B and the related kinase Dyrk1A exhibit 54% amino acid identity with 90% identity or homology within the conserved kinase domain. Several lines of evidence indicate that Dyrk1A mediates neuronal differentiation. Dyrk1A has been mapped to the Down's syndrome critical region of chromosome 21, overexpression of Dyrk1A has been found in the Down's syndrome fetal brain (6), and transgenic mice overexpressing Dyrk1A exhibit cognitive deficits and motor abnormalities characteristic of Down's syndrome (7). Dyrk1A has been shown to phosphorylate the cAMP-response element-binding protein (CREB) in vivo, leading to the stimulation of subsequent cAMP response element-mediated ...

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