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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Minibrain-related kinase (Mirk)/Dyrk1B is an arginine-directed serine/threonine kinase that is active in skeletal muscle development but is also expressed in various carcinomas. In the current study, the Met adaptor protein Ran-binding protein M (

BackgroundAmyloid-β precursor protein (APP) is a highly conserved single transmembrane protein that has been linked to Alzheimer disease. Recently, the increased expression of APP in multiple types of cancers has been reported where it has significant correlation with the cancer cell proliferation. However, the function of APP in the pathogenesis of breast cancer has not previously been determined. In this study, we studied the pathological role of APP in breast cancer and revealed its potential mechanism.MethodsThe expression level of APP in multiple breast cancer cell lines was measured by Western blot analysis and the breast cancer tissue microarray was utilized to analyze the expression pattern of APP in human patient specimens. To interrogate the functional role of APP in cell growth and apoptosis, the effect of APP knockdown in MDA-MB-231 cells were analyzed. Specifically, multiple signal transduction pathways and functional alterations linked to cell survival and motility were examined in in vivo animal model as well as in vitro cell culture with the manipulation of APP expression.ResultsWe found that the expression of APP is increased in mouse and human breast cancer cell lines, especially in the cell line possessing higher metastatic potential. Moreover, the analysis of human breast cancer tissues revealed a significant correlation between the level of APP and tumor development. Knockdown of APP (APP-kd) in breast cancer cells caused the retardation of cell growth in vitro and in vivo, with both the induction of p27kip1 and caspase-3-mediated apoptosis. APP-kd cells also had higher sensitivity to treatment of chemotherapeutic agents, TRAIL and 5-FU. Such anti-tumorigenic effects shown in the APP-kd cells partially came from reduced pro-survival AKT activation in response to IGF-1, leading to activation of key signaling regulators for cell growth, survival, and pro-apoptotic events such as GSK3-β and FOXO1. Notably, knock-down of APP in metastatic breast cancer cells limited cell migration and invasion ability upon stimulation of IGF-1.ConclusionThe present data strongly suggest that the increase of APP expression is causally linked to tumorigenicity as well as invasion of aggressive breast cancer and, therefore, the targeting of APP may be an effective therapy for breast cancer.

Mirk/Dyrk1B protein kinase was shown in an earlier study to function as a transcriptional activator of HNF1␣, which Mirk phosphorylates at Ser 249 within its CREB (cAMP-response element-binding protein)-binding protein (CBP) binding domain (1). The MAPK kinase MKK3 was also shown to activate Mirk as a protein kinase, implicating Mirk in the biological response to certain stress agents. Another MKK3 substrate, p38MAPK, is now shown to inhibit the function of Mirk as a transcriptional activator in a kinase-independent manner. Co-immunoprecipitation experiments demonstrated that kinase-inactive p38AF, as well as wild-type p38, sequestered Mirk and prevented its association with MKK3. Only the p38␣ and p38␤ isoforms, but not the ␥ or ␦ isoforms, complexed with Mirk. p38␣MAPK blocked Mirk activation of HNF1␣ in a dose-dependent manner, with high levels of kinase-inactive p38␣AF completely suppressing the activity of Mirk. Size fractionation by fast protein liquid chromatography on Superdex 200 demonstrated that Mirk is not found as a monomer in vivo, but is found within 150 -700 kDa subnuclear complexes, which co-migrate with the nuclear body scaffolding protein PML. Endogenous Mirk, p38, and MKK3 co-migrate within 500 -700-kDa protein complexes, which accumulate when nuclear export is blocked by leptomycin B. Stable overexpression of Mirk increases the fraction of Mirk protein and p38 protein within these 500 -700 kDa complexes, suggesting that the complexes act as nuclear depots for Mirk and p38. Sequestration of Mirk by p38 may occur within these subnuclear complexes. Synchronization experiments demonstrated that Mirk levels fluctuate about 10-fold within the cell cycle, while p38 levels do not, leading to the speculation that endogenous p38 could only block Mirk function when Mirk levels were low in S phase and not when Mirk levels were elevated in G 0 /G 1 . These data suggest a novel cell cycle-dependent function for p38, suppression of the function of Mirk as a transcriptional activator only when cells are proliferating, and thus limiting Mirk function to growth-arrested cells.Mirk 1 (minibrain-related kinase) 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 is a serine/threonine protein kinase, which is expressed at elevated levels in normal skeletal muscle and certain carcinoma cell lines and is expressed at low levels in many normal tissues (6). Mutant minibrain/dyrk1A flies are characterized by a marked reduction in size of the optic lobes and central brain hemispheres (7), so it is reasonable to hypothesize that minibrain/dyrk1A plays a role in neuronal survival and/or proliferation. In cell culture systems dyrk1A mediates neuronal differentiation by phosphorylating CREB in vivo, leading to the stimulation of subsequent CRE-mediated transcription during neuronal differentiation in hippocampal progenitor cells (8).Mirk/dyrk1B functions as a transactivator of a different transcription factor, HNF1␣,...