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Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H2O2 and performed biochemical, live cell imaging, and chromatin immunoprecipitation sequencing analyses to determine their response to the treatment. We show that the mismatch repair processing of oxidative DNA damage involves cohesive interactions between mismatch recognition protein MutSa, histone mark H3K36me3, and H3K36 trimethyltransferase SETD2, which activates the ATM DNA damage signaling pathway. We found that cells depleted of MutSa or SETD2 accumulate 8-oxoG adducts and fail to trigger H2O2-induced ATM activation. Furthermore, we show that SETD2 physically interacts with both MutSa and ATM, which suggests a role for SETD2 in transducing DNA damage signals from lesion-bound MutSa to ATM. Consistently, MutSa and SETD2 are highly coenriched at oxidative damage sites. The data presented here support a model wherein MutSa, SETD2, ATM, and H3K36me3 constitute a positive feedback loop to help cells cope with oxidative DNA damage. [ABSTRACT FROM AUTHOR]

Acyl-CoA: lysocardiolipin acyltransferase- I (ALCAT 1) catalyzes acylation of lysocardiolipin back to cardiolipin, an important step in cardiolipin remodeling. The present study reports the catalytic properties of ALCAT1 in vitro and its regulation by thyroid hormone status in mouse liver and heart. Recombinant ALCAT1 expressed in Sf9 cells preferred basic pH conditions and did not require divalent cations or integrity of the subcellular membrane for its enzymatic activity. Recombinant ALCAT1 was potently inhibited by ADP and ATP, but not by adenosine nucleotide analogs or other nucleotides, such as UTP and GTP, suggesting that ALCAT1 does not require ATP hydrolysis for its enzyme activity. In addition to cardiolipin, ALCAT1 also catalyzed acylation of other members of the polyglycerophospholipid family, including phosphatidylglycerol, a precursor for cardiolipin synthesis, and bis(monoacylglycero)phosphate, a structural isomer of lysophosphatidylglycerol and a metabolic intermediate of cardiolipin. These findings suggest that ALCAT1 plays a role in the remodeling of other polyglycerophospholipids. In support of a regulatory role of ALCAT1 in cardiolipin remodeling in response to oxidative stress, ALCAT1 expression in liver and heart was significantly downregulated in mice with hypothyroidism and upregulated in mice treated with thyroid hormone, which is known to stimulate mitochondrial activity, oxidative stress, and cardiolipin remodeling. [ABSTRACT FROM AUTHOR]

DNA mismatch repair (MMR) maintains genome stability primarily by correcting replication errors. MMR deficiency can lead to cancer development and bolsters cancer cell resistance to chemotherapy. However, recent studies have shown that checkpoint blockade therapy is effective in MMR-deficient cancers, thus the ability to identify cancer etiology would greatly benefit cancer treatment. MutS homolog 2 (MSH2) is an obligate subunit of mismatch recognition proteins MutSa (MSH2-MSH6) and MutSß (MSH2-MSH3). Precise regulation of MSH2 is critical, as either over- or underexpression of MSH2 results in an increased mutation frequency. The mechanism by which cells maintain MSH2 proteostasis is unknown. Using functional ubiquitination and deubiquitination assays, we show that the ovarian tumor (OTU) family deubiquitinase ubiquitin aldehyde binding 1 (OTUB1) inhibits MSH2 ubiquitination by blocking the E2 ligase ubiquitin transfer activity. Depleting OTUB1 in cells promotes the ubiquitination and subsequent degradation of MSH2, leading to greater mutation frequency and cellular resistance to genotoxic agents, including the common chemotherapy agents N-methyl-N'-nitro-N-nitrosoguanidine and cisplatin. Taken together, our data identify OTUB1 as an important regulator of MSH2 stability and provide evidence that OTUB1 is a potential biomarker for cancer etiology and therapy. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an autoimmune disease associated with synovial hyperplasia and bone and cartilage destruction. T cells, notably T helper (Th)-1 and Th17 cells, play a critical role in the pathologic process of RA. However, it remains unclear how Th1 and Th17 cells are regulated during RA. In this study, we report that the small ubiquitin-like protein X-linked gene in the G6PD cluster at Xq28 (GdX) regulates the balance of Th17 and regulatory T (Treg) cells during collagen-induced arthritis (CIA). We discovered that the splenocytes of GdX-knockout (KO) mice were insensitive to T-cell stimulants. Correspondingly, GdX-KO mice showed alleviative Th1-mediated delayed-type hypersensitivity and were resistant to CIA compared with wild-type mice. GdX-KO mice showed fewer swollen paws, lower serum proinflammatory cytokine and anti-collagen IgG levels, and decreased synovial hyperplasia. Mechanistically, we observed that deletion of GdX decreased the transcription of proinflammatory cytokines and impaired the Th1 and Th17 differentiation but increased the Treg cell proliferation. Consistently, deletion of GdX decreased the transcription level of T-cell-specific T-box transcription factor and RAR-related orphan receptor-transcription factor but increased that of forkhead box P3 after being challenged with type-II collagen. These findings suggested that GdX functions as an important regulator of Th1 or Th17 and Treg cell balance during the inflammatory responses. Therefore, GdX may be a potential target for the therapy of RA.--Fu, Y., Liu, S., Wang, Y., Ren, F., Fan, X., Liang, J., Liu, C., Li, J., Ju, Y., Chang, Z. GdX/UBL4A-knockout mice resist collagen-induced arthritis by balancing the population of Th1/Th17 and regulatory T cells. [ABSTRACT FROM AUTHOR]

The tumor suppressor phosphatase and tensin homolog (PTEN) plays a central role in regulating phosphatidylinositol 3-kinase (PI3K) signaling, and its gene is very frequently mutated in various human cancers. Numerous studies have revealed that PTEN levels are tightly regulated by both transcriptional and posttranslational modifications, with especially ubiquitylation significantly regulating PTEN protein levels. Although several ubiquitin ligases have been reported to mediate PTEN ubiquitylation in vitro, the ubiquitin ligase that promotes PTEN degradation in vivo has not been reported. Here we took advantage of specific knockout mouse models to demonstrate that WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) promotes PTEN degradation under physiological conditions, whereas another ubiquitin ligase, carboxyl terminus of Hsp70-interacting protein (CHIP), had no such effect. WWP2 knockout mice exhibited reduced body size, elevated PTEN protein levels, and reduced phosphorylation levels of the serine/threonine kinase and PTEN target AKT. In contrast, we observed no elevation of PTEN protein levels in CHIP knockout tissues and mouse embryonic fibroblasts. Furthermore, PTEN protein levels in CHIP/WWP2 double knockout mice were very similar to those in WWP2 single knockout mice and significantly higher than in WT and CHIP knockout mice. Our results demonstrate that WWP2, rather than CHIP, is an ubiquitin ligase that promotes PTEN degradation in vivo. Considering PTEN's significant role in tumor development, we propose that WWP2 may be a potential target for fine-tuning PTEN levels in anticancer therapies. [ABSTRACT FROM AUTHOR]

We previously demonstrated that p15RS, a newly discovered tumor suppressor, inhibits Wnt/β-catenin signaling by interrupting the formation of β-catenin·TCF4 complex. However, it remains unclear how p15RS helps exert such an inhibitory effect on Wnt signaling based on its molecular structure. In this study, we reported that dimerization of p15RS is required for its inhibition on the transcription regulation of Wnt-targeted genes. We found that p15RS forms a dimer through a highly conserved leucine zipper-like motif in the coiled-coil terminus domain. In particular, residues Leu-248 and Leu-255 were identified as being responsible for p15RS dimerization, as mutation of these two leucines into prolines disrupted the homodimer formation of p15RS and weakened its suppression of Wnt signaling. Functional studies further confirmed that mutations of p15RS at these residues results in diminishment of its inhibition on cell proliferation and tumor formation. We therefore concluded that dimerization of p15RS governed by the leucine zipper-like motif is critical for its inhibition of Wnt/β-catenin signaling and tumorigenesis. [ABSTRACT FROM AUTHOR]

We previously reported that p15RS (p15INK4b-related sequence), a regulation of nuclear pre-mRNA domain containing protein, inhibited Wnt signaling by interrupting the formation of the β-catenin ⋅ TCF4 complex. However, how p15RS functions as an intrinsic repressor to repress transcription remains unclear. In this study, we show that p15RS, through a specific interaction with HDAC2 (histone deacetylase 2), a deacetylase that regulates gene transcription, maintains histone H3 in a deacetylated state in the promoter region of Wnt-targeted genes where β-catenin ⋅ TCF4 is bound. We observed that histone deacetylase inhibitors impair the ability of p15RS in inhibiting Wnt/β-catenin signaling. Depletion of HDAC2 markedly disabled p15RS inhibition of Wnt/β-catenin-mediated transcription. Interestingly, overexpression of p15RS decreases the level of acetylated histone H3 in the c-MYC promoter. Finally, we demonstrate that p15RS significantly enhances the association of HDAC2 and TCF4 and enhances the occupancy of HDAC2 to DNA, resulting in the deacetylation of histone H3 and the failure of β-catenin interaction. We propose that p15RS acts as an intrinsic transcriptional repressor for Wnt/β-catenin-mediated gene transcription at least partially through recruiting HDAC2 to occupy the promoter and maintaining deacetylated histone H3. [ABSTRACT FROM AUTHOR]

TNF receptor 2 (TNFR2) exerts diverse roles in the pathogenesis of inflammatory and autoimmune diseases. Here, we report that TNFR2 but not TNFR1 forms a heteromer with interleukin-17 receptor D (IL-17RD), also named Sef, to activate NF-κB signaling. TNFR2 associates with IL-17RD, leading to mutual receptor aggregation and TRAF2 recruitment, which further activate the downstream cascade of NF-κB signaling. Depletion of IL-17RD impaired TNFR2-mediated activation of NF-κB signaling. Importantly, IL-17RD was markedly increased in renal tubular epithelial cells in nephritis rats, and a strong interaction of TNFR2 and IL-17RD was observed in the renal epithelia. The IL-17RD·TNFR2 complex in activation of NF-κB may explain the role of TNFR2 in inflammatory diseases including nephritis. [ABSTRACT FROM AUTHOR]

CREPT (cell cycle-related and expression elevated protein in tumor)/RPRD1B (regulation of nuclear pre-mRNA domaincontaining protein 1B), highly expressed during tumorigenesis, was shown to enhance transcription of CCND1 and to promote cell proliferation by interacting with RNA polymerase II. However, which signaling pathway is involved in CREPT-mediated activation of gene transcription remains unclear. In this study, we reveal that CREPT participates in transcription of the Wnt/ β-catenin signaling activated genes through the β-catenin and the TCF4 complex. Our results demonstrate that CREPT interacts with both β-catenin and TCF4, and enhances the association of β-catenin with TCF4, in response to Wnt stimulation. Furthermore, CREPT was shown to occupy at TCF4 binding sites (TBS) of the promoters of Wnt-targeted genes under Wnt stimulation. Interestingly, depletion of CREPT resulted in decreased occupancy of β-catenin on TBS, and over-expression of CREPT enhances the activity of the β-catenin∙TCF4 complex to initiate transcription of Wnt target genes, which results in up-regulated cell proliferation and invasion. Our study suggests that CREPT acts as an activator to promote transcriptional activity of the β-catenin∙TCF4 complex in response to Wnt signaling. [ABSTRACT FROM AUTHOR]

The carboxyl terminus of Hsc70-interacting protein (CHIP, also named Stub1), a U-box containing E3 ubiquitin ligase, is involved in degradation of certain oncogenic proteins. Recent studies indicated that CHIP suppresses tumor progression in human cancers by targeting Src-3, hypoxia inducible factor 1α, NF-κB, ErbB2 and c-Myc. Here, we report that CHIP was downregulated, predominantly, in the late stages of human colorectal cancer (CRC), and that the CHIP promoter was hypermethylated in CRC specimens. Overexpression of CHIP in HCT-116 cells resulted in impaired tumor growth in nude mice and decreased abilities of tumor cell migration and invasion. Conversely, depletion of CHIP in HCT-116 cells promoted tumor growth and increased tumor cell migration and invasion. CHIP was further found to negatively regulate NF-κB signaling in HCT-116 cells by promoting ubiquitination and degradation of p65, a subunit of the NF-κB complex. The suppressive effect of CHIP led to decreased expression of NF-κB-targeted oncogenes including Cyclin D1, c-Myc, MMP-2, VEGF and IL-8. We proposed that CHIP inhibits the malignancy of CRC cells, possibly through targeting NF-κB signaling. This study provides functional evidence for CHIP as a potential tumor suppressor in CRC, and CHIP expression may be a marker for stages of CRC. [ABSTRACT FROM AUTHOR]

Wnt signaling plays a pivotal role in cell proliferation, tissue homeostasis,andtumorigenesis. Dishevelled (Dvl) is a centralnode of Wnt signaling. Insulin receptor substrates (IRSs), as a critical component of insulin signaling, are involved in cell proliferation, metabolism, and cancer development. In this study, we report that IRS1/2 promotes Wnt/β-catenin signaling by stabilizing Dvl2.We found that IRS1/2 interacts with Dvl2. Overexpression of IRS1/2 increased the protein level of Dvl2 and promoted canonical Wnt signaling, as evidenced by the increased T cell-specific factor 4 transcriptional activity and the up-regulation of expression of CYCLIN D1 and c-MYC, two Wnt target genes critical for cell growth, whereas depletion of IRS1/2 reduced the level of Dvl2 and attenuated Wnt/β-catenin signaling. Biochemical analyses revealed that IRS1/2 decreased Lys-63-linked ubiquitination of Dvl2 and stabilized Dvl2 protein via suppressing its autophagy-mediated degradation. We further revealed that IRS1/2 blocks autophagy- induced formation of the Dvl2-p62/SQSTM1 complex, resulting in disabled association of Dvl2 to autophagosomes. We demonstrated that IRS1/2 promoted the induction of epithelialmesenchymal transition (EMT) and cell proliferation in response to Wnt stimulation, whereas depletion of Dvl2 impaired the IRS1/ 2-mediated EMT and cell growth. Our findings revealed that IRS1/2 promotesEMTand cell proliferation through stabilizing Dvl2. [ABSTRACT FROM AUTHOR]

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis. [ABSTRACT FROM AUTHOR]

Elf3 belongs to the Ets family of transcription factors and has been implicated in inflammation. Elf3 is highly expressed in the lungs, and Elf3-/- mice are impaired in IL-6 production after intranasal LPS exposure. To identify the role of Elf3 in Th17-driven pulmonary inflammation, we have performed epicutaneous sensitization of Elf3-/- mice with OVA followed by airway OVA challenge and have identified Elf3-/- mice to be impaired in induction of Th17 response, attributable to impairment of IL-6 production by dendritic cells (DCs). However, increased serum levels of OVA-specific IgG1 and IgE were observed, pointing toward an exaggerated Th2 response. To study Th2 response, we performed i.p. sensitization of Elf3-/- mice with OVA and confirmed loss of Elf3 to result in an aggravated Th2 response, characterized by increased generation of IL-4-producing T cells, increased levels of OVA-specific IgE and IgG1 Ab titers, and increased serum levels of Th2 cytokines, together with extensive inflammation and mucus production in airways. Elf3-/- DCs were impaired in priming Th1 differentiation, which, in turn, promoted Th2 differentiation. This was mediated by the ability of Elf3-/- DCs to undergo hypermaturation but secrete significantly lower levels of IL-12 in response to inflammatory stimuli. The impairment of IL-12 production was due to impairment of IL-12p40 gene induction in Elf3-/- DCs in response to inflammatory stimuli. Taken together, our study identifies a novel function of Elf3 in regulating allergic airway inflammation by regulating DC-driven Th1, Th2, and Th17 differentiation. [ABSTRACT FROM AUTHOR]

The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner. [ABSTRACT FROM AUTHOR]

The formation of a β-catenin·TCF4 complex in the nucleus of cells is well known as a prerequisite for the transcription of Wnt target genes. Although many co-factors have been identified to regulate the activity of the β-catenin·TCF4 complex, it remains unclear how the complex association is negatively regulated. In this study, we report that p15RS, a negative regulator of the cell cycle, blocks β-catenin·TCF4 complex formation and inhibits Wnt signaling. We observed that p15RS interacts with β-catenin and TCF4. Interestingly, whereas the interaction of p15RS with β-catenin is increased, its interaction with TCF4 is decreased upon Wnt1 stimulation. Moreover, overexpression of p15RS reduces the interaction of β-catenin with TCF4, whereas the depletion of p15RS enhances their interaction. We further demonstrate that overexpression of p15RS suppresses canonical Wnt signaling and results in retarded cell growth, whereas depletion of p15RS shows an enhanced effect on Wnt signaling. We analyzed that inhibition of Wnt signaling by p15RS leads to decreased expression of CYCLIN D1 and c-MYC, two Wnt targeted genes critical for cell growth. Our data suggest that p15RS inhibits Wnt signaling by interrupting β-catenin·TCF4 complex formation and that Wnt signaling initiates downstream gene expression by removing p15RS from promoters. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation. [ABSTRACT FROM AUTHOR]

Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat PLCδ1-PH (pleckstrin homology domain of phospholipase C-δ1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat PLCδ1-PH were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of PLCδ1-PH (ERPH1) was found to bind EPOR with high affinity (KD of 24 nM and an 1C50 of 5.7 μM) both in vitro and in a cell-based assay, respectively, although the WT PLCδ1-PH did not show any detectable binding under the assay conditions. The in vitro binding affinities of the PLCδ1-PH mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting protein-protein interfaces. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) signaling is critical in a variety of biological processes such as cell proliferation, differentiation, and apoptosis. TGF-β signaling is mediated by a group of proteins including TGF-β receptors and Smads. It is known that different cells can exhibit different sensitivities to TGF-β. Several molecular mechanisms, such as the differential expression of the receptor levels, have been suggested as contributing to these differences. Here, we report evidence for a novel mechanism of regulating TGF-β sensitivity that depends on the role of CHIP (carboxyl terminus of Hsc70-interacting protein) in regulating the basal level of Smad3 via the ubiquitin-dependent degradation pathway. First, using a luciferase assay we found that overexpression of CHIP inhibited TGF-β signaling, whereas silencing CHIP expression by small interfering RNAs led to increased TGF-β signaling sensitivity. Second, based on the results of cell proliferation assays and JunB expression, we found that TGF-β signaling could be abolished by stably overexpressing CHIP. Third, in those cell lines with stably expressed CHIP, we observed that the Smad3 protein level was dramatically decreased. Finally, we demonstrated that CHIP served as a U-box dependent E3 ligase that can directly mediate ubiquitination and degradation of Smad3 and that this action of CHIP was independent of TGF-β signaling. Taken together, these findings suggest that CHIP can modulate the sensitivity of the TGF-β signaling by controlling the basal level of Smad3 through ubiquitin-mediated degradation. [ABSTRACT FROM AUTHOR]

Transforming growth factor beta (TGF-β)/bone morphogenetic protein (BMP) family ligands interact with specific membrane receptor complexes that have serine/threonine kinase activities. The receptor phosphorylation and activation induced by the ligands leads to phosphorylation of the Smad proteins, which translocate to the nucleus, controlling gene expression. Thus, regulation of Smad proteins is a key step in TGF-β/BMP-induced signal transduction. Here we report a novel mechanism of the regulation of SMAD-mediated signaling, by which the Smad1 protein level is controlled through expression of the CHIP protein. CHIP is a U-box-dependent E3 ubiquitin ligase, previously identified as a cochaperon protein. However, we have isolated CHIP as a Smad-interacting protein in a yeast two-hybrid screen using Smad1 as bait. Furthermore we have shown CHIP-Smad interaction using the [sup 35]S-labeled CHIP protein, which can interact with glutathione S-transferase (GST)-Smad1 and GST-Smad4 in an in vitro protein-binding assay. The CHIP-Smad interaction has been confirmed in vivo in mammalian cells through coimmunoprecipitation. Interestingly, we demonstrate that the coexpression of Smad1 and Smad4 with the CHIP protein results in the degradation of the Smad proteins through a ubiquitin-mediated process. Consistent with the observation that CHIP induces Smad1 degradation, we further show that the expression of CHIP can inhibit the transcriptional activities of the Smad1/Smad4 complex induced by BMP signals. Intriguingly, pBS/U6/CHIPi, which diminishes CHIP expression, significantly enhanced Smad1/Smad4- or BMPRIB(QD)-induced gene transcription. These results suggest that CHIP can interact with the Smad1/Smad4 proteins and block BMP signal transduction through the ubiquitin-mediated degradation of Smad proteins. [ABSTRACT FROM AUTHOR]

Growth factor signaling by receptor tyrosine kinases regulates several cell fates, such as proliferation and differentiation. Sef was genetically identified as a negative regulator of fibroblast growth factor (FGF) signaling. Using bioinformatic methods and rapid amplification of eDNA ends-PCR, we isolated both the mouse and the human Sef genes, which encoded the Sef protein and Sef-S isoform that was generated through alternative splicing. We provide evidence that the Sef gene products were located mainly on the cell membrane. Co-immunoprecipitation and immunostaining experiments indicate that hSef interacts with FGFR1 and FGFR2 but not FGFR3. Our results demonstrated that stably expressed hSef strongly inhibits FGF2- or nerve growth factor-induced PC-12 cell differentiation. The intracellular domain of hSef is necessary for the inhibitory effect on FGF2-induced PC-12 cell differentiation. Furthermore, our data suggested Sef exerted the negative effect on FGF2-induced PC-12 cell differentiation through the prevention of Ras-mitogen-activated protein kinase signaling, possibly functioning upstream of the Ras molecule. These findings suggest that Sef may play an important role in the regulation of PC-12 cell differentiation. [ABSTRACT FROM AUTHOR]

The small GTPases Rab5 and Rab21 are closely related, and play essential roles in endocytic trafficking. Rab5 is regulated by VPS9-domain-containing guanine nucleotide exchange factors. Here, we describe a new VPS9-domain protein with ankyrin repeats, the VPS9-ankyrin-repeat protein (Varp). Varp interacts preferentially with GDP-bound Rab21 and has a much stronger guanine nucleotide exchange activity towards Rab21 than Rab5. Furthermore, RNAi-mediated depletion of endogenous Varp significantly disrupts the activity of Rab21 in HeLa cells. Ectopically expressed Varp mainly localizes to early endosomes and causes enlargement of early endosomes and giant late endosomes. Both the VPS9 domain and ankyrin-repeats are required for the endosomal localization and the activity of Varp in vivo. These results suggest that Varp is a potential Rab21 guanine nucleotide exchange factor and might regulate endosome dynamics in vivo. [ABSTRACT FROM AUTHOR]