Molecular cell biology research communications : MCBRC最新文献

Recently we identified novel plant Ser/Thr phosphatases, termed PP7, which belong to the PPP family and have no known close homologs in other kingdoms. We now addressed the intracellular location of Arabidopsis thaliana PP7 using GFP fusions and confocal laser scanning microscopy. PP7 · GFP fusion was expressed transiently or stably in Nicotiana benthamiana. PP7 · GFP was found to be a predominantly nuclear protein. Effects of cytoskeleton-disrupting drugs indicate that cytoskeleton may be required for efficient PP7 · GFP delivery to the nucleus. Deletion of a potential nuclear localization signal in the first insert in the catalytic domain, as well as exposure to the dark, cold, high salinity and abscisic acid failed to prevent nuclear localization of PP7 · GFP. Deletion of the 44 C-terminal amino acids resulted in a fusion protein located exclusively in the cytoplasm. The results suggest a possible similarity of the nuclear targeting signals in PP7 and the PP5/PPT subfamily.

Members of transforming growth factor β (TGF-β) family are potent regulators of multiple cellular functions, including cell proliferation, differentiation, migration, organization, and death. Yet the signaling pathways underpinning a wide array of biological activities of TGF-β appear to be deceptively simple. At every step from TGF-β secretion to activation of its target genes, the activity of TGF-β is regulated tightly, both positively and negatively. Biologically active TGF-β is cleaved from a precursor protein (latent form) and multiple process factors control the levels of active TGF-β. The efficient secretion, correct folding and deposition to the extracellular matrices require the cosecretion of latent TGF-β binding proteins (LTBPs). Once activated, TGF-β ligand signals through a heteromeric receptor complex of two distinct type I and type II serine/threonine kinase receptors TβRI and TβRII. Many factors appear to influence the formation of the active ligand–receptor complex. The relative orientation of TβRI and TβRII in the ligand–receptor complex is critical for activation: through TβRI, the activated ligand–receptor complex directly binds and phosphorylates downstream intracellular substrates, called Smads. Inhibitory Smads, Smad6 and 7, can antagonize this process. The phosphorylation of Smads leads to the formation of complexes which translocate to the nucleus. Other signaling systems can modulate the activity of the Smads: e.g., ras activity can prevent Smad complexes from entering the nucleus and specific ubiquitin ligases can target Smad for degradation. In the nucleus, the Smad complexes associate with other transcription activators or suppressors to regulate gene expression, either positively or negatively. The combined effects of the positive and/or negative TGF-β controlled gene expression together with the endogenous protein set of the target cell are responsible for the multiplicity of biological functions.

The expression of CAS is reported to be upregulated in a variety of human tumor cells, and such expression correlates with the development of tumors. CAS also plays a role in apoptosis. We investigated whether CAS expression affects the susceptibility of tumor cells to IFN-γ-induced apoptosis. Our data show that IFN-γ treatment induces CAS expression in HT-29 tumor cells. IFN-γ-induced gene expression is primarily mediated by the transcriptional factor, IRF-1. Our data show that IRF-1 mediates IFN-γ-induced CAS expression. Transfection of HT-29 cells with CAS expression vector did not induce apoptosis of cells; nevertheless, CAS overexpression greatly enhanced IFN-γ-induced apoptosis of cells. CPP32 is regarded as one of the central apoptosis executioner molecules. CAS overexpression enhances IFN-γ-induced CPP32 expression. These results indicate that tumor cells highly expressing CAS may be more susceptible to apoptosis induced by reagents that are capable of inducing CAS expression. Thus, CAS may be a target for the elimination of tumors.

The asialoglycoprotein (ASGP) receptor is expressed on hepatocytes and liver-derived cell lines and is responsible for the endocytosis of galactose-terminal glycoproteins via the coated pit pathway. Prior data showed that tyrosine kinase activity plays an important role in this endocytic process, though the critical kinase(s) responsible for this effect are unknown. We have detected a 60-kDa protein which coprecipitates with ASGP receptor in detergent-solubilized lysates of HepG2 cells. This protein autophosphorylates and binds radioactive ATP. It comigrates with authentic pp60 c-src and is recognized by a specific anti-src monoclonal antibody. The kinase associated with the ASGP receptor retains the ability to phosphorylate exogenous substrates on tyrosine. In conclusion, the tyrosine kinase c-src associates with the ASGP receptor, a protein of the coated pit pathway of endocytosis.

LP54 is an RNA-binding protein involved in localization of maternal messengers in sea urchin egg and embryos. Using a polyclonal antibody directed against Paracentrotus lividus LP54 we detected a 66-kDa cross-reacting antigen in undifferentiated and differentiated SH-SY5Y human neuroblastoma cells. After treatment of undifferentiated cells with detergent, the 66-kDa antigen was found to be enriched in the cytoskeletal fraction. By Western blot the expression of this antigen was also analyzed in regions of the CNS and in tissues of the adult rat and its exclusive presence in the hippocampus and thalamus was revealed. The immunoreactivity with P. lividus antibody against LP54 in hippocampal lysate was also confirmed throughout anti-LP54 immunoaffinity column and competition experiments. The results indicates that a related protein to the sea urchin LP54 is evolutionary conserved in mammalian CNS.

p38, a member of the mitogen-activated protein kinase (MAPK) superfamily, is activated in response to a variety of cellular stresses and ligands. Since the genome of the nematode C. elegans has been sequenced, we sought to identify and characterize the nematode homolog of mammalian p38. By sequence analysis and RT-PCR, we isolated cDNAs encoding three kinases, PMK-1, PMK-2, and PMK-3, which we call p38 map kinases due to their high sequence identity with p38. The three genes are contiguous on chromosome IV and comprise an operon. By use of a GFP reporter, we found that the promoter of the pmks is active throughout the intestine. An active form of MAPK/ERK kinase 6 (MEK6) phosphorylated and activated recombinant PMK-1 and PMK-2 in vitro. PMK-1 and PMK-2 phosphorylated activating transcription factor-2 (ATF-2), indicating an activity similar to mammalian p38. When transfected into mammalian cells, these kinases, like p38, are stimulated by osmotic stresses.

The tuberous sclerosis 2 (TSC2) gene has been genetically mapped to a disease characterized by abnormal cell proliferation that results in the production of tumorous lesions in a variety of tissues. The molecular mechanism for TSC2 mediation of tuberous sclerosis is unclear but it appears to be related to its ability to cytoplasmically interact with a second gene, TSC1, mapping to the disease. These proteins are linked to constraints on cell cycle signaling pathways and therefore envisioned to function as tumor suppressor genes. In previous studies we have demonstrated TSC2 associations with steroid receptor family members and modulation of their gene expression capabilities. Here we provide evidence for TSC2 translocation to the nucleus and a possible role for phosphorylation in both TSC2 translocation and TSC2 modulation of steroid receptor-mediated transcription.

The fibroblast growth factor receptors (FGFRs) are a family of transmembrane tyrosine kinases that play a key role in cell growth and tumorigenesis in response to FGFs. FGFR complexity is increased by the existence of additional isoforms generated by alternative mRNA splicing. We identified that the transcript FGFR3ΔTM, an alternatively spliced isoform of FGFR3 lacking exons encoding the C-terminal half of Ig III (IIIb) and transmembrane domains, is expressed in the human squamous carcinoma cell line DJM-1. To determine whether FGFR3ΔTM has the potential to be secreted, we analyzed the protein expression in CHOK1 cells transfected with FGFR3ΔTM cDNA and DJM-1 cells. Western blot analysis revealed that FGFR3ΔTM protein was secreted, N-glycosylated, and dimerized by an intermolecular disulfide bond. Cross-linking experiments showed that FGF1 and FGF2 were able to bind to FGFR3ΔTM, suggesting that the loss of the Ig IIIb domain may confer upon FGFR3ΔTM the ability to bind to FGF2.