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Intrinsic GTPase Activity (intrinsic + gtpase_activity)
Selected AbstractsRab6 family proteins interact with the dynein light chain protein DYNLRB1CYTOSKELETON, Issue 3 2008Bas Wanschers Abstract The small GTPase Rab6 is a key regulator in the retrograde transfer from endosomes via the Golgi to the ER. Three isoforms of Rab6 have been identified, the ubiquitously expressed Rab6A and Rab6A,, and the brain specific Rab6B. Recent studies have shown that Rab6A, is the major isoform regulating this retrograde transport. Cytoplasmic dynein is the main motor protein complex for this transport. Dynein consists of two heavy chains, two intermediate chains, four light intermediatechains and several light chains, called roadblock/LC7 proteins or DYNLRB proteins. In mammalian cells two light chain isoforms have been identified, DYNLRB1 and DYNLRB2. We here show with yeast-two-hybrid, co-immunoprecipitation and pull down studies that DYNLRB1 specifically interacts with all three Rab6 isoforms and co-localises at the Golgi. This is the first example of a direct interaction between Rab6 isoforms and the dynein complex. Pull down experiments showed further preferred association of DYNLRB1 with GTP-bound Rab6A and interestingly GDP-bound Rab6A, and Rab6B. In addition DYNLRB1 was found in the Golgi apparatus where it co-localises with EYFP-Rab6 isoforms. DYNLRB is a putative modulator of the intrinsic GTPase activity of GTP-binding proteins. In vitro we were not able to reproduce this effect on Rab6 GTPase activity. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Diverting a protein from its cellular location by intracellular antibodiesFEBS JOURNAL, Issue 4 2000The case of p21Ras We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic. [source] A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formationGLIA, Issue 11 2007Therese M. Roth Abstract The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/,), or null (,/,) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant. © 2007 Wiley-Liss, Inc. [source] Gs, Mutations in Fibrous Dysplasia and McCune-Albright Syndrome,JOURNAL OF BONE AND MINERAL RESEARCH, Issue S2 2006Lee S Weinstein Abstract Fibrous dysplasia (FD) is a focal bone lesion composed of immature mesenchymal osteoblastic precursor cells. Some FD patients also have hyperpigmented skin lesions (café-au-lait spots), gonadotropin-independent sexual precocity, and/or other endocrine and nonendocrine manifestations (McCune-Albright syndrome [MAS]). MAS results from somatic mutations occurring during early development, resulting in a widespread mosaic of normal and mutant-bearing cells, which predicts that the clinical presentation of each patient is determined by the extent and distribution of abnormal cells. These mutations encode constitutively active forms of Gs,, the ubiquitously expressed G protein ,-subunit that couples hormone receptors to intracellular cAMP generation. These mutations lead to substitution of amino acid residues that are critical for the intrinsic GTPase activity that is normally required to deactivate the G protein. This leads to prolonged activation of Gs, and its downstream effectors even with minimal receptor activation. This explains why MAS patients have stimulation of multiple peripheral endocrine glands in the absence of circulating stimulatory pituitary hormones and increased skin pigment, which is normally induced by melanocyte-stimulating hormone through Gs,/cAMP. Similar mutations are also present in 40% of pituitary tumors in acromegaly patients and less commonly in other endocrine tumors. FD results from increased cAMP in bone marrow stromal cells, leading to increased proliferation and abnormal differentiation. Parental origin of the mutated allele may also affect the clinical presentation, because Gs, is imprinted and expressed only from the maternal allele in some tissues (e.g., pituitary somatotrophs). [source] Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2PIGMENT CELL & MELANOMA RESEARCH, Issue 3 2010Norihiko Ohbayashi Summary Human Griscelli syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2-a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2-a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The Rab27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a. [source] Regulation of ADL6 activity by its associated molecular networkTHE PLANT JOURNAL, Issue 5 2002Bernard C.-H. Summary Plant dynamin-like proteins consist of a group of high molecular weight GTPase with diverse structural arrangements and cellular localizations. In addition, unlike animal dynamins, there was no evidence for the involvement of any plant dynamin-like protein in clathrin-mediated vesicle trafficking. In this study we demonstrate that ADL6 (Arabidopsis dynamin-like protein 6), due to its domain arrangement, behaves similarly to the animal dynamins. The association of ADL6 with clathrin-coated vesicles was demonstrated by co-fractionation and immunocytochemical studies. ADL6 also interacted via its C-terminus with ,-adaptin, an adaptor protein of clathrin-coated vesicles. Our results suggest that ADL6 participates in clathrin-mediated vesicle trafficking originating from the Golgi. In addition, our studies demonstrate that ADL6 intrinsic GTPase activity is regulated by its association with acidic phospholipids and an SH3 (Src homology 3)-containing protein. [source] | ||||||||||