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1.
BMC Cell Biol ; 3: 25, 2002 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-12323076

RESUMO

BACKGROUND: Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity. RESULTS: The functions of other Rab proteins have been defined through the use of dominant-negative mutants with amino acid substitutions in the conserved N(T)KxD nucleotide binding motif. Surprisingly, when such Rab24 constructs were expressed in cultured cells, they accumulated in nuclear inclusions which disrupted the integrity of the nuclear envelope. The inclusions reacted positively with antibodies against ubiquitin and Hsp70, similar to protein aggregates observed in polyglutamine disorders. They also appeared to sequester importin-beta and GFP-coupled glucocorticoid receptor. Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form. Studies with Rab24/Rab1B chimeras indicated that targeting of the mutant protein to inclusions requires the unique C-terminal domain of Rab24. CONCLUSION: These studies demonstrate that mutations in Rab24 can trigger a cytopathic cellular response involving accumulation of nuclear inclusions. If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope. However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.


Assuntos
Núcleo Celular/química , Corpos de Inclusão/química , Mutação/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Células 3T3/química , Transporte Ativo do Núcleo Celular/genética , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Humanos , Corpos de Inclusão/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Rim/química , Rim/citologia , Rim/embriologia , Rim/ultraestrutura , Substâncias Macromoleculares , Camundongos , Peptídeos/genética , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Ubiquitina/química , Ubiquitina/genética , Ubiquitina/imunologia , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/imunologia
2.
Biochem Biophys Res Commun ; 312(3): 670-5, 2003 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-14680817

RESUMO

Several members of the large family of Rab GTPases have been shown to function in vesicular trafficking in mammalian cells. However, the exact role of Rab24 remains poorly defined. Rab24 differs from other Rab proteins in that it has a low intrinsic GTPase activity and is not efficiently prenylated. Here we report an additional unique property of Rab24; i.e., the protein can undergo tyrosine phosphorylation when overexpressed in cultured cells. Immunoblot analyses with specific anti-phosphotyrosine monoclonal antibodies revealed the presence of phosphotyrosine (pTyr) on myc-Rab24 in whole cell lysates and immunoprecipitated samples. No pTyr was detected on other overexpressed myc-tagged GTPases (H-Ras, Rab1b, Rab6, Rab11 or Rab13). Comparisons of myc-Rab24 in the soluble and particulate fractions from HEK293 and HEp-2 cells indicated that the cytosolic pool of Rab24 was more heavily phosphorylated than the membrane pool. Treatment of transfected cells with the broad-spectrum tyrosine kinase inhibitor, genistein, as well as the specific Src-family kinase inhibitor, PP2, eliminated the pTyr signal from Rab24. In contrast the receptor tyrosine kinase inhibitor, tyrphostin A25, had no effect. Tyrosine phosphorylation of Rab24 was reduced by alanine substitution of two unique tyrosines, one found in a strong consensus phosphorylation motif (Y [Formula: see text] ) in the hypervariable domain (Y172) and the other falling within the GXXXGK(S/T) motif known as the P-loop (Y17). The latter region is known to influence GTP hydrolysis in Rab proteins, so the phosphorylation of Y17 could contribute to the low intrinsic GTPase activity of Rab24. This is the first report of tyrosine phosphorylation in any member of the Ras superfamily and it raises the possibility that this type of modification could influence Rab24 targeting and interactions with effector protein complexes.


Assuntos
Rim/metabolismo , Neoplasias Laríngeas/enzimologia , Tirosina/química , Tirosina/metabolismo , Quinases da Família src/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Ativação Enzimática , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Humanos , Rim/embriologia , Neoplasias Laríngeas/química , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Relação Estrutura-Atividade , Especificidade por Substrato , Quinases da Família src/química
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