RESUMO
Son of Sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the small GTPases RAC and RAS. Although the molecular mechanisms of RAS GEF catalysis have been unveiled, how SOS1 acquires RAC GEF activity and what is the physio-pathological relevance of this activity is much less understood. Here we show that SOS1 is tyrosine phosphorylated on Y1196 by ABL. Phosphorylation of Y1196 controls SOS1 inter-molecular interaction, is required to promote the exchange of nucleotides on RAC in vitro and for platelet-derived growth factor (PDGF) activation of RAC- and RAC-dependent actin remodeling and cell migration. SOS1 is also phosphorylated on Y1196 by BCR-ABL in chronic myelogenous leukemic cells. Importantly, in these cells, SOS1 is required for BCR-ABL-mediated activation of RAC, cell proliferation and transformation in vitro and in a xenograft mouse model. Finally, genetic removal of Sos1 in the bone marrow-derived cells (BMDCs) from Sos1fl/fl mice and infected with BCR-ABL causes a significant delay in the onset of leukemogenesis once BMDCs are injected into recipient, lethally irradiated mice. Thus, SOS1 is required for full transformation and critically contribute to the leukemogenic potential of BCR-ABL.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Fusão bcr-abl/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína SOS1/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia/genética , Leucemia/metabolismo , Camundongos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tirosina/metabolismo , Proteínas rac de Ligação ao GTP , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Despite many decades of study, mitotic chromosomes remain poorly characterized with respect to their structure and composition. Here, we have purified mitotic chromosomes from nocodazole-treated chicken DT40 cells. These chromosomes have a 0.7:1:1 ratio of nonhistone proteins to histones to DNA. They also contain a significant content of RNAs that have yet to be characterized. Overall, the isolated chromosomes contained >4000 polypeptides, >500 of which are either novel or uncharacterized. Elsewhere, we have developed an approach for comparing the results of multiple proteomics experiments. As a validation of this approach, one of 13 novel centromere proteins identified was found to occur in a complex with the previously described proteins Ska1 and Ska2. This novel protein, now known as Ska3/Rama1, occupies a unique domain in the outer kinetochore and was revealed by RNA interference (RNAi) experiments to be essential for cell cycle progression in human cells. The approach presented here offers a powerful way to define the functional proteome of complex organelles and structures whose composition is not simple or fixed.
Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Proteômica/métodos , Animais , Linhagem Celular , Galinhas , DNA/metabolismo , Histonas/metabolismo , Humanos , Ligação Proteica , Proteoma/metabolismoRESUMO
Recent studies indicate that splicing of pre-messenger RNA and export of mRNA are normally coupled in vivo. During splicing, the conserved mRNA export factor Aly is recruited to the spliced mRNA-protein complex (mRNP), which targets the mRNA for export. At present, it is not known how Aly is recruited to the spliced mRNP. Here we show that the conserved DEAD-box helicase UAP56, which functions during spliceosome assembly, interacts directly and highly specifically with Aly. Moreover, UAP56 is present together with Aly in the spliced mRNP. Significantly, excess UAP56 is a potent dominant negative inhibitor of mRNA export. Excess UAP56 also inhibits the recruitment of Aly to the spliced mRNP. Furthermore, a mutation in Aly that blocks its interaction with UAP56 prevents recruitment of Aly to the spliced mRNP. These data suggest that the splicing factor UAP56 functions in coupling the splicing and export machineries by recruiting Aly to the spliced mRNP.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas Nucleares , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Fatores de Transcrição/fisiologia , Animais , Transporte Biológico , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/fisiologia , XenopusRESUMO
Stathmin/OP18 is a regulatory phosphoprotein that controls microtubule (MT) dynamics. The protein does not have a defined three-dimensional structure, although it contains three distinct regions (an unstructured N-terminus, N: 1-44; a region with high helix propensity, H 1: 44-89; and a region with low helix propensity, H 2: 90-142). The full protein and a combination of H 1 and H 2 inhibits tubulin polymerization, while the combination of H 1 and the N-terminus is less efficient. None of the individual three regions alone are functional in this respect. However, all of them cross-link to alpha-tubulin, but only full-length stathmin produces high-molecular-weight products. Mass spectrometry analysis of alpha-tubulin-stathmin/OP18 and its truncation products shows that full-length stathmin/OP18 binds to the region around helix 10 of alpha-tubulin, a region that is involved in longitudinal interactions in the MT, sequestering the dimer and possibly linking two tubulin heterodimers. In the absence of the N-terminus, stathmin/OP18 binds to only one molecule of alpha-tubulin, at the top of the free tubulin heterodimer, preventing polymerization.