RESUMEN
Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, the kinase-specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on the Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here, we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5 in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity on BRAFV600E and CRAF, reveal how PP5 is activated by recruitment to HSP90 complexes. PP5 comprehensively dephosphorylates client proteins, removing interaction sites for regulatory partners such as 14-3-3 proteins and thus performing a 'factory reset' of the kinase prior to release.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Humanos , Proteínas de Ciclo Celular/genética , Chaperoninas/genética , Proteínas HSP90 de Choque Térmico/genética , Chaperonas Moleculares , Monoéster Fosfórico Hidrolasas , Proteómica , Proteínas Proto-Oncogénicas B-rafRESUMEN
The pairing of sister chromatids in interphase facilitates error-free homologous recombination (HR). Sister chromatids are held together by cohesin, one of three Structural Maintenance of Chromosomes (SMC) complexes. In mitosis, chromosome condensation is controlled by another SMC complex, condensin, and the type II topoisomerase (Top2). In prophase, cohesin is stripped from chromosome arms, but remains at centromeres until anaphase, whereupon it is removed via proteolytic cleavage. The third SMC complex, Smc5/6, is generally described as a regulator of HR-mediated DNA repair. However, cohesin and condensin are also required for DNA repair, and HR genes are not essential for cell viability, but the SMC complexes are. Smc5/6 null mutants die in mitosis, and in fission yeast, Smc5/6 hypomorphs show lethal mitoses following genotoxic stress, or when combined with a Top2 mutant, top2-191. We found these mitotic defects are due to retention of cohesin on chromosome arms. We also show that Top2 functions in the cohesin cycle, and accumulating data suggests this is not related to its decatenation activity. Thus the SMC complexes and Top2 functionally interact, and any DNA repair function ascribed to Smc5/6 is likely a reflection of a more fundamental role in the regulation of chromosome structure.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Inestabilidad Genómica , Mitosis , Mutación , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , CohesinasRESUMEN
The function of the essential cohesin-related Smc5-Smc6 complex has remained elusive, though hypomorphic mutants have defects late in recombination, in checkpoint maintenance, and in chromosome segregation. Recombination and checkpoints are not essential for viability, and Smc5-Smc6-null mutants die in lethal mitoses. This suggests that the chromosome segregation defects may be the source of lethality in irradiated Smc5-Smc6 hypomorphs. We show that in smc6 mutants, following DNA damage in interphase, chromosome arm segregation fails due to an aberrant persistence of cohesin, which is normally removed by the Separase-independent pathway. This postanaphase persistence of cohesin is not dependent on DNA damage, since the synthetic lethality of smc6 hypomorphs with a topoisomerase II mutant, defective in mitotic chromosome structure, is also due to the retention of cohesin on undamaged chromosome arms. In both cases, Separase overexpression bypasses the defect and restores cell viability, showing that defective cohesin removal is a major determinant of the mitotic lethality of Smc5-Smc6 mutants.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Cromosomas/metabolismo , Mitosis/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Animales , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Daño del ADN , Reparación del ADN , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Separasa , CohesinasRESUMEN
We describe two RING finger proteins in the fission yeast Schizosaccharomyces pombe, Rfp1 and Rfp2. We show that these proteins function redundantly in DNA repair. Rfp1 was isolated as a Chk1-interacting protein in a two-hybrid screen and has high amino acid sequence similarity to Rfp2. Deletion of either gene does not cause a phenotype, but a double deletion (rfp1Deltarfp2Delta) showed poor viability and defects in cell cycle progression. These cells are also sensitive to DNA-damaging agents, although they maintained normal checkpoint signaling to Chk1. Rfp1 and Rfp2 are most closely related to human Rnf4, and we showed that Rnf4 can substitute functionally for Rfp1 and/or Rfp2. The double mutants also showed significantly increased levels of protein SUMOylation, and we identified an S. pombe Ulp2/Smt4 homolog that, when overexpressed, reduced SUMO levels and suppressed the DNA damage sensitivity of rfp1Delta rfp2Delta cells.
