RESUMEN
The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as "methylation"). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3ß bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Espectrometría de Masas , Metilación , Ratones , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Fosforilación , Dominios Proteicos , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteómica/métodosRESUMEN
Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.
Asunto(s)
Proteína BRCA2/metabolismo , Neoplasias de la Mama/genética , Neoplasias Ováricas/genética , Proteína Fosfatasa 2/metabolismo , Reparación del ADN por Recombinación , Proteína BRCA2/genética , Roturas del ADN de Doble Cadena , Femenino , Predisposición Genética a la Enfermedad , Células HeLa , Humanos , Mutación , Fosforilación/genética , Unión Proteica/genética , Proteína Fosfatasa 2/genética , Recombinasa Rad51/metabolismoRESUMEN
The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A-B56 phosphatase through a coiled-coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A-B56-hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A-B56 substrates containing a canonical B56 binding motif. We find that PP2A-B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.
Asunto(s)
Proteínas de Ciclo Celular , Proteína Fosfatasa 2 , Proteína Quinasa CDC2 , Proteínas de Ciclo Celular/genética , Centrómero , Humanos , Meiosis , Mitosis , Proteína Fosfatasa 2/genéticaRESUMEN
The reciprocal regulation of phosphoprotein phosphatases (PPPs) by protein kinases is essential to cell cycle progression and control, particularly during mitosis for which the role of kinases has been extensively studied. PPPs perform much of the serine/threonine dephosphorylation in eukaryotic cells and achieve substrate selectivity and specificity through the interaction of distinct regulatory subunits with conserved catalytic subunits in holoenzyme complexes. Using a mass spectrometry-based chemical proteomics approach to enrich, identify, and quantify endogenous PPP holoenzyme complexes combined with kinase profiling, we investigated the phosphorylation-dependent regulation of PPP holoenzymes in mitotic cells. We found that cyclin-dependent kinase 1 (CDK1) phosphorylated a threonine residue on the catalytic subunit of the phosphatase PP2A, which disrupted its holoenzyme formation with the regulatory subunit B55. The consequent decrease in the dephosphorylation of PP2A-B55 substrates promoted mitotic entry. This direct phosphorylation by CDK1 was in addition to a previously reported indirect mechanism, thus adding a layer to the interaction between CDK1 and PP2A in regulating mitotic entry.
