RESUMEN
Kinetochores link chromosomes to spindle microtubules and are essential for accurate chromosome segregation during cell division. Kinetochores assemble at the centromeric region of chromosomes as a multiprotein complex. However, the molecular mechanisms of kinetochore assembly have not yet been fully elucidated. In this study, we identified pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) as a regulatory phosphatase that facilitates proper kinetochore assembly. We found that PHLPP1 interacted with the essential outer-kinetochore protein SGT1 and stabilized its protein levels. Loss of PHLPP1 from cells led to SGT1 degradation and thereby caused defective kinetochore assembly. We also found that the ring finger protein 41 (RNF41) as an E3 ligase ubiquitinated and degraded SGT1 in a phosphorylation-dependent manner. PHLPP1 dephosphorylated SGT1 at four conserved residues (Ser-17, Ser-249, Ser-289, and Thr-233) and thereby prevented SGT1 from associating with RNF41, in turn, countering SGT1 degradation. Importantly, depletion of RNF41 or expression of a non-phosphorylatable SGT1 mutant rescued the kinetochore defects caused by the loss of PHLPP1. Taken together, our results suggest that PHLPP1 plays an important role in the assembly of kinetochores by counteracting RNF41-mediated SGT1 degradation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Sustitución de Aminoácidos , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Células HEK293 , Células HeLa , Humanos , Cinetocoros/química , Microscopía Confocal , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosforilación , Mutación Puntual , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteolisis , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Imagen de Lapso de Tiempo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Nutritional abundance associated with chronic inflammation and dyslipidemia impairs the functioning of endoplasmic reticulum (ER) thereby hampering cellular responses to insulin. PHLPP1 was identified as a phosphatase which inactivates Akt, the master regulator of insulin mediated glucose homeostasis. Given the suggestive role of PHLPP1 phosphatase in terminating insulin signalling pathways, deeper insights into its functional role in inducing insulin resistance are warranted. Here, we show that PHLPP1 expression is enhanced in skeletal muscle of insulin resistant rodents which also displayed ER stress, an important mediator of insulin resistance. Using cultured cells and PHLPP1 knockdown mice, we demonstrate that PHLPP1 facilitates the development of ER stress. Importantly, shRNA mediated ablation of PHLPP1 significantly improved glucose clearance from systemic circulation with enhanced expression of glucose transporter 4 (GLUT-4) in skeletal muscle. Mechanistically, we show that endogenous PHLPP1 but not PP2Cα interacts with and directly dephosphorylates AMPK Thr172 in myoblasts without influencing its upstream kinase, LKB1. While the association between endogenous PHLPP1 and AMPK was enhanced in ER stressed cultured cells and soleus muscle of high fat diet fed mice, the basal interaction between PP2Ac and AMPK was minimally altered. Further, we show that PHLPP1α is phosphorylated by ERK1/2 at Ser932 under ER stress which is required for its ability to interact with and dephosphorylate AMPK and thereby induce ER stress. Taken together, our data position PHLPP1 as a key regulator of ER stress.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Estrés del Retículo Endoplásmico , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Células HEK293 , Humanos , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismo , Ratas , Ratas WistarRESUMEN
PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1) is a protein-serine/threonine phosphatase and a negative regulator of the PI3-kinase/Akt pathway. Although its function as a suppressor of tumor cell growth has been established, the mechanism of its regulation is not completely understood. In this study, by utilizing the tandem affinity purification approach we have identified WDR48 and USP12 as novel PHLPP1-associated proteins. The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability. Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis. Functionally, we show that WDR48 and USP12 suppress proliferation of tumor cells. Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression. Together, our results reveal WDR48 and USP12 as novel PHLPP1 regulators and potential suppressors of tumor cell survival.
Asunto(s)
Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína Oncogénica v-akt/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Apoptosis/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias/genética , Neoplasias/patología , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteínas/genética , Transducción de Señal , Ubiquitina Tiolesterasa/genéticaRESUMEN
Mitotic progression is regulated by co-ordinated action of several proteins and is crucial for the maintenance of genomic stability. CHFR (Check point protein with FHA and RING domains) is an E3 ubiquitin ligase and a checkpoint protein that regulates entry into mitosis. But the molecular players involved in CHFR mediated mitotic checkpoint are not completely understood. In this study, we identified TOPK/PBK, a serine/threonine kinase and PTEN, a lipid phosphatase to play an important role in CHFR mediated mitotic transitions. We demonstrated that CHFR ubiquitinates and regulates TOPK levels, which is essential for its checkpoint function. Moreover, TOPK phosphorylates and inactivates PTEN, which in turn activates Akt that leads to proper G2/M progression. Collectively, our results reveal TOPK and PTEN as new players in CHFR mediated mitotic checkpoint.