RESUMEN
Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)-based motor. Dynactin binds both dynein and MTs via its p150(Glued) subunit, but little is known about the 'pointed-end complex' that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin-dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo-like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore-MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left-handed ß-helix domain, with the phosphorylation site located within a disordered, C-terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Bovinos , Proteínas de Ciclo Celular/genética , Línea Celular , Embrión de Pollo , Complejo Dinactina , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Proteínas Proto-Oncogénicas/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Quinasa Tipo Polo 1RESUMEN
Dynactin is an essential part of the cytoplasmic dynein motor that enhances motor processivity and serves as an adaptor that allows dynein to bind cargoes. Much is known about dynactin's interaction with dynein and microtubules, but how it associates with its diverse complement of subcellular binding partners remains mysterious. It has been suggested that cargo specification involves a group of subunits referred to as the "pointed-end complex." We used chemical cross-linking, RNA interference, and protein overexpression to characterize interactions within the pointed-end complex and explore how it contributes to dynactin's interactions with endomembranes. The Arp11 subunit, which caps one end of dynactin's Arp1 filament, and p62, which binds Arp11 and Arp1, are necessary for dynactin stability. These subunits also allow dynactin to bind the nuclear envelope prior to mitosis. p27 and p25, by contrast, are peripheral components that can be removed without any obvious impact on dynactin integrity. Dynactin lacking these subunits shows reduced membrane binding. Depletion of p27 and p25 results in impaired early and recycling endosome movement, but late endosome movement is unaffected, and mitotic spindles appear normal. We conclude that the pointed-end complex is a bipartite structural domain that stabilizes dynactin and supports its binding to different subcellular structures.