EGFP-Plk1 localized to the centrosomes in subpanels a, b, and c, as shown by arrowheads

EGFP-Plk1 localized to the centrosomes in subpanels a, b, and c, as shown by arrowheads. G2 delay, followed by a prominent prometaphase arrest, as a LIPG consequence of defective spindle formation and Vitamin D4 activation of the spindle checkpoint. The dynamic release of Plk1 from early mitotic structures is thus crucial for mid- to late-stage mitotic events and demonstrates the importance of a fully dynamic Plk1 at the centrosome for proper cell cycle progression. This dependence on dynamic Plk1 was further Vitamin D4 observed during the mitotic reentry of cells after a DNA damage G2 checkpoint, as this process was significantly delayed upon centrosomal tethering of Plk1. These results indicate that mitotic progression and control of mitotic reentry after DNA damage resides, at least in part, on the dynamic behavior of Plk1. Polo-like kinases (Plks) are serine/threonine protein kinases that play essential roles during the cell cycle. Mammalian Plks are subdivided into four family members: Plk1, Plk2, Plk3, and Plk4 (51). Plks have a highly conserved N-terminal kinase domain name and a relatively divergent C-terminal domain name, called the Polo-box domain name (PBD), which contains one (Plk4) or two (Plk1 to Plk3) Polo-boxes. Of the four Plks, the best characterized member is usually Plk1 (10). Plk1 functions in a diverse number of processes that are crucial for proper progression through multiple stages of mitosis, including mitotic entry, centrosome maturation, bipolar spindle formation, chromosome congression and segregation, cytokinesis, and mitotic exit (3, 10). The kinase domain name of Plk1 is usually regulated in part by phosphorylation at Thr-210 within the activation loop (26, 32), likely through the actions of the upstream kinase Aurora-A in complex with the adaptor protein Bora (45, 60). Mutation of Thr-210 to Asp (T210D) mimics T-loop phosphorylation and stimulates kinase activity (26, 32, 56). Expression of an activated Plk1 T210D mutant can override the G2 arrest induced by DNA damage (62, 72) and allow cells to enter and progress completely through mitosis, albeit with a slight spindle checkpoint-dependent mitotic delay (71). The kinase domain name of Plk1, in its nonphosphorylated Vitamin D4 less active state, appears to be negatively regulated through direct conversation with the PBD (25, 48). We have shown previously that this PBDs of Plk1 function as a phosphoserine/phosphothreonine-binding module, recognizing the optimal recognition sequence motif Ser-[pSer/pThr]-[Pro/X] (15, 16). The structural basis for this phosphopeptide-specific binding has been revealed through two high-resolution X-ray structures of Plk1 PBD:phosphopeptide complexes (7, 16). In addition, we have shown that binding of phosphopeptides by the PBD in full-length Plk1 relieves its inhibitory function on kinase activity (16). These findings imply that priming phosphorylations on substrates or docking proteins by other mitotic kinases, such as cyclin-dependent kinases (Cdks), may target Plk1 to these substrates, while simultaneously activating its kinase activity. In agreement with Vitamin D4 this, a mass spectrometry study revealed that many known and potential Plk1 substrates specifically interact with the PBD of Plk1 in vitro in a phosphorylation- and mitosis-specific manner (44), while numerous studies from the Nigg, Barr, Lee, and Erikson groups, as well as others, have demonstrated that this PBD plays a crucial function in both substrate conversation and subcellular targeting of Plk1 in vivo (5, 26, 32, 33, 35, 48, 50, 54, 61). Plk1 dynamically localizes to various mitotic structures as cells progress through different stages of mitosis (3, 10) in a manner that depends on PBD function (16, 28, 33, 61, 64). During interphase and early prophase Plk1 is found at the centrosome, where it facilitates -tubulin recruitment (11) and centrosome maturation, separation, and microtubule nucleation during late prophase and prometaphase (2, 3, 8, 31). By metaphase, a fraction of Plk1 specifically localizes to the kinetochores, where it seems to be involved in regulating aspects of spindle checkpoint function and the metaphase-anaphase transition (1, 21). During anaphase, Plk1 is concentrated in the spindle midzone, where it likely facilitates microtubule sliding (42, 50), while after chromosome segregation in late anaphase, Plk1 remains located in the central spindle and midbody, where it participates in ingression of the cleavage furrow during cytokinesis (42, 50). Plk1 also resides Vitamin D4 at the Golgi apparatus, presumably linking Golgi fragmentation with mitotic entry by interacting with the structural Golgi protein GRASP65 (54). Details of the movement and exchange of Plk1 between these mitotic structures remains poorly characterized, and whether Plk1 molecules exhibit comparable or different dynamic behaviors at these distinct mitotic structures is largely unknown. What controls.