Spindle placement and spindle elongation are critical for proper cell division.

Spindle placement and spindle elongation are critical for proper cell division. manner. We demonstrate that during metaphase CDK1-mediated phosphorylation at T2055 negatively regulates NuMA cortical localization and that AR7 this phosphorylation is counteracted by PPP2CA phosphatase activity. We establish that this tug of war is essential for proper levels of cortical dynein and thus spindle positioning during metaphase. Moreover we find that upon CDK1 inactivation in anaphase the rise in dephosphorylated NuMA at the cell cortex leads to cortical dynein enrichment and thus to strong spindle elongation. Our findings uncover a mechanism whereby the status of NuMA phosphorylation coordinates mitotic progression with proper spindle function. and kinase assays and found that CDK1 can phosphorylate a C-terminal fragment of NuMA whereas this phosphorylation is usually severely impaired in the presence of the CDK1 inhibitor RO-3306 (Physique 3C). To identify the phosphorylated residue(s) we performed mass spectrometry analysis which established that phosphorylation occurs AR7 at T2015 T2055 S2087 and T2106 corresponding to the four CDK1 consensus sites (Physique 3B). We conclude that CDK1 can directly phosphorylate NuMA phosphorylation by CDK1 but not a T2055A mutant version (NuMA-C-ter(T>A)). Moreover western blot analysis of whole-cell lysates from synchronized populations revealed that phospho-T2055 antibodies recognize a single band at the expected size primarily during metaphase (Physique 3E). This band disappears in metaphase cells treated with siRNAs against NuMA or incubated with the CDK1 inhibitor RO-3306 (Physique 3F and Supplementary Physique S4A) indicating specificity for the phosphorylated form of T2055. Immunofluorescence analysis uncovered phospho-T2055 accumulation in the nucleus just before NEBD in prophase (Supplementary Physique S1G) mirroring the distribution of active CDK1 (Gavet and Pines 2010 Importantly in addition we found that phospho-T2055 is usually enriched at spindle poles in prometaphase and metaphase but not at the AR7 cell cortex in contrast to total NuMA (compare Physique 3G and Supplementary Physique S1H-I with Supplementary Physique S1B and C). Moreover we found that phospho-T2055 is essentially absent during anaphase and telophase when CDK1 is usually inactive (Physique 3I and Supplementary AR7 Physique S1J-K). Furthermore brief incubation with the CDK1 inhibitor RO-3306 drastically reduces phospho-T2055 staining in metaphase (compare Physique 3H with Physique 3G). Overall we conclude that CDK1 phosphorylates NuMA at T2055 during metaphase and that a nonphosphorylated T2055 NuMA species is present at the cell cortex weakly during metaphase and more prominently during anaphase. The phosphorylation status at T2055 governs NuMA distribution during mitosis We set out to address the importance of NuMA phosphorylation by CDK1. Importantly we found that inhibiting CDK1 using RO-3306 results in excess cortical localization of NuMA and p150Glued during metaphase (Physique 4B compare with Physique 4A). Similar results were obtained with RO-3306 in MEFs (data not shown) as well as by using Roscovitine a distinct CDK1 inhibitor in HeLa cells (Supplementary Physique S4C Rabbit Polyclonal to PEK/PERK. compare with Supplementary Physique AR7 S4B). In addition we found that cortical DYNC1H1-GFP enrichment also increases following RO-3306 treatment (Physique 4C). Physique 4 CDK1 negatively regulates NuMA/dynein cortical distribution by phosphorylating NuMA at T2055. (A B) Metaphase HeLa cells treated with 0.1% DMSO (Control) (A) or RO-3306 (9?μM) (B) and stained for NuMA (red) as well AR7 as p150Glued … To further investigate the importance of NuMA phosphorylation at T2055 by CDK1 we generated fusion proteins between GFP and nonphosphorylatable (T>A) or phosphomimetic (T>E) mutants of NuMA for the 2055 residue and compared their distribution compared to that of GFP fused towards the wild-type proteins. Interestingly we discovered that as opposed to GFP-NuMA (Body 4D and E) in nearly all cells GFP-NuMA(T>E) will not localize towards the cortex in either metaphase or anaphase (Body 4F and G). Furthermore.