Gels were stained using coomassie (SimplyBlue Safe stain; Invitrogen) and the Tiam1-TAP band excised, destained and tryptically digested
Gels were stained using coomassie (SimplyBlue Safe stain; Invitrogen) and the Tiam1-TAP band excised, destained and tryptically digested. timelapse confocal microscopy as described in Methods. Left panel: a-tubulin-RFP, middle panel: H2BGFP, right panel: merge (-tubulin-RFP= green, H2B-GFP=blue). ncomms8437-s4.avi (278K) GUID:?3F87C56D-ADB3-4F14-925F-4C260C75D8FF Supplementary Movie 4 Tiam1 siRNA cell during live imaging in monastrol. MDCK II cells expressing histone-2B-GFP (H2B-GFP) and a-tubulin-RFP were transiently transfected with Tiam1 siRNA for 2 days then treated with 25 m monastrol before being imaged using time-lapse confocal microscopy as described in Methods. Left panel: a-tubulin-RFP, middle panel: H2B-GFP, right panel: merge (-tubulin-RFP= green, H2B-GFP=blue). ncomms8437-s5.avi (381K) GUID:?E7A6E3CE-94E5-4071-8454-463A974AB0AD Abstract Centrosome separation is critical for bipolar spindle formation and the accurate segregation of chromosomes during mammalian cell mitosis. Kinesin-5 (Eg5) is usually a microtubule motor essential for centrosome separation, and Tiam1 and its substrate Rac antagonize Eg5-dependent centrosome separation in early mitosis promoting efficient chromosome congression. Here we identify S1466 of Tiam1 as a novel Cdk1 site whose phosphorylation is required for the mitotic function of Tiam1. We find that this phosphorylation of Tiam1 is required for the activation of group I p21-activated kinases (Paks) on centrosomes in prophase. Further, we show that both Pak1 and Pak2 counteract centrosome separation in a kinase-dependent manner and demonstrate that they act downstream of Tiam1. We also show that depletion of Pak1/2 allows cells to escape monopolar arrest by Eg5 inhibition, highlighting the potential importance of this signalling pathway for the AGN 210676 development of Eg5 inhibitors as cancer therapeutics. Accurate segregation of chromosomes during mitosis requires formation of a bipolar spindle, which in mammalian cells relies to a large extent around the centrosomes1. Following initial Nek2-dependent centrosome disjunction in late G2 (ref. 2), the centrosomes can individual before nuclear envelope breakdown (NEBD) in prophase and post-NEBD in prometaphase. Many mechanisms appear to contribute to centrosome separation after NEBD3, but most AGN 210676 notable is the plus-end-directed kinesin Eg5, whose microtubule (MT)-sliding activity is essential for centrosome separation in prometaphase across many species4 and which also functions in the less-understood prophase pathway in mammalian cells5,6,7. The importance of Eg5 for centrosome separation in both phases is usually demonstrated by the monopolar spindles and mitotic arrest resulting from its inhibition8,9, making Eg5 a stylish candidate for anticancer therapy10. Over FN1 recent years it has become apparent that forces that oppose centrosome separation are also important to create the correct balance to allow efficient bipolar spindle assembly and chromosome alignment7,11. Proteins known to produce these forces after NEBD include the minus-end directed kinesins HSET12 and dynein5, whose inhibition or depletion allows cells to more easily form bipolar spindles under Eg5 inhibition. More recently, we identified the guanine-nucleotide exchange factor (GEF) Tiam1 and its substrate Rac as the first signalling module to counteract Eg5 in prophase7. AGN 210676 Tiam1 has multiple cellular functions including migration, cell-cell adhesion and survival13, and is required for Ras-induced tumorigenesis kinase assay with ATP and GST-tagged Cdk1-cyclin B1 complex as indicated. Following SDSCPAGE, phosphorylation was measured by immunoblotting with anti-P*-Thr-Pro antibody (P*S/T-P). (e) Purified Tiam1-His was used for kinase assay with GST-tagged Cdk1-cyclin B1 and analysed as in d. (f) Tiam1-HA (either WT or the S1466A mutant) was immunoprecipitated from HEK293T cells arrested in mitosis (STLC) and analysed by immunoblotting with P*S/T-P antibody. Quantitation shows mean P*S/T-P normalized to HA signal+s.e.m. (with WT set as 1) (kinase assay with addition of ATP and (d) GST-tagged Cdk1-cyclin B1 complex or (e) GST-tagged Cdk1-cyclin A complex where indicated. Phosphorylation was analysed by immunoblotting with an anti-P*S1466 antibody. (f) MDCK II cells were either left untreated (Asy) or treated for 16?h with monastrol (100?M) to induce monopolar spindles, then released for the indicated occasions, lysed and analysed by immunoblotting AGN 210676 with the indicated antibodies. Approximate mitotic stage for the time course is usually indicated. Graph shows mean P*S1466 normalized to total Tiam1 for the time course from three impartial replicates+s.e.m. In (a,c,f) -tubulin was used as a loading control. (g,h) MDCK II cells were fixed and stained by immunofluorescence (IF) with the indicated antibodies and DAPI. Merge: Tiam1=red, P*S1466=green, -tubulin=blue. Representative prophase (g) and prometaphase (h) cells are shown. White arrowhead indicates cell-cell junction. Scale bars, 5?m. S1466 phosphorylation regulates centrosome separation We next sought to determine whether phosphorylation of Tiam1 at S1466 was required for its function in mitosis. In addition to the non-phosphorylatable S1466A mutant, we mutated S1466 to aspartic acid (S1466D) to attempt to mimic phosphorylation and expressed HA-tagged forms of S1466A, S1466D and WT AGN 210676 Tiam1 in MDCK II cells. Analysis of the localization of the.