translated ASK1 further showed a direct interaction between these two proteins (Figure 4e). Open in a separate window Figure 4 ASK1 interacts with EB1 both in cells and kinase assays using ASK1 or ASK1KD immunoprecipitate from 293? T cells and bacterially purified GST-EB1. are stabilized and therefore capable of mediating spindle interaction with the cell cortex, a requirement for spindle movement. These findings reveal a previously undiscovered function of ASK1 in cell division by regulating spindle orientation and positioning, and point to the importance of protein phosphorylation in the regulation of spindle behavior. projection. projection. translated ASK1 further showed a direct interaction between these two proteins (Figure 4e). Open in a separate window Figure 4 ASK1 interacts with EB1 both in cells and kinase assays using ASK1 or ASK1KD immunoprecipitate from 293?T cells and bacterially purified GST-EB1. Immunoblotting of the reaction mixture with phosphoserine and phosphothreonine antibodies revealed that EB1 was phosphorylated at both serine and threonine residues by ASK1 but not ASK1KD (Figure 5a). Furthermore, ASK1-induced EB1 phosphorylation at serine and threonine residues was abrogated when GST-EB1 pulled down from the above reaction mixture was treated with PPase (Figure 5b), confirming EB1 phosphorylation by ASK1. Open in a separate window Figure 5 ASK1 phosphorylates EB1 at S40, T154 and T206. (a) Kinase assays were performed by using ASK1 or ASK1KD immunoprecipitate from 293?T cells, with bacterially purified GST-EB1 as a substrate. The reaction mixture was then subjected to immunoblotting with phosphoserine (pSer) and phosphothreonine (pThr) antibodies. (b) Kinase assays were performed as in (a), and GST-EB1 was pulled down from the reaction mixture and treated with PPase. Immunoblotting was then performed with the WJ460 indicated antibodies. (c) Kinase assays were performed as in (a), and EB1 phosphorylation sites were identified by mass spectrometry. (d, e) Kinase assays were performed by using ASK1 immunoprecipitate and bacterially purified GST-EB1 wild-type (WT) or mutants. The reaction mixture was then subjected to immunoblotting with pSer (d) and pThr (e) antibodies. (f) Immunoprecipitation and immunoblotting showing the level of EB1 phosphorylation in 293?T cells transfected with Flag-EB1-WT or -3A, together with HA or HA-ASK1. 3A, S40/T154/T206A. To identify the residues of EB1 phosphorylated by ASK1, GST-EB1 pulled down from the above reaction mixture was subjected to SDS-PAGE and in-gel tryptic digestion. Subsequent mass spectrometric analysis of the peptides identified six potential phosphorylation sites, among which S40 is located in the CH domain, T154, S155, S156 and S157 in the linker region, and T206 in the linker region adjacent to the EBH WJ460 domain (Figure 5c and Supplementary Figure S4). For the consecutive linker-region residues T154, S155, S156 and S157, tandem mass spectrometric analysis indicated that only one of the residues was phosphorylated, but the exact phosphorylation site could not be unambiguously assigned (Figure 5c and Supplementary Figure S4). To further characterize the phosphorylation sites of EB1, we compared by kinase assays the p300 level of phosphorylation of different EB1 mutants. While EB1 serine phosphorylation by ASK1 was not affected by mutation of S155, S156 and S157 to alanines, it was completely lost when S40 alone was mutated to alanine or when S40, S155, S156 and S157 were WJ460 all mutated to alanines (Figure 5d). In addition, EB1 threonine phosphorylation by ASK1 was partially reduced by mutation of either T154 or T206 to alanine, and was completely lost when both T154 and T206 were mutated to alanines (Figure 5e). These results thus reveal S40, T154 and T206 as the residues of EB1 phosphorylated by ASK1. We then investigated whether ASK1 phosphorylates EB1 at these three residues in cells. We transfected 293?T cells with HA or HA-ASK1, together with Flag-tagged wild-type EB1 or WJ460 the phospho-deficient 3A mutant (mutation of S40, T154 and T206 to alanines). As shown in Figure 5f, overexpression of ASK1 in 293?T cells remarkably increased serine/threonine phosphorylation of wild-type EB1, but not the 3A mutant. By using an antibody against EB1 phosphorylated at T206, we further found that overexpression of wild-type ASK1 significantly enhanced EB1 phosphorylation in 293?T cells and that the kinase-dead, dominant-negative mutant of ASK1 (ASK1KD) had an opposite effect (Supplementary Figure S5). We also found.