217F and Y217D mutants, and time-lapse pictures of GFP-EB1 have been taken by confocal microscopy at 2-second intervals. (G) Experiments were performed as in (F), along with the relative fluorescence intensity of the GFP-EB1 comets was measured. *P 0.05, **P 0.01, ***P 0.001. Error bars indicate SEM.of microtubules, S27D slightly promoted the development speed and development length of microtubules and STDD promoted the dynamics, growth speed, and development length of microtubules (Fig. 1C ). These final results suggested that the phosphorylation of Y71, TSSS, and T206 is necessary for EB1 to control microtubule dynamics. Moreover, by time-lapse microscopy, we discovered that the Y217F and Y217D mutants had drastically?The Author(s) 2014. This short article is published with open access at Springerlink and journal.hep.cnW S2 T 7 S2 A 7D T3 three T3 A 3D TS SS Y71 AA F AA TS SS Y7 D 1D D D ST D A ST A D T2 D 0 T2 6A 06 DG 15 10 * 5 *** 0 WT Y217F Y217DW S2 T 7 S2 A 7D T3 three T3 A 3D TS SS Y71 AA F AA TS SS Y7 D 1D D D ST D A ST A D T2 D 0 T2 6A 06 D***W S2 T 7 S2 A 7D T3 three T3 A 3D TS SS Y71 AA F AA TS SS Y7 D 1D D D ST D A ST A D T2 D 0 T2 six A 06 D*** ** ***15 /min 9s15 /min 9sdecreased ability to track microtubule plus ends (Fig. 1F and 1G). Since EB1 interacts with +TIPs by means of its carboxyl terminus, we chose T206, Y217, and a further web-site near the carboxyl terminus, ST, for experiments investigating EB1 interaction with +TIPs. We selected adenomatous polyposis coli (APC) and mitotic centromere-associated kinesinPhosphoregulation of EB1 dimerization and functionsLETTERT206DT206AVectorVectorT206DT206AWT Y217FY217DVectorSTDDSTAAMCAK Vector Vector Vector STDD STAA WT WTCLIP170 PD Lysate p150Glued CLIP170 p150GluedY217FWTWTWTY217DPD Lysate PDADGFP GFPGST APC T206D T206A Vector Vector STDD WT WT WT Y217F Vector STAAPD GSTY217DT206A + T206AWT + WT WT + T206AWT + T206DWT + VectorT206D + T206DGFP GFPPD LysateVector + WTVector + WT WT + Vector WT + WTWT + Y217F Y217F + Y217F WT + Y217D Y217D + Y217DEGSTPD GFP PDB LysateGFPGST FYF ‘ C MCAK 89 APC 2794 MACF2 5468 * * 111 2816PDFFF ‘F(Y-Pi)F ‘Figure 2. EB1 phosphorylation at Y217 regulates its interaction with other +TIPs at the same time as its dimerization. (A) Cells have been transfected with GFP-MCAK or GST-APC, with each other with GST, GST-EB1 wild-type, or various mutants. GST pulldown and immunoblotting were then performed using the indicated antibodies to analyze the interaction of EB1 with MCAK or APC. (B) Schematic model displaying the interaction of the hydrophobic cavity of EB1 with the SxIP motif. The yellow peptide in the upper-left model indicates the IP residues within the SxIP motif. A hydrogen bond is formed between Y217 within the hydrophobic cavity of EB1 and the P residue inside the SxIP motif.Buy1374653-45-8 When Y217 is replaced by F or modified by phosphorylation, the hydrogen bond is disrupted.581063-34-5 Formula (C) Alignment from the SxIP motifs along with the adjacent sequences of MCAK, APC, and MACF2.PMID:24580853 The negatively charged residues are indicated with asterisks. (D) Cells have been transfected with GST, GST-EB1 wild-type, or several mutants. GST pulldown and immunoblotting have been then performed using the indicated antibodies to analyze the interaction of EB1 with CLIP170 and p150Glued. (E) Cells were transfected with GST, GST-EB1 wild-type, or the indicated mutants, collectively with GFP, GFP-EB1 wild-type, or the mutants. GST pulldown and immunoblotting have been then performed to examine EB1 dimerization.(MCAK) as examples for SxIP motif-containing proteins (Honnappa et al., 2009). Y217D, but not T206D.
