Engel U , Zhan Y , Long JB , Boyle SN , Ballif BA , Dorey K , Gygi SP , Koleske AJ , Vanvactor D .

R01 NS035909 NINDS NIH HHS , R01-NS35909 NINDS NIH HHS , T32 CA009361 NCI NIH HHS , T32-CA009361-30 NCI NIH HHS , BB/J005983/1 Biotechnology and Biological Sciences Research Council

	Fig. 1. CLASP2 is tyrosine-phosphorylated by Abl kinase. GFP-CLASP2α was immunoprecipitated from transfected HEK293T cells following starvation and serum treatment for 30 min in the presence or absence of STI-571 (1 µM), a specific Abl inhibitor. Abl-PP is a constitutively active Abl kinase and was co-transfected with GFP-CLASP2α as indicated. Tyrosine phosphorylation of CLASP2α was identified by anti-phosphotyrosine antibody 4G10. The lower band visible in the 4G10 blot is the IgG heavy chain. Re-blotting for GFP shows an equal amount of GFP-CLASP2α immunoprecipitated in each lane.
	Fig. 2. CLASP2 phosphorylation and interaction with Abl are enhanced by PDGF treatment. A: CLASP2α is associated with endogenous Abl. Lysates from HEK293T cells transfected with either GFP-CLASP2α or GFP (as indicated above lanes) were immunoprecipitated with anti-GFP antibody, probed with anti-CLASP2 (a gift from Anna Akhmanova left panel), stripped and reprobed with anti-Abl antibody (right panel). 1/20th of lysates from cells transfected with GFP-CLASP2α were loaded as controls. B: Increased phosphorylation of Abl after PDGF stimulation. Abl was immunoprecipitated from GFP-CLASP2α transfected 293T cells, which were serum starved and subsequently treated with PDGF for 5 min. A similar level of Abl was immunoprecipitated with and without stimulation (upper panel anti-Abl) while its phosphorylation was increased significantly after PDGF treatment as revealed by anti-phosphotyrosine antibody 4G10 (lower panel). C: PDGF increases association between Abl and CLASP2. Upper panel shows CLASP2 immunoprecipitated by Abl antibody. Lower panel shows phosphorylated CLASP2 by 4G10. GFP transfection alone does not change 4G10 signal (data not shown).
	Fig. 3. Phosphorylation of CLASP2γ by Abl in cells and in vitro. A: CLASP2γ is phosphorylated by Abl-PP in Cos-7 cells. Co-expression of the constitutive active Abl-PP reduces the mobility of CLASP2γ-myc in SDS-electrophoresis. This mobility is slightly enhanced by treatment of cells with STI-571. Treatment of cell lysates with CIP enhances the mobility in SDS-gels even further, suggesting CIP further de-phosphorylate CLASP2γ. B: In vitro phosphorylation of CLASP (25 nM) by purified Abl (10 nM) was assayed by 4G10 signal. Both recombinant proteins were produced and purified using a baculoviral expression system. C: Increasing concentrations of purified His-CLASP2γ were incubated with 10 nM recombinant Abl in the presence of γ-p32ATP. D: Quantification of concentration dependence by normalized pixel intensity shows a sigmoidal relationship between substrate concentration and phosphorylation. Data from six independent experiments are shown. Data were fit to the Hill equation with R2 = 0.96.
	Fig. 4. Identification of CLASP2 tyrosine phosphorylation sites. A: Schematic representation of human CLASP2 constructs and levels of tyrosine phosphorylation. Several N-terminal and C-terminal truncations of CLASP2γ fused to EGFP were tested for in vitro phosphorylation in Cos-7 cells (see B) and the region between the SacI and BamHI restriction sites was identified to be necessary for efficient phosphorylation. This contains the domain necessary for plus end binding (CLASP2-M, [Mimori-Kiyosue et al., 2005]) which was also shown to bind actin [Tsvetkov et al., 2007]. Within this domain the phospho-tyrosines identified by mass spec analysis of full length CLASPα, after serum induction Y800 and Y807, are highlighted in red. Significant phosphorylation of serine 790 or threonine 791 (shown in green) was also observed. The tyrosines identified in mass spec are downstream of two cassettes of GSK-phosphorylation sites (here shown in blue [Kumar et al., 2009]). B: GFP-CLASP fusions were expressed in Cos-7 cells together with Abl-PP and analyzed after IP for tyrosine phosphorylation levels by 4G10 antibody. The results for several truncations of CLASP2 are summarized in A. C: The region containing phosphotyrosines Y800 and Y807 is conserved across many mammalian CLASP2 isoforms and the individual sequences are shown here. Also shown is the predicted region for mouse CLASP2β. Total RNA was harvested from a variety of embryonic mouse tissues, reverse transcribed, and PCR performed for CLASP2. In all neuronal tissues tested (brain, spinal cord, dorsal root ganglion), sequencing of the amplified region identified both a short and long isoform that differed in this phosphotyrosine containing region (data not shown).
	Fig. 5. Modulation of CLASP2α binding to MTs by Abl kinase. Purified CLASP2α-myc-His was incubated with taxol-stabilized MT bundles (see Materials and Methods) for 30 min with the components indicated above the lanes. The mixture was centrifuged and the pellet was collected and analyzed by Western blot using an antibody directed against myc to detect CLASP2α. Blots were re-probed with the anti-phosphotyrosine antibody 4G10 and anti-tubulin (anti-tub). Increased amounts of CLASP2α were pelleted with stabilized MTs in the presence of Abl (Lane 2) compared to the absence of Abl (Lane 1), STI-571 treatment (Lane 3), or protein tyrosine phosphatase 1B (PTP1B) (1 µg/µl) treatment (Lane 4). In Lanes 3 and 4 more stabilized MTs were present in the reaction than in Lanes 1 and 2. Additional stabilized MTs were added to the reaction and pelleted in Lanes 3 and 4. Sup: supernatants. B: Quantification of blots shown in (A). Error bars represent standard error of the mean, **P < 0.01. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]
	Fig. 6. Abl regulates CLASP2 binding to microfilaments. A: F-actin filaments were incubated with purified CLASP-myc-His with or without Abl and other reagents as indicated above lanes. After centrifugation, equal amounts of pellet (top panel) and the input (lower panel) were analyzed for each treatment with antibodies to actin, anti-myc for detection of CLASP2α-myc-His, and anti-tubulin. B: Purified CLASP2α-myc-His was incubated with taxol-stabilized MT bundles (see Materials and Methods) for 30 min with the components indicated above lanes. Monomeric (G) actin was added to the reaction instead of filamentous (F) actin. The mixture was centrifuged and the pellet was collected and analyzed by Western blot using antibodies directed against actin, tubulin, and myc (to detect CLASP2α). G-actin pellets with MTs in the presence of CLASP2α (Lane 1), but not in the absence of CLASP2 (Lane 2). Abl enhances the association of G-actin with MTs (Lane 3) and this association is abolished with STI-571 (Lane 4). Bottom panels show the input (1/10 of the total reaction volume) of both CLASP and G-actin.
	Fig. 7. High Abl-activity relocalizes CLASP to Abl induced growth cone adhesive plaques. CLASP2 localization in neuronal growth cones depends on Abl activity. Live growth cones expressing indicated constructs were subjected to fast optical sectioning by spinning disk confocal microscopy followed by deconvolution for better localization accuracy. The resulting image stacks are displayed as single optical slices (z distance to substrate in µm is indicated) and as maximum projection through the stack (max. proj.); the result of a 3D thresholding is overlaid in red (iso-data). A: GFP-CLASP2γ (GFP-CLASP) co-expression with constitutive active Abl (Abl-PP) results in accumulation of GFP-CLASP2γ in the middle of the growth cone (red arrows) close to the substrate (z = 0.2 and z = 0.7), only weak residual MT plus end tracking is detected (white arrows, see also Supporting Information Movie S2). B: Enlarged insert of A (plane z = 0.7) indicates GFP-CLASP2γ accumulation in adhesive plaques (red arrow) and localization to plus ends (white arrows). C: Quantification of GFP-CLASP2γ-positive comets per volume in wild type (wt) versus Abl-PP expressing neurons. Error bars represent standard error of the mean. (P < 0.05). D: Dynamic localization of GFP-CLASP2γ in the same growth cone as shown in A. Arrow follows CLASP2γ-positive growing MT end (see also Supporting Information Movie S1). E: GFP-CLASP2γ expressing wild-type growth cone shows typical MT plus end tracking in the periphery (white arrows in insert, see also Supporting Information Movie S2). F: GFP-CLASP2γ localization of Abl-PP expressing growth cones treated with 10 µM STI571 is indistinguishable from the localization of GFP-CLASP2γ in the control growth cone in E. Inserts show plus end tracking (see also Supporting Information Movie S3). Scale bars in A, E, and F: 10 µm; in B and D: 2 µm. Magnified inserts in E and F are 10 µm × 5 µm and 9 µm × 5 µm in size, respectively.
	Fig. 8. Actin accumulates in Abl-induced adhesion plaques. A–D: Differential interference contrast (left side) and wide field fluorescence of GFP-actin of live growth cones which express GFP-actin only (A), GFP-actin co-expressed with constitutive active Abl (Abl-PP; B) or GFP-actin with Xenopus Abl (Xabl; C and D). The presence of Abl-induced adhesive plaques is indicated in bright field images (black arrows) and accumulation of GFP-actin (B, D) with white arrows. E: Quantification of growth cone advance in ctl, and Xabl growth cones which show adhesive plaques (as in D; Xabl strong), or weak actin accumulation (as in C; Xabl weak). Error bars represent standard error of the mean (*P < 0.025, **P < 0.01.) F–G: Immunolocalization of Abl (red) in growth cones expressing GFP-CLASP2γ (green) and Abl-PP with corresponding bright field images. Abl induced structures (white arrows) appear as round adhesive plaques (F) or as half circular fronts of Abl and CLASP2 accumulation (G, insert in I). H,I: Fluorescence intensity distribution of Abl and CLASP2 evaluated by intensity profiles (10 pixel integration). H: Intensity profiles shown in F as p1, p2. I: Profile across the ridge of the circular structure in G (region and direction of profile shown as arrow in insert). Scale bars are 10 μm.

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