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doi: 10.1242/10.1242/jcs.00205

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Autonomous and phosphorylation-responsive microtubule-regulating activities of the N-terminus of Op18/stathmin

¹ Department of Molecular Biology, Umeå University, Sweden
² Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA

View larger version (18K): [in a new window]   Fig. 1. The N-terminus of Op18 is sufficient to inhibit in vitro tubulin-tubulin interaction-dependent GTP hydrolysis. (A) Schematic representation of Op18 truncation derivatives. At the top, native Op18 is depicted with an unstructured N-terminus and two repeats of weakly homologous -helical regions according to previous reports (Gigant et al., 2000; Wallon et al., 2000). Phosphorylation sites are indicated with a `P' (Ser-16, Ser-25, Ser-38 and Ser-63). The positions of the two longitudinally arranged tubulin heterodimers along the two helical repeats are depicted according to the low resolution X-ray structure (Gigant et al., 2000), and the orientation of the N-terminus towards the -tubulin end of the tandem tubulin dimer complex is as suggested by cross-linking experiments (Muller et al., 2001). Each truncated Op18 derivative is denoted by the numbers within brackets that indicate the amino-acid residues present. The NR-helix corresponds to a 149 residue of a -helical portion from the rod region of non-muscle myosin heavy chain. (B) Tubulin (5 µM in PEM, pH 6.8) preloaded with -[32P]-GTP was incubated at 37°C with increasing concentrations of each of the Op18 derivative outlined in A. Initial single-turnover hydrolysis rates were evaluated as described in Materials and Methods. Data are means of two independent experiments ±s.e.m. The experiment has also been performed using GST derivatives tagged at the N-terminus and the same result was produced (data not shown).

View larger version (24K): [in a new window]   Fig. 2. Regulation of in vitro MT assembly by the Op18(1-57)-NR-helix. The upper panel shows elongation velocity at plus ends for MTs assembled at pH 7.5 with 11 µM tubulin alone or 11 µM tubulin plus the indicated concentrations of Op18(1-57)-NR-helix or Op18(25-57)-NR-helix. Catastrophe frequency at MT plus ends in the same experiment is shown in the lower panel. Data represent means±s.d. The number of events analyzed for elongation velocity and catastrophe rates, respectively, were Co, n=37 and 34; Op18(1-57)-NR-Helix 30 µM, n=14 and 30; 60 µM, n=23 and 25; Op18(25-57)-NR-helix 60 µM, n=35 and 50.

View larger version (27K): [in a new window]   Fig. 3. Ectopic expression and phosphorylation of the Op18 N-terminus fused to a non-related -helical region. (A) KA8 cells were transfected with 12 µg DNA of each of the indicated pMEP4 derivatives, and hygromycin-resistant cell lines were selected and induced for the indicated time period (0 hours, 6 hours and 24 hours) with Cd2+ (0.5 µM). Expression levels were determined as described in Materials and Methods and expressed as a ratio relative to endogenous Op18 (the endogenous Op18 level in KA8 cells is about 10 µM). The mean of two independent transfection experiments is shown. (B) KA8 transfected with the indicated pMEP4 derivative were Cd2+ induced for 24 hours in the presence of the MT-disrupting drug nocodazole (0.5 µM), and phosphorylation stoichiometry of the expressed NR-derivatives was determined by native PAGE. Non-P Op18(1-57)-NR-helix correspond to non-phosphorylated E. coli-produced protein.

View larger version (21K): [in a new window]   Fig. 4. Ectopic expression of the Op18 N-terminus is sufficient to partially destabilize the interphase MT system in KA8 cells. (A) Cells were transfected with the indicated NR-helix derivative or vector alone (Vec-Co) and induced for 6 hours as in Fig. 3. The fraction of polymerized tubulin was determined as described in Materials and Methods and the mean of two independent transfection experiments is shown. (B) A histogram derived from flow cytometric analysis of MT content in cells induced to express wild-type Op18 and Op18(1-57)-NR-helix or vector alone for 6 hours as in A. Open graphs depict -tubulin-specific fluorescence of cells transfected with the indicated pMEP4 derivative and the filled graph show control staining, in the absence of anti--tubulin but in the presence of fluorescein-conjugated rabbit anti-mouse immunoglobulin. The data are representative for three independent experiments.

View larger version (22K): [in a new window]   Fig. 5. A block in cell division reveals phosphorylation-mediated regulation of the autonomous activity of the Op18 N-terminus. Cells were transfected with the indicated NR-helix derivative or vector alone (Vec-Co) as in Fig. 3. Cells were either non-induced or induced to express ectopic proteins for 24 hours. DNA was stained with propidium iodide and analyzed by flow cytometry. The inserts shows the effect of nocodazole (0.5 µM) on Op18(1-57)-NR-helix-, Op18(25-57)-NR-helix- and NR-helix-expressing cells. Data are representative of three independent experiments.

View larger version (47K): [in a new window]   Fig. 6. The mitotic phenotypes of Op18-NR-helix derivatives. KA8 cells were transfected and hygromycin-resistant cell lines were selected and induced with Cd2+ as in Fig. 3. After 24 hours, cells were fixed and double stained with anti--tubulin (green color) and propidium iodide DNA staining (red color). Representative examples of normal and abnormal metaphase cells observed using epifluorescence are shown. Type I mitotic figures lack most, but not all, kinetochore MTs and condensed chromosomes appeared unorganized. Type II mitotic figures completely lack kinetochore MTs, and condensed chromosomes appeared unorganized and in some cases even aggregated. MTs appeared as small star-like asters with dense but short MTs. Most cells contained two asters, suggesting that the centrosomes have separated.

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