Op18/stathmin mediates multiple region-specific tubulin and microtubule-regulating activities

Abstract

Oncoprotein18/stathmin (Op18) is a regulator of microtubule (MT) dynamics that binds tubulin heterodimers and destabilizes MTs by promoting catastrophes (i.e., transitions from growing to shrinking MTs). Here, we have performed a deletion analysis to mechanistically dissect Op18 with respect to (a) modulation of tubulin GTP hydrolysis and exchange, (b) tubulin binding in vitro, and (c) tubulin association and MT-regulating activities in intact cells. The data reveal distinct types of region-specific Op18 modulation of tubulin GTP metabolism, namely inhibition of nucleotide exchange and stimulation or inhibition of GTP hydrolysis. These regulatory activities are mediated via two-site cooperative binding to tubulin by multiple nonessential physically separated regions of Op18. In vitro analysis revealed that NH(2)- and COOH-terminal truncations of Op18 have opposite effects on the rates of tubulin GTP hydrolysis. Transfection of human leukemia cells with these two types of mutants result in similar decrease of MT content, which in both cases appeared independent of a simple tubulin sequestering mechanism. However, the NH(2)- and COOH-terminal-truncated Op18 mutants regulate MTs by distinct mechanisms as evidenced by morphological analysis of microinjected newt lung cells. Hence, mutant analysis shows that Op18 has the potential to regulate tubulin/MTs by more than one specific mechanism.

Figure 1

Tubulin binding and modulation of tubulin GTPase activity by Op18 deletion mutants reveal region specific tubulin-directed activities. (A) Schematic representation of Flag-tagged deletion derivatives of Op18. (B) Tubulin binding to the indicated Op18 Flag-epitope–tagged derivatives was analyzed at equimolar concentration (10 μM) in PEM, pH 6.8, containing 17% glycerol. The Op18–tubulin mixture was incubated at 37°C together with anti-Flag coated beads for 30 min and bead-bound material was pelleted through a sucrose/glycerol cushion. The percent of the tubulin recovered in complex with Op18 was determined by densitometric scanning of Coomassie blue–stained SDS-PAGE gels. (C and D) Tubulin (10 μM) GTPase activity was determined in the presence of the indicated Op18 derivatives (15 μM). Samples were incubated at 37°C for 90 min with α-[³²P]GTP (100 μM) in PEM, pH 6.8, containing 17% glycerol in the absence (C) or presence (D) of nocodazole (33 μM). To facilitate comparison, GTPase activity in the absence (Co) or presence of Op18-wt-F (wt) are indicated by dashed lines. The means of duplicate determinations of hydrolyzed GTP are shown and data are representative for at least three independent experiments.

Figure 2

Independent regions of Op18 regulate the rate of tubulin GTP hydrolysis and nucleotide exchange. (A) Modulation of tubulin (5 μM, in PEM with 17% glycerol, pH 6.8) GTP hydrolysis rates were determined in the presence of the indicated Op18 derivatives (15 μM). Hydrolysis of α-[³²P]GTP preloaded onto tubulin (i.e., a single turnover event) was followed over time in the presence of nocodazole (33 μM). (B) Modulation of tubulin GTP hydrolysis rates in the absence of nocodazole was followed for 20 min with different Op18 derivatives (conditions and symbols as in A). After 21 min, the reaction was chased by addition of 200 μM of cold GTP and the effect on hydrolysis of α-[³²P]GTP was followed over time. (C) GTP exchange dependent α-[³²P]GTP hydrolysis was analyzed by mixing tubulin (5 μM, buffer as in A and B and 33 μM nocodazole) with the indicated Op18 derivatives (15 μM, same symbols as in A) and unlabeled GTP (100 μM). After 10 min at 37°C, α-[³²P]GTP was added to the reaction mixtures and GTP hydrolysis was followed over time. The means of duplicate determinations are shown. (D) A schematic representation of the importance of distinct Op18 regions for modulation of tubulin GTP metabolism.

Figure 3

Complexes between Op18 and two tubulin heterodimers are generated via two-site positive cooperativity. Op18–tubulin equilibrium binding curves at pH 6.8 (A) and 7.4 (B) were determined for the indicated Op18 derivatives (2 mM) at 8°C as described in Materials and Methods. The contribution of nonspecific binding (3% of free tubulin) is subtracted. Curves assuming two-site positive cooperative binding were fitted to the data points. Scatchard conversion of the binding curves are shown below (note difference in scales in C and D). Data are representative for two independent experiments.

Figure 4

Glycerol increases Op18–tubulin binding affinity in accordance with a two-site positive cooperativity model. (A) Op18–tubulin equilibrium binding curves using the indicated Op18 derivatives (2 μM) was determined in the presence of 17% glycerol as in Fig. 3. Scatchard conversions of binding curves are shown in (B).

Figure 6

Expression of truncated Op18 proteins destabilize MTs in K562 cells. K562 cells were transfected with the pMEP4 shuttle vector without (Vec-Co) or with insertion of the indicated Op18 derivatives. Transfected cell lines were selected during 5 d in hygromycin and expression from the hMTIIa promoter was induced as described in Materials and Methods. (A) Op18 expression levels, expressed as fold induction over the endogenous Op18 level determined as in Table . (B) The level of polymerized tubulin, as determined by Western blot analysis of the soluble and particulate fraction of lysed cells. (C and D) Flow cytometric analysis of MTs in the same cell populations shown in A and B (induced to express Op18 for 4.5 h). Open graphs show α-tubulin–specific fluorescence and filled graphs show control staining in the absence of anti–α-tubulin but in the presence of fluorescein-conjugated rabbit anti–mouse immunoglobulin. Histograms depicting control staining and cells expressing vector control are shown in both panels. Median fluorescence signals: control staining, 9; vector control, 698; Op18-wt-F, 168; Op18-Δ5-9-F, 286; Op18-Δ5-25-F, 382; and Op18-Δ100-147-F, 385. Data are representative for at least two independent experiments.

Figure 5

Truncated Op18 proteins fail to compete with Op18-wt for tubulin complex formation. (A) The indicated Op18 derivatives (20 μM) were mixed with bovine tubulin (10 μM) and, thereafter, transferred to pelleted glutathione beads coated with GST-Op18-wt (amount corresponding to 4 μM in the final mixture). After 20 min at 37°C, beads were separated and the GST-Op18/tubulin ratio in complexes was determined as in Fig. 1. The means of three independent experiments are shown. (B) Complex formation between endogenous tubulin and Flag-tagged Op18 was analyzed in extracts of K562 cells induced for 6 h to express the indicated Op18 derivatives. Cell extracts (8 mg of protein/ml) were added to anti-Flag–coupled beads within 10 min of cell lysis (PEM, pH 6.8, 5% glycerol and 0.5% Triton X-100) and incubated for 15 min at 8°C. Bead-bound and soluble material was separated as in Fig. 1 and Op18–tubulin was quantified by Western blot as in Table . The means of duplicate determinations are shown.

Figure 7

MT appearance in newt lung cells microinjected with low concentrations of Op18 reveal a lamellar clearing phenotype that requires the NH₂-terminal region. Cells were microinjected with the indicated Op18 protein derivative (160 μM needle concentration, 8–16 μM estimated intracellular concentration), fixed, and stained with antitubulin after 3 h, and the MT-network was observed by epiimmunofluorescence. Microinjection of all Op18 derivatives resulted in some loss of MT polymer, but the results from Op18-wt or Op18-Δ100-147 were most pronounced where the lamella regions often lacked MTs, most likely because of MT shortening. Two independent preparations of the recombinant Op18 proteins, expressed with or without the Flag-tag, were used with similar results in microinjection experiments. In the data shown, Op18 proteins without the Flag-tag were used.

Figure 8

The lamellar clearing phenotype of microinjected newt lung cells is dependent on the extreme NH₂-terminal region of Op18. Cells were microinjected with the indicated Op18 protein derivative as in Fig. 7. The data presented were generated from 2 to 5 coverslips per Op18 derivative. The percentage of cells with 25% or more of the lamella region empty of MTs was determined from 67–245 cells per Op18 derivative. Data are means ± SD.