Stathmin/Op18 phosphorylation is regulated by microtubule assembly

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of "mitotic chromatin." Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.     

Stathmin/Op18 is a novel mediator of vinblastine activity

Microtubule (MT) dynamic instability is tightly regulated by stabilizing and destabilizing proteins, the latter being exemplified by stathmin/Op18, a protein known to destabilize MTs. Studies in cells have indicated that the level of stathmin expression modifies the cytotoxicity of antimicrotubule drugs, such as vinblastine (VLB). Using isothermal titration calorimetry and analytical ultracentrifugation, we show that VLB increases the affinity of stathmin for tubulin 50-fold (and vice versa). These results are the first biochemical evidence of the direct relationship between stathmin and an antimitotic drug, and reveal a new mechanism of action for VLB.     

Stathmin/Oncoprotein 18(Op18)基因在人骨肉瘤中的表达及其意义

目的:探讨stathmin/oncoprotein 18(Op18)基因在人骨肉瘤中的表达规律及意义。方法:分别采用免疫组化法和Western blot法检测10例正常骨组织和56例骨肉瘤中stathmin蛋白的表达。结果:免疫组化法检测stathmin蛋白在正常骨组织、低级别骨肉瘤(G1级组)及高级别骨肉瘤(G2级组)中阳性表达率分别为20%、65%、100%。正常骨组织分别与G1级组、G2级组比较,均有显著性差异(P﹤0.05);G1级组与G2级组比较,有显著差异(P<0.05)。Western blot法检测显示,stathmin蛋白在正常骨组织、G1级组、G2级组表达相对值分别为(0.34±0.15)、(0.68±0.21)、(0.90±0.17)。正常骨组织分别与G1级级组、G2级组比较,差异均有显著性(P<0.01),G1级组与G2级组比较,差异有显著性(P<0.01)。结论:Stathmin在骨肉瘤中过表达,与骨肉瘤的发生及发展密切相关。     

Deciphering the cellular functions of the Op18/Stathmin family of microtubule-regulators by plasma membrane-targeted localization

The Op18/stathmin family of microtubule regulators includes the ubiquitous cytosolic Op18/stathmin (Op18) and the neuronal, primarily Golgi-associated proteins SCG10 and RB3, which all form ternary complexes with two head-to-tail-aligned tubulin heterodimers. To understand the physiological significance of previously observed differences in ternary complex stability, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. Herein, we show that, in contrast to constitutively active CD2-Op18-tetraA, both the CD2-SCG10 and CD2-RB3 chimeras sequestered tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels and consequent delayed formation of mitotic spindles. However, all three CD2 chimeras, including the tubulin sequestration-incompetent CD2-Op18-tetraA, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during interphase, but not during mitosis, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. However, the differences in tubulin binding observed in cells also indicate conceptual differences between the functions of low-abundance neural family members, which will accumulate tubulin at specific cellular compartments, and the abundant cytosolic Op18 protein, which will not.     

Stathmin regulates microtubule dynamics and microtubule organizing center polarization in activated T cells

Polarization of T cells involves reorientation of the microtubule organizing center (MTOC). Because activated ERK is localized at the immunological synapse, we investigated its role by showing that ERK activation is important for MTOC polarization. Suspecting that ERK phosphorylates a regulator of microtubules, we next focused on stathmin, a known ERK substrate. Our work indicates that during T cell activation, ERK is recruited to the synapse, allowing it to phosphorylate stathmin molecules near the immunological synapse. Supporting an important role of stathmin phosphorylation in T cell activation, we showed that T cell activation results in increased microtubule growth rate dependent on the presence of stathmin. The significance of this finding was demonstrated by results showing that CTLs from stathmin(-/-) mice displayed defective MTOC polarization and defective target cell cytolysis. These data implicate stathmin as a regulator of the microtubule network during T cell activation.     

Spindle microtubule dynamics: modulation by metabolic inhibitors

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.     

MAPs in plant cells: delineating microtubule growth dynamics and organization

Microtubule-associated proteins (MAPs) serve a wide variety of functions, from constructing and maintaining the microtubule cytoskeleton to using this cytoskeleton to transport cargo and to tether molecules that are involved in numerous cellular processes. Throughout the cell cycle, distinct microtubule arrays carry out specific roles in cytokinesis, karyokinesis, and cell expansion. Recent findings have shed new light on the importance of MAPs in controlling microtubule growth dynamics as well as in cross-linking microtubules to facilitate the formation and function of these cytoskeletal arrays.     

Op18/Stathmin counteracts the activity of overexpressed tubulin-disrupting proteins in a human leukemia cell line

Op18/stathmin (Op18) is a phosphorylation-regulated and differentially expressed microtubule-destabilizing protein in animal cells. Op18 regulates tubulin monomer-polymer partitioning of the interphase microtubule system and forms complexes with tubulin heterodimers. Recent reports have shown that specific tubulin-folding cofactors and related proteins may disrupt tubulin heterodimers. We therefore investigated whether Op18 protects unpolymerized tubulin from such disruptive activities. Our approach was based on inducible overexpression of two tubulin-disrupting proteins, namely TBCE, which is required for tubulin biogenesis, and E-like, which has been proposed to regulate tubulin turnover and microtubule stability. Expression of either of these proteins was found to cause a rapid degradation of both alpha-tubulin and beta-tubulin subunits of unpolymerized, but not polymeric, tubulin heterodimers. We found that depletion of Op18 by means of RNA interference increased the susceptibility of tubulin to TBCE or E-like mediated disruption, while overexpressed Op18 exerted a tubulin-protective effect. Tubulin protection was shown to depend on Op18 levels, binding affinity, and the partitioning between tubulin monomers and polymers. Hence, the present study reveals that Op18 at physiologically relevant levels functions to preserve the integrity of tubulin heterodimers, which may serve to regulate tubulin turnover rates.     

Comparison of the microtubule formation induced by cytosynalin and MAPs

A comparison was made of cytosynalin- and MAPs-induced microtubule formation. Cytosynalin (a 35 kDa cytoskeleton-interacting and calmodulin-binding protein) facilitated more rapid and extensive microtubule formation than MAPs. Electron microscopic examination revealed that the cytosynalin-induced microtubules were 24 nm tubules surrounded by a periodic nap structure along their length. Consequently, the apparent diameter of microtubules was seen as 34 nm. Cytosynalin maximally bound to tubulin dimer at a molar ratio of 1:1. The effect of cytosynalin on the microtubule formation was found to be more potent than that of MAP2 or tau factor as determined by electron microscopy and cosedimentation assay.     

Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1

In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1- and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation.     

Spindle microtubule dynamics following ultraviolet-microbeam irradiations of mitotic diatoms

Lesions ("ARBs") generated in metaphase and anaphase central spindles of Hantzschia by an ultraviolet microbeam are devoid of microtubules previously present. In vivo, the poleward transverse edge of the lesion invariably loses birefringence poleward, until this segment has vanished; the loss is slow during metaphase and faster at anaphase. The other transverse edge, proximal to the overlap, remains stable until disassembly of the whole spindle. We conclude that the central spindle microtubules are not in flux during metaphase to telophase, and that depolymerization of these microtubules takes place only from the end distal to the pole, as during normal spindle disassembly. Microtubule polarity and the creation of free ends may determine which microtubules are disassembled during later mitosis and how disassembly proceeds.     

Thermodynamics of the Op18/stathmin-tubulin interaction

Op18/stathmin (stathmin) is an intrinsically disordered protein involved in the regulation of the microtubule filament system. One function of stathmin is to sequester tubulin dimers into assembly incompetent complexes, and recent studies revealed two tubulin binding sites per stathmin molecule. Using high sensitivity isothermal titration calorimetry, we document that at 10 degrees C and under the conditions of 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP these two binding sites are of equal affinity with an equilibrium binding constant of K0 = 6.0 x 10(6) m(-1). The obtained large negative molar heat capacity change of deltaCp0 = -860 cal mol(-1) K(-1) (referring to tubulin) for the tubulin-stathmin binding equilibrium suggests that the hydrophobic effect is the major driving force of the binding reaction. Replacing GTP by GDP on beta-tubulin had no significant effect on the thermodynamic parameters of the tubulin-stathmin binding equilibrium. The proposed pH-sensitive dual function of stathmin was further evaluated by circular dichroism spectroscopy and nuclear magnetic resonance. At low temperatures, stathmin was found to be extensively helical but devoid of any stable tertiary structure. However, in complex with two tubulin subunits stathmin adopts a stable conformation. Both the stability and conformation of the individual proteins and complexes were not significantly affected by small changes in pH. A 4-fold decrease in affinity of stathmin for tubulin was revealed at pH 7.5 compared with pH 6.8. This decrease could be attributed to a weaker binding of the C terminus of stathmin. These findings do not support the view that stathmin works as a pH-sensitive protein.     

Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities

The guanosine tri-phosphatase Ran stimulates assembly of microtubule spindles. However, it is not known what aspects of the microtubule cytoskeleton are subject to regulation by Ran in mitosis. Here we show that Ran-GTP stimulates microtubule assembly by increasing the rescue frequency of microtubules three- to eightfold. In addition to changing microtubule dynamics, Ran-GTP also alters the balance of motor activities, partly as a result of an increase in the amount of motile Eg5, a plus-end-directed microtubule motor that is essential for spindle formation. Thus, Ran regulates multiple processes that are involved in spindle assembly.     

Regulation of Op18 during spindle assembly in Xenopus egg extracts

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.     

Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics

Knockout mice deficient in the gap junction gene connexin43 exhibit developmental anomalies associated with abnormal neural crest, primordial germ cell, and proepicardial cell migration. These migration defects are due to a loss of directional cell movement, and are associated with abnormal actin stress fiber organization and a loss of polarized cell morphology. To elucidate the mechanism by which Cx43 regulates cell polarity, we used a wound closure assays with mouse embryonic fibroblasts (MEFs) to examine polarized cell morphology and directional cell movement. Studies using embryonic fibroblasts from Cx43 knockout (Cx43KO) mice showed Cx43 deficiency caused cell polarity defects as characterized by a failure of the Golgi apparatus and the microtubule organizing center to reorient with the direction of wound closure. Actin stress fibers at the wound edge also failed to appropriately align, and stabilized microtubule (Glu-tubulin) levels were markedly reduced. Forced expression of Cx43 with deletion of its tubulin-binding domain (Cx43dT) in both wildtype MEFs and neural crest cell explants recapitulated the cell migration defects seen in Cx43KO cells. However, forced expression of Cx43 with point mutation causing gap junction channel closure had no effect on cell motility. TIRF imaging revealed increased microtubule instability in Cx43KO cells, and microtubule targeting of membrane localized Cx43 was reduced with expression of Cx43dT construct in wildtype cells. Together, these findings suggest the essential role of Cx43 gap junctions in development is mediated by regulation of the tubulin cytoskeleton and cell polarity by Cx43 via a nonchannel function.     

Antimitotic sulfonamides inhibit microtubule assembly dynamics and cancer cell proliferation

Several sulfonamides have antitumor activities and are currently undergoing clinical evaluation for the treatment of cancer. In this study, we have elucidated the antiproliferative mechanism of action of five indole sulfonamides. The indole sulfonamides inhibited the polymerization of microtubule protein into microtubules in vitro. In addition, three representative derivatives, ER-68378 (2), ER-68384 (4) and ER-68394 (5), suppressed the dynamic instability behavior at the plus ends of individual steady-state microtubules in vitro. The analogues inhibited HeLa cell proliferation with half-maximal inhibitory concentrations in the range of 6-17 microM. The compounds blocked cell cycle progression at mitosis. At their lowest effective antimitotic concentrations, they depolymerized the spindle microtubules and disorganized the chromosomes but did not affect the microtubules in interphase cells. However, at relatively high concentrations, interphase microtubules were also depolymerized by these sulfonamides. Furthermore, all five compounds were found to induce apoptosis in the cells in association with the phosphorylation of bcl-2. The results suggest that the indole sulfonamides inhibit cell proliferation at mitosis by perturbing the assembly dynamics of spindle microtubules and that they can kill cancer cells by inducing apoptosis through the bcl-2-dependent pathway.     

Vav1 controls DAP10-mediated natural cytotoxicity by regulating actin and microtubule dynamics

The NK cell-activating receptor NKG2D recognizes several MHC class I-related molecules expressed on virally infected and tumor cells. Human NKG2D transduces activation signals exclusively via an associated DAP10 adaptor containing a YxNM motif, whereas murine NKG2D can signal through either DAP10 or the DAP12 adaptor, which contains an ITAM sequence. DAP10 signaling is thought to be mediated, at least in part, by PI3K and is independent of Syk/Zap-70 kinases; however, the exact mechanism by which DAP10 induces natural cytotoxicity is incompletely understood. Herein, we identify Vav1, a Rho GTPase guanine nucleotide exchange factor, as a critical signaling mediator downstream of DAP10 in NK cells. Specifically, using mice deficient in Vav1 and DAP12, we demonstrate an essential role for Vav1 in DAP10-induced NK cell cytoskeletal polarization involving both actin and microtubule networks, maturation of the cytolytic synapse, and target cell lysis. Mechanistically, we show that Vav1 interacts with DAP10 YxNM motifs through the adaptor protein Grb2 and is required for activation of PI3K-dependent Akt signaling. Based on these findings, we propose a novel model of ITAM-independent signaling by Vav downstream of DAP10 in NK cells.