Stathmin Function Research Articles

Immunohistochemical expression of stathmin in oral dysplasia: An original study with an insight of its action on microtubules.

Background and Objectives:Stathmin is a phosphoprotein, which in its phosphorylated/unphosphorylated states plays a major role in polymerization/depolymerization of microtubules, respectively. Assembly of microtubules is an important aspect of cell division called mitosis. Hinderance in the function of stathmin could lead to damage in the mitotic process resulting in aneuploidy which is common manifestation of malignancies. Hence, stathmin could be used as a tumor marker for oral dysplasias and cancers. The purpose of the study is to compare the expression of stathmin in normal subjects to the patients with oral leukoplakia and to correlate its expression with different histopathological grades of oral leukoplakia This is the first ever study conducted to examine the expression of stathmin in oral dysplasia.Methodology:Thirty histopathologically confirmed cases of oral dysplasia were selected for the study. These tissues were evaluated immunohistochemically for stathmin. To enumerate the stathmin stained cells, 300 cells were examined manually in at least 5 areas and a mean percentage of positive–stained slides were determined. Then, each sample was assigned to one of the following staining scores: (0) – (<10% of stained cells); (1) – (11%–25% of stained cells); (2) – (26%–50% of stained cells); (3) – (51%–75% of stained cells); (4) – (76%–90% of stained cells) and (5) – (91%–100% of stained cells). The results were analyzed statistically using ANNOVA test.Results:When comparison was made with respect to staining scores of stathmin between normal and dysplasia groups, the results were found to be statistically significant with a P = 0.0001. A statistically significant difference was observed between various histopathological grades of dysplasia with respect to stathmin immunohistochemistry scores with a P = 0.0001.Conclusion:These results suggest stathmin as a tumor marker and prognostic indicator.

Role of stathmin microtubule-destabilizing activity in Shigella flexneri motility and tunneling

The infection of the intestinal mucosa by Shigella bacteria is a global health issue resulting in a variety of potentially life-threatening gastrointestinal complications. Their unique method of intracellular motility depends on microtubule destabilization to clear the dense host cytoskeletal network in a process called tunneling. It is hypothesized that the host protein stathmin may play a role in this process, due to its tubulin-sequestering capability. This proposal aims to provide potential methodologies to elucidate the function of stathmin with respect to Shigella flexneri motility. Three experiments are proposed, involving comparisons between human intestinal epithelial cell strains under varying levels of stathmin expression, each infected with S. flexneri. Respectively, the experiments examine tunnel widths via electron microscopy, microtubule densities via imaging fluorescence correlation spectroscopy, and bacterial movement patterns via live fluorescence microscopy. If microtubule destabilization and movement is impaired in null stathmin strains, as predicted, such findings may inform a novel therapeutic target for Shigellosis by preventing internal spreading. This is particularly significant in our current landscape, as antibiotic-resistant strains of Shigella are growing increasingly prevalent.

Synthetic lethality of RB1 and aurora A is driven by stathmin-mediated disruption of microtubule dynamics

RB1 mutational inactivation is a cancer driver in various types of cancer including lung cancer, making it an important target for therapeutic exploitation. We performed chemical and genetic vulnerability screens in RB1-isogenic lung cancer pair and herein report that aurora kinase A (AURKA) inhibition is synthetic lethal in RB1-deficient lung cancer. Mechanistically, RB1−/− cells show unbalanced microtubule dynamics through E2F-mediated upregulation of the microtubule destabilizer stathmin and are hypersensitive to agents targeting microtubule stability. Inhibition of AURKA activity activates stathmin function via reduced phosphorylation and facilitates microtubule destabilization in RB1−/− cells, heavily impacting the bipolar spindle formation and inducing mitotic cell death selectively in RB1−/− cells. This study shows that stathmin-mediated disruption of microtubule dynamics is critical to induce synthetic lethality in RB1-deficient cancer and suggests that upstream factors regulating microtubule dynamics, such as AURKA, can be potential therapeutic targets in RB1-deficient cancer.

Stathmin is overexpressed and regulated by mutant p53 in oral squamous cell carcinoma

BackgroundThe aim of this study was to investigate the oncogenic function and regulatory mechanism of stathmin in oral squamous cell carcinoma (OSCC).MethodsTwo-dimensional electrophoresis and liquid chromatography-tandem mass chromatography were applied to screen differentiated proteins during carcinogenesis in OSCC. Cell Counting Kit-8 (CCK-8) assays, colony formation, migration, flow cytometry, immunofluorescence and a xenograft model were used to detect the function of stathmin. The correlation between stathmin and p53 expression was analyzed using immunohistochemistry. Mutant/wild type p53 plasmids and small interfering RNA were used to examine the regulation of stathmin. Chromatin immunoprecipitation assays and luciferase assays were performed to detect the transcriptional activation of stathmin by p53.ResultsOverexpression of stathmin was screened and confirmed in OSCC patients and cell lines. Silencing expression of stathmin inhibited proliferation, colony formation and migration and promoted apoptosis. Poly ADP ribose polymerase (PARP) and cyclin-dependent kinase 1 (cdc2) were activated after silencing the expression of stathmin. Suppression of tumorigenicity was also confirmed in vivo. Mutant p53 transcriptionally activated the expression of stathmin in HN6 and HN13 cancer cells, but not in HN30 cells harboring wild type p53.ConclusionsThese results suggest that stathmin acts as an oncogene and is transcriptionally regulated by mutant p53, but not by wild-type p53. Stathmin could be a potential anti-tumor therapeutic target in OSCC.

Expression and phosphorylation of stathmin correlate with cell migration in esophageal squamous cell carcinoma

Microtubules play extensive roles in cellular processes, including cell motility. Stathmin is an important protein which destabilizes microtubules. The essential function of stathmin is closely associated with its phosphorylation status. Stathmin is overexpressed in many human cancers and has a significant relationship with clinical characteristics such as grade, tumor size and prognosis. We demonstrated that stathmin was overexpressed in ESCC tissues using both 2-DE and immunohistochemistry analysis. In addition, overexpression of stathmin was significantly correlated with histological grade in ESCC. However, no correlation was found with age, gender and lymph node metastasis. Knockdown of stathmin with siRNA impaired cell migration in KYSE30 and KYSE410 cells. When EC0156 cells were treated with paclitaxel, stathmin was stably phosphorylated and migration was impaired. These observations suggest that stathmin may have a more important function in ESCC development and migration. The present study provides further understanding of the importance of stathmin in ESCC therapy or diagnosis.

Abstract 2792: Novel anti-microtubule agents for breast cancer

Abstract Background and Objectives: Stathmin is a founding member of a family of microtubule-destabilizing proteins and is one of the key regulators of the microtubule cytoskeleton. Interestingly, stathmin is expressed at high levels in breast cancer and its overexpression is linked with disease progression. Recent studies identified numerous short peptides from the N-terminal region of stathmin family that were shown to impede tubulin polymerization. Here, we tested the hypothesis whether intracellular delivery of stathmin-like peptide(s) would inhibit the malignant proliferation of breast cancer cells by disrupting microtubule assembly in in vitro models of human breast cancer. Methods and Results: The peptide(s) that we designed consisted of a short stathmin-like domain that was fused to a TAT transduction domain, which served as a carrier to facilitate entry into breast cancer cells. We also included a hemagglutinin (HA) epitope tag to track the peptide intracellularly. Since stathmin could be phosphorylated &amp; inactivated intracellularly by p34cdc2 kinase, we also designed another peptide in which Ser-16 was mutated to an alanine to prevent its inactivation by phosphorylation. Previous studies had shown that this substitution results in a more potent form of stathmin that cannot be inactivated by phosphorylation. As a control, we had generated a similar peptide in which the stathmin-like domain was scrambled. We tested the effects of these peptides in different breast cancer cell lines (MDA-MB-231, T47D, &amp; SKBR-3). Immunostaining of breast cancer cells exposed to different peptides showed a vast majority of cells (&amp;gt;95%) positive for anti-HA staining suggesting that the designed peptides can be efficiently taken up by the cells. Proliferation assays in different breast cancer cell lines showed growth inhibitory effects in cells exposed to both, wild type or mutant peptides compared to cells exposed to a control peptide. However, the mutant peptide mediated a more profound anti-proliferative effect as expected. Immunofluorescence analysis of the microtubule organization of breast cancer cells exposed to different peptides using an anti-tubulin antibody showed a marked reduction in the density of microtubules. We also quantified the levels of polymerized and unpolymerized tubulin in breast cancer cells exposed to the different peptides using a sensitive biochemical assay. Immunoblot analysis showed a 1.8 fold decrease in the ratio of polymerized to soluble tubulin in cells exposed to wild-type peptide relative to control cells. The ratio of polymerized to soluble tubulin was further decreased by 3.7 fold in cells exposed to the mutant peptide. This suggests that the observed anti-proliferative effect is likely a result of inhibition of microtubule assembly. Thus, peptide(s) could be used to modulate stathmin function and inhibit malignant proliferation of breast cancer cells by interfering with the microtubule equilibrium. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2792. doi:1538-7445.AM2012-2792

Stathmin is Required for Stability of theDrosophilaNeuromuscular Junction

Synaptic connections can be stably maintained for prolonged periods, yet can be rapidly disassembled during the developmental refinement of neural circuitry and following cytological insults that lead to neurodegeneration. To date, the molecular mechanisms that determine whether a synapse will persist versus being remodeled or eliminated remain poorly understood. Mutations in Drosophila stathmin were isolated in two independent genetic screens that sought mutations leading to impaired synapse stability at the Drosophila neuromuscular junction (NMJ). Here we demonstrate that Stathmin, a protein that associates with microtubules and can function as a point of signaling integration, is necessary to maintain the stability of the Drosophila NMJ. We show that Stathmin protein is widely distributed within motoneurons and that loss of Stathmin causes impaired NMJ growth and stability. In addition, we show that stathmin mutants display evidence of defective axonal transport, a common feature associated with neuronal degeneration and altered synapse stability. The disassembly of the NMJ in stathmin includes a predictable sequence of cytological events, suggesting that a common program of synapse disassembly is induced following the loss of Stathmin protein. These data define a required function for Stathmin during synapse maintenance in a model system in which there is only a single stathmin gene, enabling future genetic investigation of Stathmin function with potential relevance to the cause and progression of neuromuscular degenerative disease.

Stathmin Regulates Centrosomal Nucleation of Microtubules and Tubulin Dimer/Polymer Partitioning

Stathmin is a microtubule-destabilizing protein ubiquitously expressed in vertebrates and highly expressed in many cancers. In several cell types, stathmin regulates the partitioning of tubulin between unassembled and polymer forms, but the mechanism responsible for partitioning has not been determined. We examined stathmin function in two cell systems: mouse embryonic fibroblasts (MEFs) isolated from embryos +/+, +/-, and -/- for the stathmin gene and porcine kidney epithelial (LLCPK) cells expressing stathmin-cyan fluorescent protein (CFP) or injected with stathmin protein. In MEFs, the relative amount of stathmin corresponded to genotype, where cells heterozygous for stathmin expressed half as much stathmin mRNA and protein as wild-type cells. Reduction or loss of stathmin resulted in increased microtubule polymer but little change to microtubule dynamics at the cell periphery. Increased stathmin level in LLCPK cells, sufficient to reduce microtubule density, but allowing microtubules to remain at the cell periphery, also did not have a major impact on microtubule dynamics. In contrast, stathmin level had a significant effect on microtubule nucleation rate from centrosomes, where lower stathmin levels increased nucleation and higher stathmin levels reduced nucleation. The stathmin-dependent regulation of nucleation is only active in interphase; overexpression of stathmin-CFP did not impact metaphase microtubule nucleation rate in LLCPK cells and the number of astral microtubules was similar in stathmin +/+ and -/- MEFs. These data support a model in which stathmin functions in interphase to control the partitioning of tubulins between dimer and polymer pools by setting the number of microtubules per cell.

Gene expression profiles in mouse embryo fibroblasts lacking stathmin, a microtubule regulatory protein, reveal changes in the expression of genes contributing to cell motility.

BackgroundStathmin (STMN1) protein functions to regulate assembly of the microtubule cytoskeleton by destabilizing microtubule polymers. Stathmin over-expression has been correlated with cancer stage progression, while stathmin depletion leads to death of some cancer cell lines in culture. In contrast, stathmin-null mice are viable with minor axonopathies and loss of innate fear response. Several stathmin binding partners, in addition to tubulin, have been shown to affect cell motility in culture. To expand our understanding of stathmin function in normal cells, we compared gene expression profiles, measured by microarray and qRT-PCR, of mouse embryo fibroblasts isolated from STMN1+/+ and STMN1-/- mice to determine the transcriptome level changes present in the genetic knock-out of stathmin.ResultsMicroarray analysis of STMN1 loss at a fold change threshold of ≥ 2.0 revealed expression changes for 437 genes, of which 269 were up-regulated and 168 were down-regulated. Microarray data and qRT-PCR analysis of mRNA expression demonstrated changes in the message levels for STMN4, encoding RB3, a protein related to stathmin, and in alterations to many tubulin isotype mRNAs. KEGG Pathway analysis of the microarray data indicated changes to cell motility-related genes, and qRT-PCR plates specific for focal adhesion and ECM proteins generally confirmed the microarray data. Several microtubule assembly regulators and motors were also differentially regulated in STMN1-/- cells, but these changes should not compensate for loss of stathmin.ConclusionApproximately 50% of genes up or down regulated (at a fold change of ≥ 2) in STMN1-/- mouse embryo fibroblasts function broadly in cell adhesion and motility. These results support models indicating a role for stathmin in regulating cell locomotion, but also suggest that this functional activity may involve changes to the cohort of proteins expressed in the cell, rather than as a direct consequence of stathmin-dependent regulation of the microtubule cytoskeleton.

Alternate Cyclin D1 mRNA Splicing Modulates p27KIP1 Binding and Cell Migration

Cyclin D1 is an important cell cycle regulator, but in cancer its overexpression also increases cellular migration mediated by p27 KIP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N terminus as the canonical cyclin D1a isoform but a distinct C terminus. In this study we show that these different isoforms have unique properties with regard to the cellular migration function of cyclin D1. Although they displayed little difference in transcriptional co-repression assays on idealized reporter genes, microarray cDNA expression analysis revealed differential regulation of genes, including those that influence cellular migration. Additionally, whereas cyclin D1a stabilized p27 KIP1 and inhibited RhoA-induced ROCK kinase activity, promoting cellular migration, cyclin D1b failed to stabilize p27 KIP1 or inhibit ROCK kinase activity and had no effect on migration. Our findings argue that alternate splicing is an important determinant of the function of cyclin D1 in cellular migration.

RETRACTED: Drosophila Stathmin Is Required to Maintain Tubulin Pools

Stathmin, or Oncoprotein 18 (Op18), is the founding member of a phosphoprotein family that can regulate the microtubule cytoskeleton by sequestering tubulin and promoting microtubule catastrophe. Stathmin is subject to spatially and temporally controlled regulatory phosphorylation, which inhibits its interaction with tubulin. Drosophila Stathmin has similar properties to the mammalian proteins. We find that Drosophila Stathmin is required for specific microtubule-dependent processes: maintenance of oocyte identity within a germline cyst and localization of polarity determinants. Unexpectedly, microtubules are less abundant in stathmin mutant cells compared to normal cells, showing that a key function of Stathmin in vivo is the long-term maintenance of the microtubule cytoskeleton. The microtubule network re-forms more slowly after coldshock in stathmin mutant follicle cells. Surprisingly, stathmin mutant animals and tissues show a marked decrease in total tubulin-protein levels, and this might explain the effect on the microtubule cytoskeleton. Stathmin overexpression also increases tubulin protein. Free alpha- and beta-tubulin have been shown to negatively autoregulate their own synthesis. We suggest that Stathmin serves to maintain a noninhibitory, soluble, and releasable tubulin pool.

Reversal of Stathmin-Mediated Resistance to Paclitaxel and Vinblastine in Human Breast Carcinoma Cells

Antimicrotubule agents are commonly used chemotherapy drugs for the treatment of breast and other cancers. However, these agents have variable activity partly because of microtubule regulatory proteins. Stathmin, an 18-kDa phosphoprotein that promotes microtubule depolymerization, was found to be frequently overexpressed in breast cancer. We previously identified stathmin-mediated mechanisms of resistance to antimicrotubule agents, including altered drug binding and delayed transit from G(2) into M phase, where these agents are effective in disrupting microtubule dynamics. We hypothesized that by reversing stathmin-mediated depolymerization of microtubules or by promoting entry into mitosis, this could increase sensitivity to antimicrotubule agents in human breast cancer cells overexpressing stathmin. We found that targeting stathmin or wee-1 expression with RNA interference can induce microtubule polymerization and promote G(2)/M progression, respectively, and sensitize stathmin-overexpressing breast cancer cells to paclitaxel and vinblastine. Furthermore, targeting wee-1 led to the phosphorylation of stathmin, which is known to attenuate its activity. Therefore, these data suggest a novel approach to improving the efficacy of certain antimicrotubule agents against breast cancer by regulating the function of stathmin.

Molecular Discovery Could Provide Good Reason to Fear

Back to table of contents Previous article Next article Clinical & Research NewsFull AccessMolecular Discovery Could Provide Good Reason to FearJoan Arehart-TreichelJoan Arehart-TreichelSearch for more papers by this authorPublished Online:3 Feb 2006https://doi.org/10.1176/pn.41.3.0023aAlthough fear is known to be processed in the amygdala, an almond-shaped structure deep within the brain, as well as in some other brain areas, very little has been known about the molecular mechanisms of learned and innate fear. Specifically, glutamate receptors and major protein kinases have been implicated, but they are important for other types of memories and behaviors besides fear.Now a fear molecule has been identified, a protein called stathmin. The identification of stathmin as a fear molecule constitutes a notable advance in understanding the molecular basis of fear, both the major investigator and senior investigator of the study told Psychiatric News. The major investigator was Gleb Shumyatsky, Ph.D., an assistant professor of genetics at Rutgers University. The senior investigator was Eric Kandel, M.D., a professor of psychiatry at Columbia University and a winner of the 2000 Nobel Prize in Medicine or Physiology (Psychiatric News, November 3, 2000).As Shumyatsky, Kandel, and their colleagues reported in the November 18, 2005, Cell, they first found that the amygdala and certain other brain areas crucial to regulating fear are very rich in stathmin. Then they found that mice without the stathmin gene showed less anxiety when they heard a tone that had previously been associated with a shock than did mice with the stathmin gene. The mice without the gene were also more prone than the controls to explore novel open spaces and maze environments. Furthermore, the mice without the stathmin gene did not show deficits in a spatial task dependent on the hippocampus, where stathmin is not normally present.“Stathmin is involved in fear function, but not in some other types of memory,” Shumyatsky said. As the scientists concluded in their study report, stathmin is “essential in regulating both innate and learned fear.”Also, Shumyatsky pointed out, “because stathmin is an inhibitor of microtubule formation, our study for the first time links microtubule function to memory for fear, or, for that matter, any kind of memory.” Microtubules, located in the cytoplasm of cells, are involved in cell structure and cell movement.A challenge now is to determine whether stathmin also plays a role in fear in humans. “One has to see first if stathmin is expressed in the [human] amygdala in a way similar to that in mice,” Shumyatsky explained.“ Then we can look for possible genomic mutations in the stathmin gene sequences in human patients with a history of genetic predisposition to anxiety disorders.”If stathmin indeed turns out to be involved in fear processing in humans, it could well lead to the discovery of new types of antianxiety drugs, both Shumyatsky and Kandel believe. For example, various compounds could be tested in mice without the stathmin gene.Shumyatsky, Kandel, and their team are planning to delineate the intricacies of stathmin function in fear and how it interacts with other proteins involved in fear behavior.The research was funded primarily by the National Institutes of Health and the National Alliance for Research on Schizophrenia and Depression.An abstract of “Stathmin, a Gene Enriched in the Amygdala, Controls Both Learned and Innate Fear” is posted at<www.cell.com/content/article/abstract?uid=PIIS0092867405008755>.▪ ISSUES NewArchived

The role of stathmin in the regulation of the cell cycle.

Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take.

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.

The Effect of Stathmin Phosphorylation on Microtubule Assembly Depends on Tubulin Critical Concentration

Stathmin is a phosphorylation-regulated tubulin-binding protein. In vitro and in vivo studies using nonphosphorylatable and pseudophosphorylated mutants of stathmin have questioned the view that stathmin might act only as a tubulin-sequestering factor. Stathmin was proposed to effectively regulate microtubule dynamic instability by increasing the frequency of catastrophe (the transition from steady growth to rapid depolymerization), without interacting with tubulin. We have used a noninvasive method to measure the equilibrium dissociation constants of the T(2)S complexes of tubulin with stathmin, pseudophosphorylated (4E)-stathmin, and diphosphostathmin. At both pH 6.8 and pH 7.4, the relative sequestering efficiency of the different stathmin variants depends on the concentration of free tubulin, i.e. on the dynamic state of microtubules. This control is exerted in a narrow range of tubulin concentration due to the highly cooperative binding of tubulin to stathmin. Changes in pH affect the stability of tubulin-stathmin complexes but do not change stathmin function. The 4E-stathmin mutant mimics inactive phosphorylated stathmin at low tubulin concentration and sequesters tubulin almost as efficiently as stathmin at higher tubulin concentration. We propose that stathmin acts solely by sequestering tubulin, without affecting microtubule dynamics, and that the effect of stathmin phosphorylation on microtubule assembly depends on tubulin critical concentration.

Effects of stathmin inhibition on the mitotic spindle.

Stathmin is a major cytosolic phosphoprotein that plays an important role in the regulation of microtubule dynamics during cell cycle progression. It has recently been proposed that the major function of stathmin is to promote depolymerization of the microtubules that make up the mitotic spindle. In this report, we tested the prediction that a deficiency in stathmin expression would result in constitutive stabilization of microtubules and lead to abnormalities in the organization of the mitotic spindle. Our studies demonstrate that antisense inhibition of stathmin expression in K562 erythroleukemic cells results in increased ratio of polymerized to depolymerized tubulin. These changes are associated with phenotypic abnormalities of the mitotic spindle and difficulty in completing mitosis. These studies also showed that inhibition of stathmin expression results in increased susceptibility of K562 leukemic cells to the pharmacological agents, like taxol, which are known to stabilize the mitotic spindle. In contrast, stathmin inhibition results in decreased sensitivity to vinblastine, an agent that destabilizes the mitotic spindle. Thus, our experimental findings are supportive of the model that stathmin is a microtubule-destabilizing factor that plays an important role in the regulation of the mitotic spindle. We also suggest a potential therapeutic approach for cancer based on the combination of stathmin inhibition with pharmacologic agents that stabilize the mitotic spindle.

Stathmin slows down guanosine diphosphate dissociation from tubulin in a phosphorylation-controlled fashion.

Stathmin is an important protein that interacts with tubulin and regulates microtubule dynamics in a phosphorylation-controlled fashion. Here we show that the dissociation of guanosine 5'-diphosphate (GDP) from beta-tubulin is slowed 20-fold in the (tubulin)(2)-stathmin ternary complex (T(2)S). The kinetics of GDP or guanosine 5'-triphosphate (GTP) dissociation from tubulin have been monitored by the change in tryptophan fluorescence of tubulin upon exchanging 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-diphosphate (S6-GDP) for tubulin-bound guanine nucleotide. At molar ratios of stathmin to tubulin lower than 0.5, biphasic kinetics were observed, indicating that the dynamics of the complex is extremely slow, consistent with its high stability. The method was used to characterize the effects of phosphorylation of stathmin on its interaction with tubulin. The serine-to-glutamate substitution of all four phosphorylatable serines of stathmin (4E-stathmin) weakens the stability of the T(2)S complex by about 2 orders of magnitude. The phosphorylation of serines 16 and 63 in stathmin has a more severe effect and weakens the stability of T(2)S 10(4)-fold. The rate of GDP dissociation is lowered only 7-fold and 4-fold in the complexes of tubulin with 4E-stathmin and diphosphostathmin, respectively. Sedimentation velocity studies support the conclusions of nucleotide exchange data and show that the T(2)S complexes formed between tubulin and 4E-stathmin or diphosphostathmin are less compact than the highly stable T(2)S complex. The correlation between the effect of phosphorylation of stathmin on the stability of T(2)S complex measured in vitro and on the function of stathmin in vivo is discussed.

Bistratene A Induces a Microtubule-Dependent Block in Cytokinesis and Altered Stathmin Expression in HL60 Cells

Bistratene A is a cyclic polyether which affects cell cycle progression and can induce phosphorylation of cellular proteins. Treatment of HL60 cells with 100 ng/ml bistratene A was found to inhibit cytokinesis but had no effect on DNA synthesis and nuclear division. Consequently, bistratene A-treated cells became polyploid and multinucleate. In association with the development of this phenotype, the cytoplasmic protein stathmin was biphasically phosphorylated and levels of expression were doubled. Immunostaining of binucleate cells (bistratene A for 24 h) revealed increased α-tubulin localization where the cleavage furrow might be expected to form, i.e., along the equatorial plane. Treatment of these binucleate cells with the microtubule depolymerizing agent nocadazole promoted cleavage furrow formation and partially ameliorated the bistratene A-induced block in cell division. These findings implicate the polymerization status of microtubules and stathmin function in the regulation of cytokinesis.

Stathmin overexpression in 293 cells affects signal transduction and cell growth

Stathmin is a ubiquitous cytoplasmic protein whose phosphorylation state changes markedly in response to extracellular signals, and during the cell cycle. To clarify the function of stathmin, its four phosphorylation sites were mutated to either alanines (4A-stathmin) or glutamates (4E-stathmin). In transfected cells, 4A-stathmin caused a strong G2/M block and also inhibited the responsiveness of a co-transfected fos promoter/luciferase reporter plasmid to serum stimulation, whereas wild type and 4E-stathmin had relatively minor effects. These results support the idea that stathmin plays a role in multiple cellular processes and indicate that the regulation of the phosphorylation state of stathmin is likely to determine its action.