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PubMed2001

Stathmin inhibition enhances okadaic acid-induced mitotic arrest: a potential role for stathmin in mitotic exit.

Stathmin is a microtubule-destabilizing phosphoprotein that plays a critical role in the regulation of mitosis. The microtubule-depolymerizing activity of stathmin is lost upon phosphorylation in mitosis. Although the role of phosphorylation of stathmin by p34(cdc2) kinase in the assembly of the mitotic spindle is well established, the role of dephosphorylation of stathmin in mitosis is unknown. In this study, we tested the hypothesis that dephosphorylation of stathmin may be critically important for the depolymerization of the mitotic spindle and the exit from mitosis. We compared the effects of okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, on different parameters of mitotic progression in the presence or absence of stathmin deficiency. Because okadaic acid prevents dephosphorylation of stathmin and results in accumulation of the inactive phosphorylated form, exposure to okadaic acid would be expected to have a more profound effect on mitosis in the presence of relative stathmin deficiency. We found that inhibition of stathmin expression results in increased sensitivity to the antimitotic effects of okadaic acid. This was reflected by increased growth inhibition associated with mitotic arrest. A vast majority of the stathmin-inhibited cells were found to be arrested in late metaphase/anaphase and had severe mitotic spindle abnormalities. Exposure to okadaic acid also resulted in a bigger ratio of polymerized/unpolymerized tubulin in stathmin-inhibited cells relative to control cells. Because the only difference between the control and the stathmin-inhibited cells is the deficiency of stathmin in the latter, the increased susceptibility of the stathmin-inhibited cells to okadaic acid-induced mitotic arrest implies a role for stathmin in the later stages of mitosis.

Cell Cycle↗

PubMed2001

Stathmin levels in growth plate chondrocytes are modulated by vitamin D3 metabolites and transforming growth factor-beta1 and are associated with proliferation.

Stathmin is a highly conserved, phosphorylated cytosolic protein that is found at decreased levels in all cells as they become more terminally differentiated, or when they decrease in their rate of proliferation. This study examined the hypothesis that stathmin levels in growth plate chondrocytes decreases as endochondral maturation increases. To test this hypothesis, we used a costochondral growth plate chondrocyte cell culture model. Cells derived from the resting zone (RC) express twice as much stathmin mRNA in culture and have twice as much stathmin protein as cells derived from the post proliferative growth zone ([GC]; prehypertrophic and upper hypertrophic cell zones). Stathmin levels in vivo were assessed by immunohistochemistry. To assess the effects of agents that modulate proliferation and differentiation, RC and GC chondrocytes were cultured in the presence of 10(-10) to 10(-8) M 1alpha,25-(OH)2D3, which regulates proliferation in both cell types but affects differentiation of only GC cells, or 10(-9) to 10(-7) M 24R,25-(OH)2D3, which regulates differentiation and maturation of RC cells but decreases proliferation of GC cells. In addition, RC cells were treated with 0.44 or 0.88 ng/mL of recombinant human transforming growth factor beta1 (rhTGF-beta1), which stimulates proliferation of RC cells and regulates proteoglycan production, but not alkaline phosphatase activity. Stathmin protein levels were determined using quantitative immunoblots, with recombinant human stathmin as a standard. The results show that stathmin levels are associated with proliferation. Proliferating chondrocytes in vivo exhibited higher levels of immunoreactive stathmin than either RC or GC cells in the growth plate. In culture, 1alpha,25-(OH)2D3 caused a dose-dependent decrease in stathmin in RC and GC cells within 24 h. 24R, 25-(OH)2D3 also reduced stathmin levels in GC cells within 24 h but only affected RC cells after prolonged exposures (96 h), at which time RC cells express a GC-like phenotype. rhTGF-beta1 caused an increase in stathmin levels in RC cells. Stathmin levels are sensitive to protein kinase C (PKC) in other cells. Inhibition of PKC with chelerythrine had no effect on the response of RC cells to 1alpha,25-(OH)2D3 but it blocked the effect of rhTGF-beta1, indicating that decreases in stathmin by vitamin D3 metabolites may not be modulated by PKC, whereas increases in stathmin via rhTGF-beta1 may be regulated via a PKC-dependent mechanism. These results support the hypothesis that constitutively expressed levels of stathmin are related to cell maturation state and that they are modulated by factors that regulate proliferation.

24,25-Dihydroxyvitamin D 3↗

PubMed2002

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.

Animals↗

PubMed1998

Overexpression of the stathmin gene in a subset of human breast cancer.

Stathmin is a highly conserved cytosolic phosphoprotein that destabilizes microtubules. Stathmin, which has been proposed as a relay protein integrating diverse cell signalling pathways, acts in vitro as a tubulin-sequestering protein, and its activity is dramatically reduced by phosphorylation. Interestingly, stathmin expression and phosphorylation are regulated during the control of cell growth and differentiation, and there is much evidence suggesting that in vivo stathmin plays a role in the control of microtubule dynamics during mitosis. Stathmin may thus be considered as one of the key regulators of cell division. We examined 50 human primary breast tumours for stathmin mRNA and protein expression and screened for abnormalities in the chromosome region harbouring the stathmin gene. Overexpression of stathmin was found in 15 tumours (30%). At the present stage, no clear correlation emerged between stathmin expression and several prognosis markers. Interestingly, perfect matching was observed between stathmin mRNA overexpression, protein overexpression and strong staining for stathmin on paraffin-embedded tumour sections when specimens were available. Furthermore, a tentative link between loss of heterozygosity (LOH) in the 1p32-1pter region and stathmin overexpression was observed. Our results suggest that stathmin might play a role in breast carcinogenesis and that stathmin-overexpressing tumours may represent a new subtype of breast cancer.

Breast↗

PubMed2002

Effect of stathmin on the sensitivity to antimicrotubule drugs in human breast cancer.

Stathmin is a p53-regulated protein known to influence microtubule dynamics. Because several chemotherapeutic agents used to treat breast cancer alter the dynamic equilibrium of tubulin polymerization, stathmin may play an important role in determining the sensitivity to these drugs. Therefore, we evaluated the effect of stathmin expression on the action of taxanes and Vinca alkaloids using a panel of human breast cancer cell lines. Cell lines harboring mutant p53 expressed high levels of stathmin. Two cell lines with different levels of endogenous stathmin expression and isogenic-paired cell lines transfected to overexpress stathmin were used to determine whether or not stathmin modulated the sensitivity to drugs. Overexpression of stathmin decreased polymerization of microtubules, markedly decreased binding of paclitaxel, and increased binding of vinblastine. Stathmin overexpression decreased sensitivity to paclitaxel and, to a lesser extent, to vinblastine. In contrast, stathmin content had no significant effect on the sensitivity to chemotherapeutic drugs that do not target microtubules. Cell lines overexpressing stathmin were more likely to enter G(2) but less likely to enter mitosis as determined by fluorescence-activated cell sorting and mitotic index. This effect was magnified when stathmin-overexpressing cells were treated with vinblastine as measured by the detection of proteins phosphorylated in early mitosis. These data suggest that the action of antimicrotubule drugs can be affected by stathmin in at least two ways: (a) altered drug binding; and (b) growth arrest at the G(2) to M boundary. Mutant p53 breast cancers exhibiting high levels of stathmin may be resistant to antimicrotubule agents.

Antineoplastic Agents, Phytogenic↗

PubMed1993

Stathmin gene family: phylogenetic conservation and developmental regulation in Xenopus.

The ubiquitous cytoplasmic phosphoprotein stathmin was proposed to play a general role as an intracellular relay integrating diverse signals regulating the proliferation, differentiation, and functions of cells (Sobel, A. (1991) Trends Biol. Sci. 16, 301-305). It was originally identified in mammalian cells and tissues, but antibodies directed against the mammalian protein also recognized a stathmin-like 19-kDa protein in all vertebrate classes. The immunoreactive protein in Xenopus laevis displayed, like mammalian stathmin, several nonphosphorylated and phosphorylated heat-soluble forms with distinct migration on two-dimensional polyacrylamide gel electrophoresis. Screening of Xenopus oocyte and brain cDNA libraries with a rat stathmin cDNA probe allowed us to isolate several stathmin-related cDNA clones, among which clone XO35 encodes the Xenopus homologue of stathmin whose deduced amino acid sequence is 79% identical to and displays most of the characteristic structural features of the mammalian protein. In particular, one of the cAMP-dependent protein kinase and the two "proline-directed" kinase-specific sites known to be phosphorylated in rat stathmin are also present in the Xenopus protein. Furthermore, two other sets of clones coding for related proteins belonging to the stathmin gene family were also isolated; clone SC15 encodes the Xenopus homologue of SCG10, a rat protein specifically related to neuronal differentiation; clone XB3 encodes a protein which, as SCG10 or SC15, possesses a stathmin-like domain and an additional N-terminal extension but is more distant from SCG10 than SC15. Interestingly, the mRNA transcripts of Xenopus stathmin (XO35) appear ubiquitous, like stathmin in mammals, whereas the SC15 and XB3 mRNAs appeared as markers of the nervous tissue in Xenopus. During Xenopus oogenesis, stathmin accumulates and remains stable as a maternal product throughout early development. Concurrently, its phosphorylation is regulated from essentially unphosphorylated forms to highly phosphorylated ones in the mature egg, which are then progressively dephosphorylated again from the midblastula to the tailbud stage. Altogether, our results demonstrate the high evolutionary conservation of stathmin together with the members of its related gene family, not only at the level of their molecular structures, but also of their biochemical and biological regulation. These observations are thus further in favor of a very general and likely essential role of stathmin in the normal control of cells throughout development and in the adult.

Amino Acid Sequence↗

PubMed1999

Stathmin interaction with HSC70 family proteins.

Stathmin is a ubiquitous cytosolic phosphoprotein participating in the relay and integration of diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation, and activities. It is phosphorylated in response to diverse extracellular signals including hormones and growth factors, and it is highly expressed during development and in diverse tumoral cells and tissues. Stathmin interacts with tubulin and other potential protein partners such as BiP, KIS, CC1 and CC2/tsg101. In our present search for further functional partners of stathmin, we identified proteins in the Hsp70 family, and in particular Hsc70, as interacting with stathmin in vitro. Hsc70 is among the proteins coimmunoprecipitated with stathmin, and it is the main protein retained specifically on stathmin-Sepharose beads identified by one- and two-dimensional electrophoresis and immunoblots. Bovine serum albumin (BSA)-Sepharose did not bind Hsc70, and anti-stathmin antisera specifically inhibited the interaction of Hsc70 with stathmin-Sepharose. The binding of Hsc70 to stathmin is dependent on the phosphorylation status of stathmin, as it did not occur with a "pseudophosphorylated" mutant form of stathmin. This interaction is further dependent on the ATP status of Hsc70. It was inhibited in the presence of ATP-Mg++ but not in the presence of ATP-Mg++ and ethylenediaminetetraacetic acid (EDTA) or of ADP. Our results suggest that the interaction of stathmin with Hsc70 is specific in both proteins and most likely biologically relevant in the context of their functional implication in the control of numerous intracellular signaling and regulatory pathways, and hence of normal cell growth and differentiation.

Adenosine Triphosphate↗

PubMed1998

Role for protein phosphatases in the cell-cycle-regulated phosphorylation of stathmin.

Stathmin is a major cytosolic phosphoprotein that regulates microtubule dynamics during the assembly of the mitotic spindle. The activity of stathmin itself is regulated by changes in its state of phosphorylation during the transition from interphase to metaphase. For a better understanding of the regulation of stathmin activity during the cell cycle, we explored the mechanism(s) responsible for the decrease in the level of phosphorylation of stathmin as cells complete mitosis and enter a new G1 phase. We show that stathmin mRNA and protein are expressed constitutively throughout the different phases of the cell cycle. This suggests that the non-phosphorylated stathmin that predominates during G1 is not generated by degradation of phosphorylated stathmin in mitosis and synthesis of new unphosphorylated stathmin as cells enter a new G1 phase. This suggested that protein phosphatases might be responsible for dephosphorylating stathmin as cells enter a new cell cycle. Okadaic acid-mediated inhibition of protein phosphatases in vivo showed a major increase in the level of phosphorylation of stathmin. Dephosphorylation studies in vitro showed differential patterns of site-specific dephosphorylaton of stathmin to protein phosphatase type 1, protein phosphatase type 2A and protein phosphatase type 2B. Thus stathmin might be a target for okadaic acid-sensitive protein phosphatase(s), and its activity in eukaryotic cells might be modulated by the sequential activity of specific protein kinases and phosphatases.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1993

Stathmin: cellular localization of a major phosphoprotein in the adult rat and human CNS.

Stathmin is a ubiquitous, 19 kDa cytoplasmic protein the phosphorylation of which is associated with many cellular signaling pathways. It is particularly abundant in neurons and reaches a peak of expression in the neonatal period, although it remains highly expressed in the adult brain. In order to determine whether this abundant expression is associated with discrete cellular populations that are still at an immature stage during adulthood, as suggested by others, the cellular localization of stathmin was investigated in the adult rat and human central nervous system. Western blotting with a specific antiserum indicated that stathmin was ubiquitous in the brain and spinal cord but that its relative concentration varied up to 2.6 times between regions. To characterize the distribution of stathmin within the brain, its cellular localization was analyzed by immunocytochemistry. Highly immunoreactive neurons and oligodendrocytes were observed, and stathmin immunoreactivity was localized to the perikaryon and all processes, but not the nucleus. Most brain and spinal cord cell groups showed stathmin immunoreactivity, although the extent and intensity of labeling differed largely from one place to another. Particularly numerous stathmin-immunoreactive neuronal cell bodies were found in the pyriform, cingulate, and neocortex, as well as in many cholinergic nuclei of the basal forebrain and brainstem, in the medial thalamus, in various brainstem nuclei, in the dorsalmost layers of the spinal cord, and in brain areas lacking a blood-brain barrier to macromolecules. In addition to neuronal populations, stathmin-antibodies intensely labeled choroid plexuses. Many other brain regions exhibited moderate neuronal immunostaining. The distribution of stathmin-immunoreactive processes was in some areas relatively heterogeneous. Intense immunoreactivity was observed in some fiber tracts (corpus callosum, anterior commissure, inferior cerebellar peduncle, etc.) but was missing in others (internal capsule, posterior commissure, etc.). Some brain areas rich in immunoreactive neurons also displayed an intense immunoreactivity of the neuropile, whereas others contained either immunoreactive cells or fibers. In the human brain, stathmin immunostaining occurred in many areas, corresponding to those identified in the rat, with the exception of the cerebral cortex, the hippocampal fascia dentata, and the substantia nigra. The present results support our suggestion that, in addition to its involvement in cell proliferation and differentiation, stathmin may also be related to regulation of differentiated cell functions, as it appears to be a major signaling protein in widespread areas of the adult brain in both rat and human.

Aged↗

PubMed2000

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.

Animals↗

PubMed1995

Stathmin is a major substrate for mitogen-activated protein kinase during heat shock and chemical stress in HeLa cells.

Stathmin is a ubiquitous, highly conserved 19-kDa cytoplasmic protein whose expression and phosphorylation are regulated in relation to cell proliferation, differentiation or activation, in many biological systems. In this report, we show that stathmin undergoes major phosphorylation in HeLa cells submitted to heat or chemical stress. Heat-shock-induced stathmin phosphorylation was very rapid, as maximal incorporation of phosphate was observed at 5 min. Phosphorylation of stathmin might, therefore, occur as a very early step in the intracellular response to heat shock. The sites of phosphorylation of stathmin involved during the stress response were identified as mostly Ser25 and, to a lesser extent, Ser38. These sites are both followed by a proline residue, and known to be good substrates in vitro for mitogen-activated protein kinase (MAP-kinase) and p34cdc2 kinase, respectively. In lysates from heat-shocked cells, an increased stathmin-kinase activity, distinct from the histone-H1-kinase activity, was found to phosphorylate stathmin mostly on Ser25, the main site for MAP-kinase in vitro. This stathmin-kinase coeluted quantitatively with the stress-activated MAP-kinase from an FPLC MonoQ column. Furthermore, a stathmin kinase activity was precipitated from lysates of heat-shocked HeLa cells by an anti-(MAP-kinase) serum. Together, these results indicate that the phosphorylation of stathmin by MAP-kinase is likely to be a significant component of the signalling array controlling the cellular response to stress, and they further underline the general involvement of stathmin in intracellular signalling.

Enzyme Activation↗

PubMed1992

Prolactin-induced proliferation of the Nb2 T-lymphoma is associated with protein kinase-C-independent phosphorylation of stathmin.

Phosphorylation of stathmin, a 19-kDa protein found in many tissues, has been linked to cell differentiation and proliferation. This protein is present in lymphocytes, and both phosphorylation and expression of stathmin are regulated by lymphotropic agents. In this study an antibody specific for stathmin was used to examine phosphorylation in response to PRL. The results suggest that PRL stimulates stathmin phosphorylation in the Nb2 lymphoma and that phosphorylation correlates with PRL-induced cell proliferation. Stathmin expression does not change substantially as PRL-stimulated Nb2 cells move through the cell cycle and enter into the S-phase. Thus, stathmin phosphorylation, but not expression, is regulated by PRL. Activation of protein kinase-C (PKC) in Nb2 cells also induces phosphorylation of stathmin, but PKC does not appear to mediate phosphorylation in response to PRL. The pattern of phosphorylation in response to 12-O-tetradecanoylphorbol-13-acetate differs from that in response to PRL, and down-regulation of PKC does not inhibit PRL-induced phosphorylation or proliferation. In addition to stathmin, PRL increases phosphorylation of a group of stathmin-like proteins. Phosphorylation of these proteins also correlates well with PRL-induced proliferation. Taken together, the results suggest that phosphorylation of stathmin and stathmin-like proteins may mediate some actions of PRL in Nb2 cells. The results further suggest that activation of PKC is not an important early event in PRL-stimulated mitogenesis in Nb2 cells.

Cell Division↗

PubMed1998

The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network.

Stathmin is a small regulatory phosphoprotein integrating diverse intracellular signaling pathways. It is also the generic element of a protein family including the neural proteins SCG10, SCLIP, RB3 and its two splice variants RB3' and RB3". Stathmin itself was shown to interact in vitro with tubulin in a phosphorylation-dependent manner, sequestering free tubulin and hence promoting microtubule depolymerization. We investigated the intracellular distribution and tubulin depolymerizing activity in vivo of all known members of the stathmin family. Whereas stathmin is not associated with interphase microtubules in HeLa cells, a fraction of it is concentrated at the mitotic spindle. We generated antisera specific for stathmin phosphoforms, which allowed us to visualize the regulation of phosphorylation-dephosphorylation during the successive stages of mitosis, and the partial localization of stathmin phosphorylated on serine 16 at the mitotic spindle. Results from overexpression experiments of wild-type and novel phosphorylation site mutants of stathmin further suggest that it induces depolymerization of interphase and mitotic microtubules in its unphosphorylated state but is inactivated by phosphorylation in mitosis. Phosphorylation of mutants 16A25A and 38A63A on sites 38 and 63 or 16 and 25, respectively, was sufficient for the formation of a functional spindle, whereas mutant 16A25A38A63E retained a microtubule depolymerizing activity. Transient expression of each of the neural phosphoproteins of the stathmin family showed that they are at least partially associated to the Golgi apparatus and not to other major membrane compartments, probably through their different NH2-terminal domains, as described for SCG10. Most importantly, like stathmin and SCG10, overexpressed SCLIP, RB3 and RB3" were able to depolymerize interphase microtubules. Altogether, our results demonstrate in vivo the functional conservation of the stathmin domain within each protein of the stathmin family, with a microtubule destabilizing activity most likely essential for their specific biological function(s).

Antibodies↗

PubMed1995

Stathmin expression is a feature of proliferating cells of most, if not all, cell lineages.

BACKGROUND: Stathmin is a phylogenetically conserved protein which was identified initially as a prominent cytosolic protein in hemopoietic cells, endocrine cells, brain, and testis. In these tissues, it has been suggested that the level of stathmin expression is important in development and cell proliferation. Furthermore, stathmin phosphorylation appears to be involved in the regulation of cell growth arrest, terminal differentiation, and hormone secretion. Elevated levels of expression of stathmin have been described in leukemia and lymphoma cells. The aim of this study was to characterize the distribution of cells that express the stathmin protein in a wide variety of normal human and rodent tissues. EXPERIMENTAL DESIGN: First, antisera against a synthetic stathmin peptide have been raised in rabbits and the specificity of these antisera confirmed by their reactivity with stathmin on 1-dimensional and 2-dimensional Western blots. Second, the most appropriate means of fixing tissues in order to stain stathmin has been investigated. Finally, using the optimized conditions of tissue fixation, the antisera have been used to immunostain sections taken from a wide variety of tissues. RESULTS: Immunopositivity was found in cells of all the lineages studied, with the stained cells present within the proliferating compartment of tissues. Conversely, most nonproliferating mature cells did not stain with the antisera to stathmin. The only nonproliferating cells that appeared to express stathmin were a subpopulation of glial cells, neurons, and anterior pituitary cells. CONCLUSIONS: It is proposed that stathmin is necessary for cell proliferation in most, or all, cell lineages and that its primary function relates to some aspect of cell division.

Animals↗

PubMed1999

Cellular and subcellular localization of stathmin during oocyte and preimplantation embryo development.

Stathmin is a 19 kDa cytosolic phosphoprotein, proposed to act as a relay integrating diverse intracellular signaling pathways involved in regulation of cell proliferation, differentiation, and function. To gain further information about its significance during early development, we analyzed stathmin expression and subcellular localization in mouse oocytes and preimplantation embryos. RT-PCR analysis revealed a low expression of stathmin mRNA in unfertilized oocytes and a higher expression at the blastocyst stage. A fine cytoplasmic punctuate fluorescent immunoreactive stathmin pattern was detected in the oocyte, while it evolved toward an increasingly speckled pattern in the two-cell and later four- to eight-cell embryo, with even larger speckles at the morula stage. In blastocysts, stathmin immunoreactivity was fine and intense in inner cell mass cells, whereas it was low and variable in trophectodermal cells. Electron microscopic analysis allowed visualization with more detail of two types of stathmin immunolocalization: small clusters in the cytoplasm of oocytes and blastocyst cells, together with loosely arranged clusters around the outer membrane of cytoplasmic vesicles, corresponding to the immunofluorescent speckles in embryos until the morula stage. In conclusion, it appears from our results that maternal stathmin is accumulated in the oocyte and is relocalized within the oocyte and early preimplantation embryonic cell cytoplasm to interact with specific cytoplasmic membrane formations. Probably newly synthesized, embryonic stathmin is expressed in the blastocyst, where it is localized more uniformly in the cytoplasm mostly of inner cell mass (ICM) cells. These expression and localization patterns are probably related to the particular roles of stathmin at the successive steps of oocyte maturation and early embryonic development. They further support the proposed physiologic importance of stathmin in essential biologic regulation.

Animals↗

PubMed1992

Expression of transfected stathmin cDNA reveals novel phosphorylated forms associated with developmental and functional cell regulation.

Stathmin is a ubiquitous, highly conserved phosphoprotein, which most likely acts as an intracellular relay integrating various transduction pathways triggered by extracellular signals. Two post-translational isoforms (alpha and beta) have been previously identified whose increasingly phosphorylated forms migrate as a set of isoelectric variant spots (molecular mass 19 kDa; pI 6.2-5.6) on two-dimensional electrophoretic gels. In parallel with the phosphorylation of these forms of stathmin, two sets of three proteins migrating with slightly higher apparent molecular masses (21 and 23 kDa respectively) also incorporated radioactive phosphate in response to cell regulation through various transduction pathways. These phosphoproteins, previously referred to as proteins '16' and '17', share several biochemical properties with stathmin and are recognized by antibodies directed to stathmin or to stathmin peptides. Furthermore, when rat stathmin cDNA was transfected into mouse myogenic C2 cells, it directed the expression of protein sets 16 and 17 together with the 19 kDa forms of stathmin, as detected with a species-specific anti-stathmin antiserum. Proteins 16 and 17 are thus novel phosphorylated derivatives of stathmin, encoded by the same cDNA as its previously identified 19 kDa forms. These results increase the known complexity and diversity of stathmin patterns, which may yield the molecular support for its proposed role as a relay integrating various signals which regulate the proliferation, differentiation and functions of cells during development and adult life.

Animals↗

PubMed2002

Drosophila stathmin: a microtubule-destabilizing factor involved in nervous system formation.

Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins in Drosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin and stathmin family genes. They interfere with microtubule assembly in vitro, and in vivo when expressed in HeLa cells. Drosophila stathmin expression is regulated during embryogenesis: it is high in the migrating germ cells and in the central and peripheral nervous systems, a pattern resembling that of mammalian stathmin. Furthermore, RNA interference inactivation of Drosophila stathmin expression resulted in germ cell migration arrest at stage 14. It also induced important anomalies in nervous system development, such as loss of commissures and longitudinal connectives in the ventral cord, or abnormal chordotonal neuron organization. In conclusion, a single Drosophila gene encodes phosphoproteins homologous to the entire vertebrate stathmin family. We demonstrate for the first time their direct involvement in major biological processes such as development of the reproductive and nervous systems.

Amino Acid Sequence↗

PubMed1999

Differential, regional, and cellular expression of the stathmin family transcripts in the adult rat brain.

Stathmin is a ubiquitous cytosolic phosphoprotein, preferentially expressed in the nervous system, and previously described as a relay integrating diverse intracellular signaling pathways. Stathmin is the generic element of a mammalian protein family including SCG10, SCLIP, and RB3 with its splice variants RB3' and RB3". In contrast with stathmin, SCG10, SCLIP, and RB3/RB3'/RB3" are exclusively expressed in the nervous system, stathmin and SCG10 being mostly expressed during cell proliferation and differentiation, and SCLIP and RB3 rather in mature neural cells. To further understand their specific roles in the CNS, we compared the localization of the stathmin, SCG10, SCLIP, and RB3 transcripts in adult rat brain. Northern blot analysis as well as in situ hybridization experiments showed that all stathmin-related mRNAs are expressed in a wide range of adult rat brain areas. At a regional level, SCG10 and SCLIP appear generally distributed similarly except in a few areas. The pattern of expression of the RB3 transcript is very different from that of the three other members of the stathmin family. Furthermore, unlike SCG10 and SCLIP, which were detected only in neurons, but like stathmin, RB3 was detected in neurons and also in glial cells of the white matter. Altogether, our results suggest distinct roles for each member of the stathmin-related phosphoprotein family, in regard to their specific regional and cellular localization in the rat brain.

Animals↗