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PubMed2004

Analysis of the regulation of microtubule dynamics by interaction of RGSZ1 (RGS20) with the neuronal stathmin, SCG10.

Regulators of G protein signaling (RGS proteins) are a diverse family of proteins that act to negatively regulate signaling by heterotrimeric G proteins; however, recent data have implied additional functions for RGS proteins. Previously, we employed the yeast two-hybrid system and identified the microtubule-destabilizing protein, superior cervical ganglia neural-specific 10 protein (SCG10), as a potential effector protein of RGSZ1. This article describes the expression and biochemical purification of both RGSZ1 and SCG10 and details the development of various in vitro assays to evaluate microtubule polymerization?depolymerization. Both turbidimetric and microscopy-based assays can be employed to study the impact that RGS proteins have on SCG10 function. The application of these in vitro assays may help identify a novel role for RGS proteins in regulating the cytoskeletal network.

Animals↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed1997

The stathmin/tubulin interaction in vitro.

Stathmin is a highly conserved ubiquitous cytoplasmic protein, phosphorylated in response to extracellular signals and during the cell cycle. Stathmin has recently been shown to destabilize microtubules, but the molecular mechanisms of this function remained unclear. We show here that stathmin directly interacts with tubulin. We assessed the conditions of this interaction and determined some its quantitative parameters using plasmon resonance, gel filtration chromatography, and analytical ultracentrifugation. The stathmin/tubulin interaction leads to the formation of a 7.7 S complex with a 60-A Stokes radius, associating one stathmin with two tubulin heterodimer molecules as determined by direct quantification by Western blotting. This interaction is sensitive to pH and ionic environment. Its equilibrium dissociation constant, determined by plasmon resonance measurement of kinetic constants, has an optimum value of 0.5 microM at pH 6.5. The affinity was lowered with a fully "pseudophosphorylated" 4-Glu mutant form of stathmin, suggesting that it is modulated in vivo by stathmin phosphorylation. Finally, analysis of microtubule dynamics by video microscopy shows that, in our conditions, stathmin reduces the growth rate of microtubules with no effect on the catastrophe frequency. Overall, our results suggest that the stathmin destabilizing activity on microtubules is related to tubulin sequestration by stathmin.

Animals↗

PubMed2001

Differential effect of two stathmin/Op18 phosphorylation mutants on Xenopus embryo development.

Stathmin/Op18 destabilizes microtubules in vitro and regulates microtubule polymerization in vivo. Both a microtubule catastrophe-promoting activity and a tubulin sequestering activity were demonstrated for stathmin in vitro, and both could contribute to microtubule depolymerization in vivo. Stathmin activity can be turned down by extensive phosphorylation on its four phosphorylatable serines, and down-regulation of stathmin activity by phosphorylation is necessary for cells to proceed through mitosis. We show here that microinjection of a nonphosphorylatable Ser to Ala (4A) quadruple mutant in Xenopus two-cell stage embryos results in cell cleavage arrest in the injected blastomeres and aborted development, whereas injection of a pseudo-phosphorylated Ser to Glu quadruple mutant (4E) does not prevent normal development. Addition of these mutants to mitotic cytostatic factor-arrested extracts in which spindle assembly was induced led to a dramatic reduction of spindle size with 4A stathmin, and to a moderate increase with 4E stathmin, but both localized to spindle poles. Interestingly, the microtubule assembly-dependent phosphorylation of endogenous stathmin was abolished in the presence of 4A stathmin, but not of 4E stathmin. Altogether, this shows that the phosphorylation-mediated regulation of stathmin activity during the cell cycle is essential for early Xenopus embryonic development.

Animals↗

PubMed1993

Molecular diversity of the SCG10/stathmin gene family in the mouse.

SCG10 is a neuronal growth-associated protein that shares an amino acid sequence similarity with an 18- to 19-kDa phosphoprotein named stathmin (also called p19, p18, Op18, pp17, prosolin, pp20, 19K, and leukemia-associated phosphoprotein, Lap18), which is more broadly expressed in a variety of cell types of the neural, immune, and reproductive systems. The sequence similarity has suggested that SCG10 and stathmin have been derived from structurally and evolutionarily related genes. To explore the structural and evolutionary relationships between these genes, we have isolated a series of cosmid and phage clones that covers the entire region of the mouse stathmin gene and most of the mouse SCG10 gene. The SCG10 transcription unit spans at least 30 kb, while the stathmin gene is 6 kb in length. Both genes consist of five exons, and many of the intron/exon boundaries fall into the homologous regions of conserved domains of these two proteins. However, the promoter-proximal regions are distinct in the two genes, suggesting that they have evolved by fusion of the duplicated coding exons to unique promoters. Southern blot analysis indicates that SCG10 mRNA is encoded by a single gene in the mouse genome, while stathmin cDNA probes detect multiple genes. Chromosome mapping experiments reveal that the SCG10 gene is localized at the proximal region of mouse chromosome 3 and is linked to Il-7, while the stathmin gene loci are distributed to three chromosomes; the authentic stathmin gene lies on chromosome 4, whereas the loci on chromosomes 9 and 17 are likely to be pseudogenes. These data are consistent with the idea that the neuron-specific SCG10 gene evolved by duplication and modification of the more broadly expressed stathmin/Lap18 gene.

Amino Acid Sequence↗

PubMed2005

Distinct and sequential upregulation of genes regulating cell growth and cell cycle progression during hepatic ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) in liver and other organs is manifested as an injury phase followed by recovery and resolution. Control of cell growth and proliferation is essential for recovery from the injury. We examined the expression of three related regulators of cell cycle progression in liver IRI: spermidine/spermine N-acetyltransferase (SSAT), p21 (a cyclin-dependent kinase inhibitor), and stathmin. Mice were subjected to hepatic IRI, and liver tissues were harvested at timed intervals. The expression of SSAT, the rate-limiting enzyme in the polyamine catabolic pathway, had increased fivefold 6 h after IRI and correlated with increased putrescine levels in the liver, consistent with increased SSAT enzymatic activity in IRI. The expression of p21, which is transactivated by p53, was undetectable in sham-operated animals but was heavily induced at 12 and 24 h of reperfusion and declined to undetectable baseline levels at 72 h of reperfusion. The interaction of the polyamine pathway with the p53-p21 pathway was shown in vitro, where activation of SSAT with polyamine analog or the addition of putrescine to cultured hepatocytes induced the expression of p53 and p21 and decreased cell viability. The expression of stathmin, which is under negative transcriptional regulation by p21 and controls cell proliferation and progression through mitosis, remained undetectable at 6, 12, and 24 h of reperfusion and was progressively and heavily induced at 48 and 72 h of reperfusion. Double-immunofluorescence labeling with antibodies against stathmin and PCNA, a marker of cell proliferation, demonstrated colocalization of stathmin and PCNA at 48 and 72 h of reperfusion in hepatocytes, indicating the initiation of cell proliferation. The distinct and sequential upregulation of SSAT, p21, and stathmin, along with biochemical activation of the polyamine catabolic pathway in IRI in vivo and the demonstration of p53-p21 upregulation by SSAT and putrescine in vitro, points to the important role of regulators of cell growth and cell cycle progression in the pathophysiology and/or recovery in liver IRI. The data further suggest that SSAT may play a role in the initiation of injury, whereas p21 and stathmin may be involved in the resolution and recovery after liver IRI.

Acetyltransferases↗

PubMed2002

Targeting of tumor associated antigens in renal cell carcinoma using proteome-based analysis and their clinical significance.

The suitability of proteome-based strategies for the targeting of tumor-associated markers along with further analysis regarding their clinical significance were investigated in human renal cell carcinoma (RCC). The immunogenic protein expression profile of normal kidney and RCC cell lines was studied by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, also termed PROTEOMEX. Employing this approach, a series of proteins reactive with either RCC patient sera and/or reactive with control sera were identified by microanalysis of tryptic peptides. Some of these candidate antigens represent members of the cytoskeletal family, such as cytokeratins, in particular cytokeratin 8, cytoskeletal tropomyosin, F-actin capping protein, gamma-actin, stathmin, tubulin-alpha, tubulin-beta and vimentin. The expression pattern and clinical significance of three of these antigens, namely cytokeratin 8, stathmin and vimentin, were further analyzed in a large series of surgically removed RCC lesions of distinct subtypes. A heterogeneous expression pattern of cytokeratin 8, stathmin and vimentin was demonstrated in the different RCC subtypes. All epithelial cells of the autologous normal kidney showed a strong cytokeratin 8 staining pattern, whereas they totally lack vimentin expression. Stathmin was expressed in 10% of tubule cells. In conclusion, PROTEOMEX could be employed for the identification of tumor-associated antigens of the cytoskeleton which are differentially expressed in RCC of distinct subtypes as well as in normal renal epithelium.

Antigens, Neoplasm↗

PubMed2005

Selective upregulation of RB3/stathmin4 by ciliary neurotrophic factor following optic nerve axotomy.

In this study, we examined the cellular responses of stathmin-related proteins in the rat retina following optic nerve (ON) axotomy. To examine the distribution of stathmin-related gene products, we performed semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemical analyses. Retrograde labeling using a fluorescein tracer, fluorogold (FG), was used for the identification of retinal ganglion cells (RGCs). RT-PCR and ISH analyses indicated that the expression of RB3 was specifically increased in the ganglion cell layer (GCL) comparing to other members of stathmin-related gene family examined 3 days following the ON axotomy. When brain-derived neurotrophic factor was administrated intravitreously, the induction of RB3 mRNA sustained up to 7 days after axotomy, although the peak induction level was unchanged. In contrast, ciliary neurotrophic factor (CNTF) administration increased the peak level of RB3 mRNA induction significantly at 3 days after axotomy. Immunohistochemistry in combination with the retrograde labeling of axotomized cells by FG revealed that RB3 was increased following axotomy in FG-labeled RGCs. These data suggest that RB3 is the unique response protein in the stathmin-related proteins following ON axotomy and the induced RB3 may play a critical role in the CNTF-induced response on the axotomized RGCs, e.g. axonal regeneration and/or neuroprotection.

Animals↗

PubMed2002

SCG10-related neuronal growth-associated proteins in neural development, plasticity, degeneration, and aging.

Neuronal growth-associated proteins (nGAPs) are in general neuron-specific gene products whose expression correlates tightly with neuronal process outgrowth and/or regeneration, and are mostly good downstream targets of neurotrophin stimulation. Expression of genes encoding nGAPs such as GAP-43, SCG10, and stathmin is upregulated following lesioning of cortical and hippocampal regions of the adult rat brain. In the brains of aged animals, however, the magnitude of the response is reduced, whereas the time course of the response is mostly unchanged when compared with that for brains of young ones. Expression of GAP-43 and stathmin is reduced by aging, and is also changed in age-related neurodegenerative conditions such as Alzheimer's disease in humans. Certain nGAPs are induced during long-term potentiation (LTP) and also during critical periods of song-learning and ocular dominance column formation in birds and cats, respectively. Recent evidence further supports the idea that functional synaptic modulation is often associated with remodeling of synaptic structures. These results suggest that neurotrophin-responsive nGAPs serve as molecular markers of neuronal plasticity during development and aging, and that the neuronal plasticity decreases, at least in certain neuronal circuits, in the aged brain and neurodegenerative diseases. Recent findings on the roles of stathmin and SCG10-related proteins in microtubule destabilization and its functional block by phosphorylation further support the importance of the SCG10 family proteins in neuronal cytoskeletal regulation, particularly as to microtubule dynamics. We summarize here a decade of research on SCG10 and its related molecules with special interests to brain aging and disease.

Aging↗

PubMedDate not supplied

SCG10, a neuron-specific growth-associated protein in Alzheimer's disease.

Neuronal growth-associated proteins (nGAPs) are markers of neuronal process outgrowth and are associated with both degenerative and sprouting responses in Alzheimer's disease (AD) brain. To study possible involvement of SCG10, an nGAP, in AD, we cloned human SCG10 cDNA and analyzed SCG-10 at mRNA and protein levels in control and AD brains. The deduced amino acid sequence of human SCG10 was 69% identical to stathmin, another nGAP. By in situ hybridization, both SCG10 and stathmin mRNAs were detected in selected neuronal populations in aged human brains. Quantitative analysis by RNase protection revealed that levels of neither SCG10 nor stathmin mRNAs were significantly altered in AD. Using an SCG10-specific antibody, Western blot analysis did not reveal any quantitative changes of SCG10 in AD. However, when the concentration of SCG10 protein was plotted against the number of tangles, a positive correlation was found. SCG10 levels did not correlate with plaque numbers. Furthermore, immunohistochemical study revealed that neuronal SCG10 protein accumulated in the cell bodies in AD-affected regions. Thus, SCG10 compartmentalization and metabolism may be altered in AD possibly due to mechanisms related to tangle formation in this disease.

Alzheimer Disease↗

PubMed2006

The "SCG10-LIke Protein" SCLIP is a novel regulator of axonal branching in hippocampal neurons, unlike SCG10.

Stathmin family phosphoproteins participate in the control of microtubule dynamics and have been proposed to be involved in the control of neuronal differentiation. We examined the biological properties and functions of SCLIP, the stathmin family protein most similar to SCG10, a widely studied and recognized neuronal differentiation marker. SCLIP, like SCG10, is present from the earliest stages of hippocampal neuron differentiation in culture at vesicle-like structures following dynamic microtubules. Its inhibition by RNA interference resulted in increased axonal branching, revealing a novel biological role for SCLIP, distinct from SCG10 whose down-regulation in the same conditions promoted growth cone expansion. The enhanced axonal branching resulted from the formation of collateral lamellar protrusions, with cytoskeleton reorganization typical of normal branching. In addition to revealing a novel function for SCLIP in axonal morphogenesis, our results demonstrate for the first time that stathmin family proteins fulfill different and complementary roles during neuronal differentiation.

Animals↗

PubMed2000

Cloning and expression of a gene encoding Sm16, an anti-inflammatory protein from Schistosoma mansoni.

The gene encoding Sm16, an anti-inflammatory, immunomodulatory protein present abundantly in secretions of the infective stages of Schistosoma mansoni was cloned and partially characterized. A data base analysis showed sequence homology to an earlier reported schistosomular stathmin-like gene sequences reported in dbEST and Genbank. The putative gene coding for Sm16 is of 500 bp with an open reading frame of 117 aa that included an N-terminal signal peptide sequence of 18 aa. There are three potential sites for phosphorylation (two serine and one tyrosine residue) but no glycosylation sites in the sequence. The coding region of Sm16 was amplified from cercarial cDNA, cloned and expressed in bacterial and insect expression systems. The purified recombinant protein showed strong immunoreactivity with a polyclonal rabbit anti-Sm16 antibody raised against the native anti-inflammatory protein Sm16. Contrary to earlier report, this gene appears to be not stage-specific. Metabolic labeling studies suggested that Sm16 is phosphorylated and is synthesized by both cercariae and schistosomula of S. mansoni. Sequence homology with human stathmin, a cell cycle regulatory phospho protein, was 30%. However, when probed with specific antibodies, no cross reactivity was observed between Sm16 and human stathmin.

Amino Acid Sequence↗

PubMed2002

Increased expression of mRNAs for microtubule disassembly molecules during nerve regeneration.

The mRNA expression of the microtubule disassembly molecules (SCG10, stathmin, SCLIP and RB3) in response to nerve injury was examined using a rat hypoglossal nerve injury model. After nerve injury prominent increase in mRNA expression of SCG10, stathmin and RB3 was observed, while only slight increase in SCLIP mRNA was observed in injured motor neurons. The increase in SCG10 and RB3 mRNA expression was quicker than that of stathmin and SCLIP. All the elevated signals decreased gradually to control levels by 4 weeks after nerve injury.

Animals↗

PubMed2003

Transcripts in pretreatment biopsies from a three-arm randomized trial in metastatic non-small-cell lung cancer.

Non-small-cell lung cancer patients with locally advanced or metastatic disease at the time of diagnosis show marginal response to chemotherapy in terms of tumor shrinkage, time to progression and median survival. The identification and implementation of predictive genetic markers of response-specific cytotoxic drugs is a priority of current research and future trials. In this study, we have used quantitative PCR to analyse expression of beta-tubulin III, stathmin, RRM1, COX-2 and GSTP1 in mRNA isolated from paraffin-embedded tumor biopsies of 75 nonsmall-cell lung cancer patients treated as part of a large randomized trial. In total, 22 patients were treated with gemcitabine/cisplatin, 25 with vinorelbine/cisplatin and 28 with paclitaxel/carboplatin. There were no differences in clinical characteristics and transcript levels in the pretreatment biopsies according to treatment arm. Patients with low beta-tubulin III levels had better response in the paclitaxel/carboplatin arm (P=0.05), and those with low RRM1 levels showed a tendency to better response in the gemcitabine/cisplatin arm. Time to progression was influenced by beta-tubulin III (P=0.03) and stathmin (P=0.05) levels in the vinorelbine/cisplatin arm, and there was a tendency toward correlation between beta-tubulin III levels and time to progression in the paclitaxel/carboplatin arm. RRM1 levels influenced time to progression (P=0.05) and even more so, survival (P=0.0028) in the gemcitabine/cisplatin arm. The predictive value of beta-tubulin III, stathmin and RRM1 should be tested in prospective customized chemotherapy trials, the results of which will help tailor chemotherapy to improve patient survival.

Adult↗

PubMed1999

OP18/stathmin binds near the C-terminus of tubulin and facilitates GTP binding.

It is has been previously suggested that the protein Op18/stathmin may interact with tubulin via the alpha-tubulin subunit [Larsson, N., Marklund, U., Melander Gradin, H., Brattsand, G. & Gullberg, M. (1997) Mol. Cell. Biol. 17, 5530-5539]. In this study we have used limited proteolysis and cross-linking analysis to localize further the stathmin-binding site on alpha-tubulin. Our results indicate that such a binding site is in a region close to the C-terminus of the molecule comprising residues 307 to the subtilisin-cleavage site on the alpha-tubulin subunit. Based on a recent model of the structure of tubulin [Nogales, E., Wolf, S.G. & Dowing, D.H. (1998) Nature (London) 391, 199-203], we found that this region contained the same areas that may be involved in longitudinal contacts of alpha-tubulin subunits within the microtubule. We also observed that the binding of stathmin to tubulin can modulate the binding of GTP to tubulin, as a consequence of a conformational change in the beta-tubulin subunit that occurs upon interaction of stathmin with tubulin.

Adenosine Triphosphate↗

PubMed2006

Overexpression of peptidyl-prolyl isomerase-like 1 is associated with the growth of colon cancer cells.

PURPOSE AND EXPERIMENTAL DESIGN: To discover novel therapeutic targets for colon cancers, we previously surveyed expression patterns among 23,000 genes in colon cancer tissues using a cDNA microarray. Among the genes that were up-regulated in the tumors, we selected for this study peptidyl-prolyl isomerase-like 1 (PPIL1) encoding PPIL1, a cyclophilin-related protein. RESULTS: Western blot analysis and immunohistochemical staining using PPIL1-specific antibody showed that PPIL1 protein was frequently overexpressed in colon cancer cells compared with noncancerous epithelial cells of the colon mucosa. Colony formation assay showed a growth-promoting effect of wild-type PPIL1 on NIH3T3 and HEK293 cells. Consistently, transfection of short-interfering RNA specific to PPIL1 into SNUC4 and SNUC5 cells effectively reduced expression of the gene and retarded growth of the colon cancer cells. We further identified two PPIL1-interacting proteins, SNW1/SKIP (SKI-binding protein) and stathmin. SNW1/SKIP is involved in the regulation of transcription and mRNA splicing, whereas stathmin is involved in stabilization of microtubules. Therefore, elevated expression of PPIL1 may play an important role in proliferation of cancer cells through the control of SNW1/SKIP and/or stathmin. CONCLUSION: The findings reported here may offer new insight into colonic carcinogenesis and contribute to the development of new molecular strategies for treatment of human colorectal tumors.

Animals↗

PubMed2006

Gene expression profiling in the mammary gland of rats treated with 7,12-dimethylbenz[a]anthracene.

Identification of molecular markers of early-stage breast cancer development is important for the diagnosis and prevention of the disease. In the present study, we used microarray analysis to examine the differential expression of genes in the rat mammary gland soon after treatment with a known chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), and prior to tumor development. Six weeks after DMBA, differential expression of multiple genes involved in cell growth, differentiation and microtubule dynamics were observed. Gene expression changes were further validated by a combination of techniques, including real-time PCR, RT-PCR, Western blotting and immunohistochemistry. An inhibition of differentiation in this early stage was suggested by the lower expression of beta-casein and transferrin and higher expression of hsp27 in glands from DMBA-treated rats. Possible cell cycle deregulation was indicated by an increased expression of cyclin D1 and hsp86, a heat shock protein associated with cyclin D1. Prior to tumor development, DMBA increased cellular proliferation as detected by Ki-67 and stathmin immunostaining in histologically normal mammary gland. Genes regulating microtubule function, including stathmin, Ran, alpha-tubulin and hsp27, were all overexpressed in the mammary gland of DMBA-treated rats, raising the possibility that disruption of microtubule dynamics and abnormal mitosis may be critical events preceding breast cancer development. Several of the altered proteins, including hsp27, hsp86 and stathmin, may ultimately serve as markers of early breast cancer development.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2005

Role of Ser50 phosphorylation in SCG10 regulation of microtubule depolymerization.

Members of the stathmin-like protein family depolymerize microtubules (MTs), probably due to the ability of each stathmin monomer to bind two tubulin heterodimers in a complex (T(2)S complex). SCG10, a member of this family, is localized in the growth cone of neurons. It has four identified sites of serine phosphorylation (S50, S63, S73, and S97). Of these, S50 and S97 are phosphorylated by cAMP-dependent protein kinase, an enzyme involved in growth cone guidance. When the equivalent sites in stathmins are phosphorylated, they lose their ability to depolymerize MTs. We investigated the specific role of the two cAMP-dependent protein kinase (PKA) phosphorylation sites in SCG10. A mutant of SCG10 phosphorylated only on S50 retained the ability to depolymerize MTs, but SCG10 phosphorylated on S97 or on both S50 and S97 lost MT-depolymerizing activity. Surface plasmon resonance studies revealed that the phosphorylation of SCG10 at these sites reduced the tubulin heterodimer binding, mainly due to a reduced rate of association. In particular, compared to the two other phosphorylated forms, SCG10 phosphorylated at S50 had a significantly smaller dissociation constant for the binding of the first tubulin heterodimer and larger association and dissociation rate constants for the binding of the second heterodimer. This indicates that the phosphorylation of S50 compensates for the effect of phosphorylation at other sites by modulating T2S complex formation. Furthermore, these results suggest that S50-P maintains MT-depolymerizing activity, which indicates that the biological functions of phosphorylation at S50 and S97 are different.

Animals↗