RESUMO
Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of "mitotic chromatin." Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.
Assuntos
Proteínas dos Microtúbulos , Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Células HeLa , Humanos , Interfase/fisiologia , Masculino , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Óvulo/metabolismo , Óvulo/ultraestrutura , Paclitaxel/farmacologia , Fosforilação , Isoformas de Proteínas , Serina/metabolismo , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Estatmina , Xenopus , Proteínas de XenopusRESUMO
We have investigated the effects of GnRH (LHRH) and of the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate on stathmin phosphorylation in the gonadotrope alphaT3-1 cell line. Stathmin expression and its phosphorylation were maximal during the exponential phase of cell growth. LHRH stimulated stathmin phosphorylation through a specific receptor in a dose- and time-dependent manner, and TPA induced a similar extensive stathmin phosphorylation. Their effects were inhibited either in PKC-depleted alphaT3-1 cells, or by the PKC inhibitor staurosporine. In the context of the known implication of PKC in LHRH-induced signal transduction, our results show that stathmin phosphorylation is involved in LHRH transduction, either as a result of direct activation of specific PKC isoforms or through a pathway involving kinases downstream to PKC activation.
Assuntos
Hormônio Liberador de Gonadotropina/farmacologia , Proteínas dos Microtúbulos , Fosfoproteínas/metabolismo , Hipófise/metabolismo , Transdução de Sinais , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Estatmina , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Stathmin is a 19 kDa cytoplasmic phosphoprotein proposed to act as a relay for signals activating diverse intracellular regulatory pathways. After two-thirds partial hepatectomy, the concentration of stathmin reached a peak between 48 and 72 hours, comparable to the levels observed in neonatal liver, at about 10 times the basal adult level. Stathmin then decreased to basal levels within 7 days, more rapidly than during postnatal tissue development (7 weeks), with no detectable change in its phosphorylation state. Interestingly, the mRNA for stathmin reached a peak much earlier than the protein, at 24 hours posthepatectomy, and decreased to a still detectable level until 96 hours after hepatectomy. Altogether, the present results further support the generatility of the implication of stathmin in regulatory pathways of cell proliferation and differentation during normal tissue development and posttraumatic regeneration.
Assuntos
Regeneração Hepática , Fígado/metabolismo , Proteínas dos Microtúbulos , Fosfoproteínas/biossíntese , Animais , Feminino , Hepatectomia , Fígado/crescimento & desenvolvimento , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , EstatminaRESUMO
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.
Assuntos
Sistema Nervoso Central/metabolismo , Proteínas dos Microtúbulos , Fosfoproteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Sistema Nervoso Central/anatomia & histologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , Ratos , Ratos Sprague-Dawley , Sistemas do Segundo Mensageiro/fisiologia , EstatminaRESUMO
Stathmin is a cytosolic protein expressed particularly in the developing nervous system, whose phosphorylation is correlated with the action of multiple extracellular stimuli regulating cell proliferation and differentiation. In this study, we used an antibody that specifically recognizes the carboxyterminal region of stathmin to analyze the distribution of this protein in the olfactory system of adult rats, and found a high and selective immunoreactivity in immature olfactory receptors of the olfactory neuroepithelium and in cells of the rostral migratory stream. These results reveal an expression of stathmin in regions of the adult nervous system characterized by striking structural plasticity and cell renewal, suggesting that this protein could play a role in the differentiation of newly generated cell populations.
Assuntos
Mapeamento Encefálico/métodos , Citosol/química , Proteínas dos Microtúbulos , Proteínas do Tecido Nervoso/análise , Condutos Olfatórios/química , Fosfoproteínas/análise , Animais , Imuno-Histoquímica , Bulbo Olfatório/química , Mucosa Olfatória/química , Prosencéfalo/química , Ratos , Ratos Wistar , EstatminaRESUMO
The purpose of the present study was to investigate whether the level of norepinephrine and its rate of disappearance after decarboxylase inhibition were modified in the spinal cord of a chronic pain model: the arthritic rat. Chromatographic studies allowing the simultaneous determination of norepinephrine and uric acid by means of HPLC with electrochemical detection are described. The norepinephrine and uric acid levels in the spinal cord were higher in arthritic rats than in normal rats. In addition the rate of disappearance of the amine was increased in the dorsal part of the cord in arthritic rats. These results agree with previous reports suggesting an activating effect of nociceptive stimuli on descending noradrenergic systems. They also indicate that studies on purine metabolism in the CNS in inflammatory and/or pain processes will be of interest.
Assuntos
Artrite Experimental/metabolismo , Artrite/metabolismo , Norepinefrina/metabolismo , Medula Espinal/metabolismo , Ácido Úrico/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Ratos , Ratos Endogâmicos , Medula Espinal/fisiopatologia , Fatores de TempoRESUMO
In a previous study, it was shown that, one month after kainic acid (KA) injection into the thalamus, afferents deprived of postsynaptic target neurons exhibit structural alteration, including the loss of synaptic vesicles. The present study was undertaken to determine whether these long-term morphological changes were associated with changes in biochemical markers of monoaminergic pathways. In situ injection of KA was performed into the right ventrobasal complex of the rat thalamus (VB). Protein content and total amount of norepinephrine, dopamine, 5-hydroxytryptamine and 5-hydroxyindolacetic acid were analyzed in the lesioned area one, two, three and four months after injection using high-performance liquid chromatography and electrochemical detection. The results were compared to those obtained in an equal volume of tissue dissected out from the opposite (intact) VB. Protein content per unit volume decreased progressively to 50% of control in the neuron-depleted area. In contrast, whatever the amine considered, its total amount remained unaltered up to 4 months after the excitotoxic lesion. 5-hydroxyindolacetic acid was also unchanged 4 months after lesion. This study suggests that (i) the quantity of monoamines in afferents to the rat VB does not depend upon the presence of postsynaptic target neurons, (ii) a non-vesicular storage compartment may compensate the loss of synaptic vesicles in afferent fibers to the lesioned area.
Assuntos
Aminas Biogênicas/fisiologia , Catecolaminas/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Neurônios/citologia , Serotonina/metabolismo , Tálamo/metabolismo , Vias Aferentes/metabolismo , Vias Aferentes/fisiologia , Animais , Contagem de Células , Cromatografia Líquida de Alta Pressão , Eletroquímica , Ácido Caínico , Tálamo/citologia , Tálamo/fisiologia , Fatores de TempoRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP70 , Proteínas dos Microtúbulos , Fosfoproteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Choque Térmico HSC70 , Humanos , EstatminaRESUMO
We investigated specific signaling events initiated after T cell triggering through the costimulatory surface receptors CD2 and CD28 as compared with activation via the Ag receptor (TCR/CD3). We therefore followed the phosphorylation of stathmin, a ubiquitous cytoplasmic phosphoprotein proposed as a general relay integrating diverse intracellular signaling pathways through the combinatorial phosphorylation of serines 16, 25, 38, and 63, the likely physiologic substrates for Ca2+/calmodulin (CaM)-dependent kinases, mitogen-activated protein (MAP) kinase, cyclin-dependent kinases (cdks), and protein kinase A, respectively. We addressed the specific protein kinase systems involved in the CD2 pathway of T cell activation through the analysis of stathmin phosphorylation patterns in exponentially growing Jurkat T cells, as revealed by phosphopeptide mapping. Stimulation via CD2 activated multiple signal transduction pathways, resulting in phosphorylation of distinct sites of stathmin, the combination of which only partially overlaps the CD3- and CD28-induced patterns. The partial redundancy of the three T cell activation pathways was evidenced by the phosphorylation of Ser25 and Ser38, substrates of MAP kinases and of the cdk family kinase(s), respectively. Conversely, the phosphorylation of Ser16 of stathmin was observed in response to both CD2 and CD28 triggering, but not CD3 triggering, with a kinetics compatible with the lasting activation of CaM kinase II in response to CD2 triggering. In vitro, Ser16 of recombinant human stathmin was phosphorylated also by purified CaM kinase II, and in vivo, CaM kinase II activity was indeed stimulated in CD2-triggered Jurkat cells. Altogether, our results favor an association of CaM kinase II activity with costimulatory signals of T lymphocyte activation and phosphorylation of stathmin on Ser16.
Assuntos
Antígenos CD2/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citosol/enzimologia , Proteínas dos Microtúbulos , Fosfoproteínas/metabolismo , Serina/metabolismo , Linfócitos T/enzimologia , Linfócitos T/imunologia , Sítios de Ligação/imunologia , Antígenos CD28/farmacologia , Complexo CD3/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Ativação Enzimática/imunologia , Humanos , Células Jurkat , Ativação Linfocitária , Fosforilação , EstatminaRESUMO
Traditional classification in the genus Capra is based mainly on horn morphology. However, previous investigations based on allozyme data are not consistent with this classification. We thus reexamined the evolutionary history of the genus by analyzing mitochondrial DNA (mtDNA) sequence variation. We collected bone samples from museums or dead animals found in the field. Thirty-four individuals were successfully sequenced for a portion of the mtDNA cytochrome b gene and control region (500 bp in total). We obtained a star-like phylogeny supporting a rapid radiation of the genus. In accordance with traditional classification, mtDNA data support the presence of two clades in the Caucasus and the hypothesis of a domestication event in the Fertile Crescent. However, in conflict with morphology, we found that C. aegagrus and C. ibex are polyphyletic species, and we propose a new scenario for Capra immigration into Europe.
Assuntos
DNA Mitocondrial/genética , Cabras/genética , Filogenia , Animais , Animais Domésticos , Ásia , Evolução Biológica , Grupo dos Citocromos b/genética , Europa (Continente) , Variação Genética , Genética Populacional , Cabras/classificação , Haplótipos , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNARESUMO
Mammary gland growth occurs essentially during pregnancy and induction of milk synthesis is triggered at parturition. Prolactin is mammogenic in vivo but only marginally in vitro. Prolactin induces milk synthesis in vivo and in cultured mammary cells. Prolactin is also strictly required for the multiplication of the rat lymphoid Nb2 cells. Stathmin is an ubiquitous and highly conserved phosphoprotein which seems to be involved in the intracellular mechanisms which trigger cell multiplication and differentiation. In the present study, the concentration of stathmin mRNA has been evaluated during the pregnancy-lactation-weaning cycle in mouse and rabbit. Stathmin mRNA appeared at its highest level during pregnancy and it was almost undetectable during lactation. Prolactin injected into mid-pregnant rabbits induced milk synthesis and this effect was not accompanied by any modification of stathmin mRNA concentration. In cultured primary rabbit mammary cells, prolactin induced casein gene expression without any alteration of stathmin mRNA concentration. In Nb2 cells, prolactin induced a progressive increase of stathmin mRNA concentration. This effect was not significant until after 4 h of prolactin action. These data suggest that stathmin is involved in mammary and Nb2 cell multiplication but may not be necessary for mammary cell differentiation.
Assuntos
Regulação da Expressão Gênica , Lactação/genética , Glândulas Mamárias Animais/metabolismo , Proteínas dos Microtúbulos , Fosfoproteínas/biossíntese , Prenhez/genética , Actinas/biossíntese , Actinas/genética , Animais , Caseínas/biossíntese , Caseínas/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Linfoma de Células T/patologia , Glândulas Mamárias Animais/efeitos dos fármacos , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/patologia , Fosfoproteínas/genética , Gravidez , Prolactina/farmacologia , RNA Mensageiro/análise , RNA Neoplásico/análise , Coelhos , Ratos , Estatmina , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
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.
Assuntos
Desenvolvimento Embrionário , Proteínas dos Microtúbulos , Oócitos/química , Fosfoproteínas/análise , Animais , Desenvolvimento Embrionário e Fetal , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Oócitos/ultraestrutura , Fosfoproteínas/genética , Gravidez , Estatmina , Frações SubcelularesRESUMO
Protein phosphorylation is involved at multiple steps of RNA processing and in the regulation of protein expression. We present here the first identification of a serine/threonine kinase that possesses an RNP-type RNA recognition motif: KIS. We originally isolated KIS in a two-hybrid screen through its interaction with stathmin, a small phosphoprotein proposed to play a general role in the relay and integration of diverse intracellular signaling pathways. Determination of the primary sequence of KIS shows that it is formed by the juxtaposition of a kinase core with little homology to known kinases and a C-terminal domain that contains a characteristic RNA recognition motif with an intriguing homology to the C-terminal motif of the splicing factor U2AF. KIS produced in bacteria has an autophosphorylating activity and phosphorylates stathmin on serine residues. It also phosphorylates in vitro other classical substrates such as myelin basic protein and synapsin but not histones that inhibit its autophosphorylating activity. Immunofluorescence and biochemical analyses indicate that KIS overexpressed in HEK293 fibroblastic cells is partly targetted to the nucleus. Altogether, these results suggest the implication of KIS in the control of trafficking and/or splicing of RNAs probably through phosphorylation of associated factors.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Compartimento Celular , Núcleo Celular/enzimologia , Clonagem Molecular , Citoplasma/enzimologia , Embrião de Mamíferos/química , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Proteínas Serina-Treonina Quinases/genética , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Ratos , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Distribuição TecidualRESUMO
We present here a first appraisal of the phosphorylation site specificity of KIS (for 'kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks.
Assuntos
Prolina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA/metabolismo , Serina/metabolismo , Animais , Sítios de Ligação , Bovinos , Cromatografia Líquida de Alta Pressão , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas , Proteína Básica da Mielina/metabolismo , Fosforilação , Ratos , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Sinapsinas/metabolismo , Treonina/metabolismo , Fatores de TempoRESUMO
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).
Assuntos
Proteínas dos Microtúbulos , Microtúbulos/química , Microtúbulos/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Anticorpos , Proteínas de Transporte , Ciclo Celular/fisiologia , Epitopos , Imunofluorescência , Células HeLa , Humanos , Proteínas de Membrana , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/metabolismo , Fosfoproteínas/imunologia , Fosforilação , EstatminaRESUMO
Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.
Assuntos
Reação em Cadeia da Polimerase/métodos , Alelos , Animais , Sequência de Bases , Simulação por Computador , Fezes/química , Genótipo , Guias como Assunto , Modelos Teóricos , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Ursidae/genéticaRESUMO
Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation and activities. It interacts with several putative downstream target and/or partner proteins. One major action of stathmin is to interfere with microtubule dynamics, by inhibiting the formation of microtubules and/or favoring their depolymerization. Stathmin (S) interacts directly with soluble tubulin (T), which results in the formation of a T2S complex which sequesters free tubulin and therefore impedes microtubule formation. However, it has been also proposed that stathmin's action on microtubules might result from the direct promotion of catastrophes, which is still controversial. Phosphorylation of stathmin regulates its biological actions: it reduces its affinity for tubulin and hence its action on microtubule dynamics, which allows for example progression of cells through mitosis. Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP and RB3/RB3'/RB3". Interestingly, the stathmin-like domains of these proteins also possess a tubulin binding activity in vitro. In vivo, the transient expression of neural phosphoproteins of the stathmin family leads to their localization at Golgi membranes and, as previously described for stathmin and SCG10, to the depolymerization of interphasic microtubules. Altogether, the same mechanism for microtubule destabilization, that implies tubulin sequestration, is a common feature likely involved in the specific biological roles of each member of the stathmin family.