RESUMO
Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose , Treonina/metabolismo , Motivos de Aminoácidos/genética , Animais , Aurora Quinase A/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina A2/genética , Ciclina A2/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Ativação Enzimática , Feminino , Humanos , Oócitos/metabolismo , Fosforilação , Prolina/genética , Prolina/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Serina/genética , Serina/metabolismo , Treonina/genética , Xenopus laevis , Quinase 1 Polo-LikeRESUMO
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.
Assuntos
Desenvolvimento Embrionário , Proteínas dos Microtúbulos , Mutação , Fosfoproteínas/metabolismo , Animais , Embrião não Mamífero/metabolismo , Humanos , Microscopia de Fluorescência , Fosfoproteínas/genética , Fosforilação , Estatmina , Xenopus/embriologia , Proteínas de XenopusRESUMO
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
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
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.
Assuntos
Proteínas dos Microtúbulos , Fosfoproteínas/fisiologia , Tubulina (Proteína)/metabolismo , Animais , Sítios de Ligação , Proteínas de Transporte , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Humanos , Proteínas de Membrana , Microtúbulos/metabolismo , Modelos Biológicos , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/fisiologia , Neurônios/fisiologia , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , EstatminaRESUMO
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
Stathmin is a ubiquitous cytoplasmic protein whose phosphorylation state changes markedly in response to extracellular signals, and during the cell cycle. To clarify the function of stathmin, its four phosphorylation sites were mutated to either alanines (4A-stathmin) or glutamates (4E-stathmin). In transfected cells, 4A-stathmin caused a strong G2/M block and also inhibited the responsiveness of a co-transfected fos promoter/ luciferase reporter plasmid to serum stimulation, whereas wild type and 4E-stathmin had relatively minor effects. These results support the idea that stathmin plays a role in multiple cellular processes and indicate that the regulation of the phosphorylation state of stathmin is likely to determine its action.
Assuntos
Ciclo Celular/fisiologia , Proteínas dos Microtúbulos , Fosfoproteínas/fisiologia , Transdução de Sinais , Alanina , Substituição de Aminoácidos , Divisão Celular , Linhagem Celular , Citometria de Fluxo , Genes Reporter , Ácido Glutâmico , Humanos , Rim , Cinética , Luciferases/biossíntese , Mutagênese Sítio-Dirigida , Fosfoproteínas/biossíntese , Fosforilação , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Estatmina , TransfecçãoRESUMO
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.
Assuntos
Proteínas dos Microtúbulos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Conformação Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Animais , Técnicas Biossensoriais , Encéfalo/metabolismo , Bovinos , Cromatografia em Gel , Concentração de Íons de Hidrogênio , Cinética , Mutagênese Sítio-Dirigida , Fosfoproteínas/isolamento & purificação , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina , Estatmina , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
Meiotic and mitotic spindles are required for the even segregation of duplicated chromosomes to the two daughter cells. The mechanism of spindle assembly is not fully understood, but two have been proposed that are not mutually exclusive. The 'search and capture' model suggests that dynamic microtubules become progressively captured and stabilized by the kinetochores on chromosomes, leading to spindle assembly. The 'local stabilization' model proposes that chromosomes change the state of the cytoplasm around them, making it more favourable to microtubule polymerization. It has been shown that Stathmin/Op18 inhibits microtubule polymerization in vitro by interaction with tubulin, and that overexpression in tissue culture cells of non-phosphorylatable mutants of Stathmin/Op18 prevents the assembly of mitotic spindles. We have used Xenopus egg extracts and magnetic chromatin beads to show that mitotic chromatin induces phosphorylation of Stathmin/Op18. We have also shown that Stathmin/Op18 is one of the factors regulated by mitotic chromatin that governs preferential microtubule growth around chromosomes during spindle assembly.
Assuntos
Cromatina/fisiologia , Proteínas dos Microtúbulos , Mitose , Fosfoproteínas/metabolismo , Animais , Sítios de Ligação , Proteína Quinase CDC2/metabolismo , Clonagem Molecular , Mutagênese Sítio-Dirigida , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/genética , Fosforilação , Fuso Acromático/metabolismo , Estatmina , Xenopus , Proteínas de XenopusRESUMO
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.