Stathmin/Op18 destabilizes microtubules in vitro and regulates microtubule polymerization in vivo... more Stathmin/Op18 destabilizes microtubules in vitro and regulates microtubule polymerization in vivo. Both a microtubule catastrophe-promoting activity and a tubu-lin sequestering activity were demonstrated for stath-min 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 stath-min activity by phosphorylation is necessary for cells to proceed through mitosis. We show here that microinjec-tion 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-phosphory-lated Ser to Glu quadruple mutant (4E) does not prevent normal development. Addition of these mutants to mi-totic 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 phosphoryla-tion-mediated regulation of stathmin activity during the cell cycle is essential for early Xenopus embryonic development. Stathmin/Op18 is a soluble, ubiquitous phosphoprotein originally proposed to act as a relay for diverse intracellular sig-naling pathway (1). Stathmin was subsequently isolated as a microtubule (MT) 1 catastrophe-promoting factor that was shown to interact directly with free tubulin (2). Further work proved that stathmin causes MT destabilization in vitro and in vivo and that its MT-destabilizing activity can be turned off by phosphorylation on its four phosphorylatable sites (3–7). Stathmin becomes extensively phosphorylated at mitosis (6, 8 –10) on its four phosphorylatable serines. These sites are targets both in vitro and in vivo of multiple kinases (for a review, see Ref. 11): Ca 2 /calmodulin-dependent kinases II and IV/Gr phosphorylate Ser 16 , mitogen-activated protein kinases phosphorylate Ser 25 and Ser38, cyclin-dependent kinases phos-phorylate Ser 25 and Ser 38 , and cAMP-dependent protein kinase phosphorylates Ser 16 and Ser 63. Overexpression of phosphoryl-ation target site-deficient mutants in mammalian somatic cells revealed that whereas wild-type phosphorylatable stathmin allows spindle formation at mitosis, a stathmin mutant in which the four phosphorylatable serines have been replaced by alanines (4A mutant) prevents formation of a normal spindle and results in a mitotic block of the cells (6, 12, 13). As the 4A mutant mimics the constitutively active form of stathmin, this shows that down-regulation of stathmin MT-destabilizing activity is likely required to allow formation of the mitotic spindle and progression through mitosis. However, it has been recently reported that a 4E pseudo-phosphorylated mutant, in which the four phosphorylation serines have been replaced by glu-tamic acid to mimic the constitutively down-regulated form, shows only a limited decrease in tubulin complex formation in vitro and in MT-destabilizing activity in transfected somatic cells (14, 15). To investigate the in vivo importance of the phosphorylation state of stathmin in an embryonic cell system, we used wild-type human stathmin and the two 4A and 4E mutants. We introduced these recombinants stathmin proteins in two-cell stage embryos and in mitotic egg extracts from Xenopus laevis to examine their effects on embryo development and on the MT network. We then performed immunofluorescence experiments to detect the specific localization of the added forms in the mitotic egg extracts and extended these observations to so-matic tissue culture cells. MATERIALS AND METHODS Preparation of Extracts and Human Stathmin Expression—Low speed (15,000 g) Xenopus egg extracts blocked in metaphase II of meiosis (CSF extracts) and permeabilized sperm heads were prepared as described previously (16). Spindles were assembled at 22 °C in the interphase to mitosis pathway as described previously (17) by adding 0.2 mM CaCl 2 for 1 h, then adding an equal volume of fresh CSF extract kept on ice to drive the extract back into mitosis. High speed extracts were prepared from CSF extracts spun at 245,000 g for 20 min at 4 °C in a TLS 55 rotor (Beckman) and were complemented with 0.05 volume of Energy Mix (150 mM creatine phosphate, 20 mM ATP, 20 mM MgCl 2). Recombinant human wild-type 4A (serine changed to alanine at positions 16, 25, 38, and 63) and 4E (serines substituted with glutamic acid) stathmin were in PBS at 10 mg/ml (18). Briefly, stathmin-expressing bacteria were sonicated, and the extracts were centrifuged at 4 °C for 5
Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both... more 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.
Stathmin/Op18 destabilizes microtubules in vitro and regulates microtubule polymerization in vivo... more Stathmin/Op18 destabilizes microtubules in vitro and regulates microtubule polymerization in vivo. Both a microtubule catastrophe-promoting activity and a tubu-lin sequestering activity were demonstrated for stath-min 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 stath-min activity by phosphorylation is necessary for cells to proceed through mitosis. We show here that microinjec-tion 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-phosphory-lated Ser to Glu quadruple mutant (4E) does not prevent normal development. Addition of these mutants to mi-totic 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 phosphoryla-tion-mediated regulation of stathmin activity during the cell cycle is essential for early Xenopus embryonic development. Stathmin/Op18 is a soluble, ubiquitous phosphoprotein originally proposed to act as a relay for diverse intracellular sig-naling pathway (1). Stathmin was subsequently isolated as a microtubule (MT) 1 catastrophe-promoting factor that was shown to interact directly with free tubulin (2). Further work proved that stathmin causes MT destabilization in vitro and in vivo and that its MT-destabilizing activity can be turned off by phosphorylation on its four phosphorylatable sites (3–7). Stathmin becomes extensively phosphorylated at mitosis (6, 8 –10) on its four phosphorylatable serines. These sites are targets both in vitro and in vivo of multiple kinases (for a review, see Ref. 11): Ca 2 /calmodulin-dependent kinases II and IV/Gr phosphorylate Ser 16 , mitogen-activated protein kinases phosphorylate Ser 25 and Ser38, cyclin-dependent kinases phos-phorylate Ser 25 and Ser 38 , and cAMP-dependent protein kinase phosphorylates Ser 16 and Ser 63. Overexpression of phosphoryl-ation target site-deficient mutants in mammalian somatic cells revealed that whereas wild-type phosphorylatable stathmin allows spindle formation at mitosis, a stathmin mutant in which the four phosphorylatable serines have been replaced by alanines (4A mutant) prevents formation of a normal spindle and results in a mitotic block of the cells (6, 12, 13). As the 4A mutant mimics the constitutively active form of stathmin, this shows that down-regulation of stathmin MT-destabilizing activity is likely required to allow formation of the mitotic spindle and progression through mitosis. However, it has been recently reported that a 4E pseudo-phosphorylated mutant, in which the four phosphorylation serines have been replaced by glu-tamic acid to mimic the constitutively down-regulated form, shows only a limited decrease in tubulin complex formation in vitro and in MT-destabilizing activity in transfected somatic cells (14, 15). To investigate the in vivo importance of the phosphorylation state of stathmin in an embryonic cell system, we used wild-type human stathmin and the two 4A and 4E mutants. We introduced these recombinants stathmin proteins in two-cell stage embryos and in mitotic egg extracts from Xenopus laevis to examine their effects on embryo development and on the MT network. We then performed immunofluorescence experiments to detect the specific localization of the added forms in the mitotic egg extracts and extended these observations to so-matic tissue culture cells. MATERIALS AND METHODS Preparation of Extracts and Human Stathmin Expression—Low speed (15,000 g) Xenopus egg extracts blocked in metaphase II of meiosis (CSF extracts) and permeabilized sperm heads were prepared as described previously (16). Spindles were assembled at 22 °C in the interphase to mitosis pathway as described previously (17) by adding 0.2 mM CaCl 2 for 1 h, then adding an equal volume of fresh CSF extract kept on ice to drive the extract back into mitosis. High speed extracts were prepared from CSF extracts spun at 245,000 g for 20 min at 4 °C in a TLS 55 rotor (Beckman) and were complemented with 0.05 volume of Energy Mix (150 mM creatine phosphate, 20 mM ATP, 20 mM MgCl 2). Recombinant human wild-type 4A (serine changed to alanine at positions 16, 25, 38, and 63) and 4E (serines substituted with glutamic acid) stathmin were in PBS at 10 mg/ml (18). Briefly, stathmin-expressing bacteria were sonicated, and the extracts were centrifuged at 4 °C for 5
Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both... more 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.
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Papers by Thomas M Küntziger