Requirement for MAPK activation for normal mitotic progression in Xenopus egg extracts

Science

Volumn 282, Issue 5392, 1998, Pages 1312-1315

  Guadagno, Thomas M ^a   Ferrell Jr , James E ^a  

^a STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

MITOGEN ACTIVATED PROTEIN KINASE;

ANIMAL CELL; ARTICLE; ENZYME ACTIVATION; ENZYME ACTIVITY; MITOSIS; NONHUMAN; PRIORITY JOURNAL; XENOPUS;

EID: 0032514880     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5392.1312     Document Type: Article

References (33)

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15. Cycling extracts were prepared from electrically activated Xenopus eggs as described [A. W. Murray and M. W. Kirschner, Nature 339, 275 (1989); A. W. Murray, Methods Cell Biol. 36, 581 (1991)]. To inhibit MEK activation, a dimethyl sulfoxide (DMSO) solution of PD98059 (Calbiochem) was added to yield a final concentration of 200 μM PD98059 and 1% DMSO. Control extracts were treated with DMSO alone. Immunodepletion of MEK was accomplished by incubating extracts for 75 to 90 min at 4°C with protein A-purified antibody 662 [K.-M. Hsiao, S.-y. Chou, S.-J. Shih, J. E. Ferrell Jr., Proc. Natl. Acad. Sci. U.S.A. 91, 5480 (1994)] prebound to protein A-Sepharose beads (Sigma). Mock depletions were carried out with rabbit immunoglobulin G (IgC) in place of antibody 662.
16. Cycling extracts were prepared from electrically activated Xenopus eggs as described [A. W. Murray and M. W. Kirschner, Nature 339, 275 (1989); A. W. Murray, Methods Cell Biol. 36, 581 (1991)]. To inhibit MEK activation, a dimethyl sulfoxide (DMSO) solution of PD98059 (Calbiochem) was added to yield a final concentration of 200 μM PD98059 and 1% DMSO. Control extracts were treated with DMSO alone. Immunodepletion of MEK was accomplished by incubating extracts for 75 to 90 min at 4°C with protein A-purified antibody 662 [K.-M. Hsiao, S.-y. Chou, S.-J. Shih, J. E. Ferrell Jr., Proc. Natl. Acad. Sci. U.S.A. 91, 5480 (1994)] prebound to protein A-Sepharose beads (Sigma). Mock depletions were carried out with rabbit immunoglobulin G (IgC) in place of antibody 662.
17. Cycling extracts were prepared from electrically activated Xenopus eggs as described [A. W. Murray and M. W. Kirschner, Nature 339, 275 (1989); A. W. Murray, Methods Cell Biol. 36, 581 (1991)]. To inhibit MEK activation, a dimethyl sulfoxide (DMSO) solution of PD98059 (Calbiochem) was added to yield a final concentration of 200 μM PD98059 and 1% DMSO. Control extracts were treated with DMSO alone. Immunodepletion of MEK was accomplished by incubating extracts for 75 to 90 min at 4°C with protein A-purified antibody 662 [K.-M. Hsiao, S.-y. Chou, S.-J. Shih, J. E. Ferrell Jr., Proc. Natl. Acad. Sci. U.S.A. 91, 5480 (1994)] prebound to protein A-Sepharose beads (Sigma). Mock depletions were carried out with rabbit immunoglobulin G (IgC) in place of antibody 662.
18. MEK function appears to be essential for mitotic fragmentation of the Golgi apparatus in normal rat kidney cells [U. Acharya, A. Mallabiabarrena, J. K. Acharya, V. Malhotra, Cell 92, 183 (1998)]. We were unable to assess the status of the Golgi apparatus because it is not well organized in extracts even in interphase.
19. This concentration of sperm (500 sperm per microliter) is substantially lower than that needed to reconstitute the spindle assembly checkpoint (about 9000 sperm per microliter) (6).
20. 2-like arrest, with Cdc2-cyclin complexes present but inactive. When MAPK is activated just before mitosis, the result is arrest in a mitotic state with cyclin destruction partially inhibited - the classic cytostatic factor arrest [A. Abrieu, D. Fisher, M. N. Simon, M. Doree, A. Picard, EMBO J. 16, 6407 (1997); S. A. Walter, T. M. Guadagno, J. E. Ferrell Jr., Mol. Biol. Cell 8, 2157 (1997); J. C. Bitangcol et al., ibid. 9, 451 (1998); M. S. Murakami and G. F. Vande Woude, Development 125, 237 (1998)].
21. 2-like arrest, with Cdc2-cyclin complexes present but inactive. When MAPK is activated just before mitosis, the result is arrest in a mitotic state with cyclin destruction partially inhibited - the classic cytostatic factor arrest [A. Abrieu, D. Fisher, M. N. Simon, M. Doree, A. Picard, EMBO J. 16, 6407 (1997); S. A. Walter, T. M. Guadagno, J. E. Ferrell Jr., Mol. Biol. Cell 8, 2157 (1997); J. C. Bitangcol et al., ibid. 9, 451 (1998); M. S. Murakami and G. F. Vande Woude, Development 125, 237 (1998)].
22. 2-like arrest, with Cdc2-cyclin complexes present but inactive. When MAPK is activated just before mitosis, the result is arrest in a mitotic state with cyclin destruction partially inhibited - the classic cytostatic factor arrest [A. Abrieu, D. Fisher, M. N. Simon, M. Doree, A. Picard, EMBO J. 16, 6407 (1997); S. A. Walter, T. M. Guadagno, J. E. Ferrell Jr., Mol. Biol. Cell 8, 2157 (1997); J. C. Bitangcol et al., ibid. 9, 451 (1998); M. S. Murakami and G. F. Vande Woude, Development 125, 237 (1998)].
23. 2-like arrest, with Cdc2-cyclin complexes present but inactive. When MAPK is activated just before mitosis, the result is arrest in a mitotic state with cyclin destruction partially inhibited - the classic cytostatic factor arrest [A. Abrieu, D. Fisher, M. N. Simon, M. Doree, A. Picard, EMBO J. 16, 6407 (1997); S. A. Walter, T. M. Guadagno, J. E. Ferrell Jr., Mol. Biol. Cell 8, 2157 (1997); J. C. Bitangcol et al., ibid. 9, 451 (1998); M. S. Murakami and G. F. Vande Woude, Development 125, 237 (1998)].
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25. T. M. Guadagno and J. E. Ferrell Jr., unpublished data.
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27. To prepare interphase extracts, we incubated eggs with cycloheximide (100 μg/ml) at room temperature for 10 min, electrically activated them, and then packed and crushed them in the presence of cycloheximide [C. Smythe and J. W. Newport, Methods Cell Biol. 35, 449 (1991)]. The resulting extracts were devoid of detectable cyclins and Mos and contained inactive p42 MAPK.
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29. The microtubule nucleation assay was done essentially as described [T. Stearns and M. Kirschner, Cell 76, 623 (1994)]. Briefly, rhodamine-labeled bovine tubulin (from T. Stearns, Stanford University) and Xenopus sperm were added to extracts to yield final concentrations of 120 μg/ml and 200 nuclei per microliter, respectively. Extracts were incubated at room temperature for 10 min. Samples were diluted in 9 vol of glutaraldehyde (0.25%), centrifuged through a 25% glycerol cushion onto coverslips, and stained with DAPI. The DAPI-stained sperm and rhodamine-labeled tubulin were examined by fluorescence microscopy.
30. Addition of purified Xenopus or rat MAPK to interphase extracts can be sufficient to produce mitotic-like microtubules under some circumstances [Y. Gotoh et al., Nature 349, 251 (1991)]. One difference between their experiment and ours is the way the interphase extracts were prepared. We used cycloheximide-soaked eggs, and prepared the interphase extracts in cycloheximide-containing buffers, which prevents cyclin synthesis (17). Apparently they prepared interphase extracts in the absence of cycloheximide, and as a result their extracts may have contained substantial amounts of cyclins. The mitotic-like microtubules they observed therefore may have resulted from a combination of MAPK and Cdc2-cyclin activities.
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33. Supported by a grant from the National Institutes of Health (GM46383). We thank T. Stearns for providing rhodamine-labeled tubulin and for advice; N. Ahn for providing MEK plasmids and for sharing unpublished data; G. Gorbsky and M. Weber for sharing unpublished data; M. Murakami and G. Vande Woude for providing Mos plasmids; and K. Cimprich and members of the Ferrell laboratory for comments on the manuscript. T.M.G. is a Special Fellow of the Leukemia Society of America. J.E.F. is a Howard Hughes Junior Faculty Scholar.