Differential Mitotic Phosphorylation of Proteins of the Nuclear Pore Complex

doi:10.1074/jbc.270.1.254

Differential Mitotic Phosphorylation of Proteins of the Nuclear Pore Complex (*)

From the Department of Biology, University of California at San Diego, La Jolla, California 92093-0347

↵^§ Current address: The Scripps Research Inst., 10666 N. Torrey Pines Rd., La Jolla, CA 92037.

^¶ To whom correspondence should be addressed.

Abstract

During each cell cycle, the nucleus of higher eukaryotes undergoes a dramatic assembly and disassembly. These events can be faithfully reproduced in vitro using cell-free extracts derived from Xenopus eggs. Such extracts contain three major N-acetylglucosaminylated proteins, p200, p97, and p60. All three become assembled into reconstituted nuclear pores. Here we show that p200, p97, and p60 exist in eggs in soluble high molecular mass complexes of 1000, 450, and 600 kDA, respectively. The bulk of p60 is stably associated with proteins of 58 and 54 kDa, while p200 is associated with a fraction of p60 in a separate complex lacking p58 and p54. Upon examining the behavior of these proteins in the cell cycle, we find that p200 and p97 are highly phosphorylated at mitosis, both in vivo and in vitro. Moreover, in extracts that cycle between interphase and mitosis, p200 and p97 are specifically phosphorylated at mitosis. Corresponding with their mitotic phosphorylation, both p200 and p97 are specific substrates for purified mitotic Cdc2 kinase, whereas nucleoporin p60 is not. Analysis indicates that the size of the complexes containing the pore N-acetylglucosamine glycoproteins does not change during mitosis, suggesting that such complexes represent stable multicomponent modules into which the nucleus disassembles at mitosis.

Footnotes

↵* This work was supported by a fellowship grant from the Medical Research Council of Canada and an independent research allowance from the Alberta Heritage Foundation for Medical Research (to C. M.) and by Grant GM33279 from the National Institutes of Health and a grant from the Pew Memorial Trust (to D. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

WGA

wheat germ agglutinin

PVDF

polyvinylidine difluoride.
↵² M. Powers, C. Macaulay, and D. J. Forbes, manuscript in preparation.
↵³ M. Powers, C. Macaulay, and D. J. Forbes, submitted for publication.
↵⁴ C. Macaulay, unpublished results.
- Received August 19, 1994.
- Revision received October 17, 1994.