期刊
MOLECULAR BIOLOGY OF THE CELL
卷 16, 期 1, 页码 117-127出版社
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E04-05-0426
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- NHLBI NIH HHS [HL61295, R29 HL061295, R01 HL044985, R01 HL061295, HL44985] Funding Source: Medline
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL044985, R01HL061295, R29HL061295] Funding Source: NIH RePORTER
The nonerythrocyte isoform of the cytoskeletal protein 4.1R (4.1R) is associated with morphologically dynamic structures during cell division and has been implicated in mitotic spindle function. In this study, we define important 4.1R isoforms expressed in interphase and mitotic cells by RT-PCR and mini-cDNA library construction. Moreover, we show that 4.1R is phosphorylated by p34(cdc2) kinase on residues Thr60 and Ser679 in a mitosis-specific manner. Phosphorylated 4.1R(135) isoform(s) associate with tubulin and Nuclear Mitotic Apparatus protein (NuMA) in intact HeLa cells in vivo as well as with the microtubule-associated proteins in mitotic asters assembled in vitro. Recombinant 4.1R(135) is readily phosphorylated in mitotic extracts and reconstitutes mitotic aster assemblies in 4.1R-immunodepleted extracts in vitro. Furthermore, phosphorylation of these residues appears to be essential for the targeting of 4.1R to the spindle poles and for mitotic microtubule aster assembly in vitro. Phosphorylation of 4.1R also enhances its association with NuMA and tubulin. Finally, we used siRNA inhibition to deplete 4.1R from HeLa cells and provide the first direct genetic evidence that 4.1R is required to efficiently focus mitotic spindle poles. Thus, we suggest that 4.1R is a member of the suite of direct cdc2 substrates that are required for the establishment of a bipolar spindle.
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