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Vol. 16, Issue 1, 117-127, January 2005

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Mitotic Regulation of Protein 4.1R Involves Phosphorylation by cdc2 Kinase

Shu-Ching Huang ^*  , Eva S. Liu ^*, Siu-Hong Chan , Indira D. Munagala ^*, Heidi T. Cho ^*, Ramasamy Jagadeeswaran ^*, and Edward J. Benz, Jr ^{*  || ¶ #}

^* Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA 02115; Department of Medicine, Harvard Medical School, Boston, MA 02115; ^# Department of Pathology, Harvard Medical School, Boston, MA 02115; Department of Medicine, Johns Hopkins University, Baltimore, MD 21205; ^|| Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115; and ^¶ Department of Pediatrics, Children's Hospital of Boston, Boston, MA 02115

Submitted May 21, 2004; Revised September 29, 2004; Accepted October 8, 2004
Monitoring Editor: Douglas Koshland

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 4.1R¹³⁵ 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 4.1R¹³⁵ 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.

Corresponding author. E-mail address: shu-ching_huang{at}dfci.harvard.edu.

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