J. Cell Biol., Volume 145, Number 1, April 5, 1999 29-43

A Nonerythroid Isoform of Protein 4.1R Interacts with the Nuclear Mitotic Apparatus (NuMA) Protein

Subhendra N. Mattagajasingh,^* Shu-Ching Huang,^* Julia S. Hartenstein,^* Michael Snyder, Vincent T. Marchesi,^§ and Edward J. Benz Jr.^*

^* Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205;  Department of Biology, Yale University, New Haven, Connecticut 06511; ^§ Boyer Center for Molecular Medicine and Department of Pathology, Yale School of Medicine, New Haven, Connecticut 06510; and  Department of Molecular Biology and Genetics, The Johns Hopkins University, Baltimore, Maryland 21205

Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH₂ terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.

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