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

doi:10.1083/jcb.145.1.29

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© The Rockefeller University Press, 0021-9525/1999//29 $5.00
The Journal of Cell Biology, Volume 145, Number 1, , 1999 29-43

Regular Articles

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 phosphoproteinthat stabilizes the spectrin/actin cytoskeleton. In nonerythroidcells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. Thisisoform contains a 209 amino acid extension at its NH₂ terminus(head piece; HP). Immunoreactive epitopes specific for HP havebeen detected within the cell nucleus, nuclear matrix, centrosomes,and parts of the mitotic apparatus in dividing cells. Usinga yeast two-hybrid system, in vitro binding assays, coimmunolocalization,and coimmunoprecipitation studies, we show that a 135-kD 4.1Risoform 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 polesearly in mitosis. Protein 4.1R associates with NuMA in theinterphase nucleus and forms a complex with spindle pole organizingproteins, NuMA, dynein, and dynactin during cell division.Overexpression of a 135-kD isoform of 4.1R alters the normaldistribution of NuMA in the interphase nucleus. The minimalsequence sufficient for this interaction has been mapped tothe amino acids encoded by exons 20 and 21 of 4.1R and residues1788–1810 of NuMA. Our results not only suggest that4.1R could, possibly, play an important role in organizingthe nuclear architecture, mitotic spindle, and spindle poles,but also could define a novel role for its 22–24-kD domain.

Key Words: protein 4.1R • NuMA • dynein • dynactin • mitotic spindle

Abbreviations used in this paper: Ab, polyclonal antibody; 4.1R, red blood cell protein 4.1; Gal4-AD, Gal4 activation domain; Gal4-BD, Gal4 DNA-binding domain; HP, head piece or the NH₂-terminal extension of 4.1R; IP, immunoprecipitation buffer; NuMA, nuclear mitotic apparatus protein.

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