A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein.
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.
Mattagajasingh
S N
SN
Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Huang
S C
SC
Hartenstein
J S
JS
Snyder
M
M
Marchesi
V T
VT
Benz
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D001665:Binding Sites; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D003598:Cytoskeletal Proteins; D004285:Dogs; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D007668:Kidney; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008938:Mitosis; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009479:Neuropeptides; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism*; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D008941:Spindle Apparatus / Q000378:metabolism*
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D000072159:Dynactin Complex; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C050045:NUMA1 protein, human; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D004398:Dyneins