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

Abstract

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.

Protein 4.1R, a Microtubule-associated Protein Involved in Microtubule Aster Assembly in Mammalian Mitotic Extract

“…In keeping with the key transitions in the cell cycle controlled by the cyclin-dependent kinases, the redistribution of 4.1R to the mitotic spindle and spindle poles is regulated by a phosphorylation event that occurs at Thr-60 and Ser-679 sites by cyclin-dependent cdc2 kinase. 2 We (20) previously showed that a 135-kDa 4.1R isoform may participate in mitosis via its association with the non-centrosomal proteins NuMA, dynein, and dynactin, which are essential to the organization of functional spindle poles and tethering of the centrosomes to the spindle pole (21). From these results, we hypothesize that 4.1R plays an important role in organizing mitotic spindle and spindle poles.…”

“…Antibody Production-Antisera to synthetic peptides of exon 13 of 4.1R (DSADRSPRPTSAPAI) (anti-exon 13) and the recombinant HP of 4.1R (anti-HP) were prepared as described previously (20). The sera were purified on immunoaffinity columns using the Sulfolink kit (Pierce) covalently cross-linked to the synthetic peptides or fusion peptides used for immunization.…”

“…Coimmunoprecipitation and Immunoblotting-Coimmunoprecipitation of 4.1R, tubulin, and NuMA was performed using HeLa mitotic extracts as described previously (20). In brief, mitotic HeLa cells were homogenized in coimmunoprecipitation buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mg/ml bovine serum albumin, 0.2 mM EDTA, 5 mM iodoacetamide, 2% CHAPS, protease inhibitors, phosphatase inhibitors), and given 20 strokes in a tight-fitting glass homogenizer.…”

“…4.1R isoforms have also been shown to be the components of the nuclear matrix (16,17) and may be involved in splicing processes (18). 4.1R is reorganized during the cell cycle; it localizes in the nucleus and cytoplasm of interphase cells and rapidly redistributes to the developing spindle poles when the nuclear envelope dissembles in prometaphase (16,17,19,20). In keeping with the key transitions in the cell cycle controlled by the cyclin-dependent kinases, the redistribution of 4.1R to the mitotic spindle and spindle poles is regulated by a phosphorylation event that occurs at Thr-60 and Ser-679 sites by cyclin-dependent cdc2 kinase.…”

Protein 4.1B expression is induced in mammary epithelial cells during pregnancy and regulates their proliferation

“…4.1B contains a putative nuclear localization signal (NLS) (KRRK), as well as an upstream casein kinase II site (SAAE), a common feature in proteins containing an NLS (Kittiniyom et al, 2004). Furthermore, 4.1B has been shown to translocate from the plasma membrane into the nucleus in PC12 cells following differentiation (Gascard et al, 2004), and the 4.1 family members merlin, ezrin, and 4.1R localize within the nucleus under some conditions (Mattagajasingh et al, 1999;Batchelor et al, 2004;Muranen et al, 2005). To exclude the possibility that the observed nuclear localization of 4.1B was due to nonspecific staining by the 4.1B antibody, we created a GFP-4.1B fusion construct (pEFGP-4.1B) in order to monitor the localization of the exogenous protein without having to rely on the antibody.…”