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.

Figure 1

Schematic representation of 4.1R and NuMA peptides found to interact in the yeast two-hybrid screening of a human brain cDNA library. (A) The organization of protein 4.1R and its 22–24-kD domain peptides employed as bait in the yeast two-hybrid assays. Different exons of 4.1R gene that correspond to its different domains are shown. The asterisks represent the alternatively spliced exons. (B) A schematic representation of the structural organization of the NuMA protein and its segments encompassed by positive clones obtained as prey in the two-hybrid assays.

Figure 3

Interaction between 4.1R and NuMA occurs through the COOH-terminal 62 amino acids of 4.1R and amino acids 1788–1810 of NuMA. (A) Schematic diagrams of the various exons within the 22–24-kD domain of 4.1 (white rectangles) fused to the DNA binding domain of GAL4 (shaded rectangles) used in the two-hybrid assay are shown. The names of the plasmids that encoded each construct are given to the right of each schematic diagram. Plasmid pAS2-1 expresses the GAL4-BD alone and was used as a negative control. These plasmids were cotransformed with pACT2-NuMA expressing amino acids 1697–2115 of NuMA fused to the activation domain of Gal4. The column on the right shows the results of filter assays for β-galactosidase (β-gal) activity as the reporter gene: (+) indicates the expression of the reporter genes (and thus the interaction between the peptides) and (−) indicates nonexpression of the reporter genes (and thus no interaction between the peptides). The last 62 amino acids of protein 4.1 encoded by exons 20 and 21 bind to NuMA. (B) Schematic diagrams of the various NuMA polypeptides (dotted rectangles) fused to the activation domain of GAL4 (shaded rectangles) used in the two-hybrid assay are shown. Plasmid pACT2, which expresses the Gal4-AD alone, was used as a negative control. These plasmids were cotransformed with pGBT9 or pAS2-1 expressing COOH-terminal domain of 4.1R or its exons 20 and 21 fused to the DNA-binding domain of GAL4. The column on the right shows the results of filter assays for β-galactosidase activity: (++) indicates strong β-gal activity, (+) indicates detectable β-gal activity, and (−) indicates no detectable β-gal activity.

Figure 2

4.1R and NuMA interact through their COOH-terminal domains. 4.1R and NuMA peptides and their segments were expressed as Gal4-BD or Gal4-AD fusion proteins, respectively, in yeast strain Y190 by cotransformation, and were assayed for the expression of the reporter genes (see Materials and Methods). (+) indicates the expression of the reporter genes, LacZ and HIS3 (and thus the interaction between the peptides) and (−) indicates nonexpression of the reporter genes (and thus no interaction between the peptides).

Figure 4

Protein 4.1R interacts with NuMA in vitro. Purified bacterially produced 4.1R/GST fusion proteins were incubated with [³⁵S]methionine-labeled NuMA. After incubation, the bound protein complexes were analyzed by SDS-PAGE and visualized by fluorography. (A) Purified 4.1R/GST fusion proteins are shown by Coomassie blue staining: HP/GST (lane 1); 30 kD/GST (lane 2); 16 kD/GST (lane 3); 10 kD/GST (lane 4); 24 kD/GST (lane 5); GST (lane 6); 135 kD/GST (lane 7); and 80 kD/ GST (lane 8). (B) Binding of 4.1R/GST to [³⁵S]methionine-labeled NuMA amino acid sequence 1697–2102 translated from NuMA1/ TOPO is shown. (C) Binding of 4.1R/GST to NuMA amino acid sequence 1697–1889 translated from NuMA2/TOPO. (D) Binding of 4.1R/GST to NuMA amino acid sequence 1831–2102 translated from NuMA3/TOPO. The input [³⁵S]methionine-labeled NuMA proteins are shown on the far left lane.

Figure 5

Subcellular localization of 4.1R and NuMA proteins. MDCK cells were fractionated and 20 μg proteins (unless mentioned otherwise) from the cytoplasm (equivalent to ∼1.6 × 10⁵ cells), nuclear (equivalent to ∼5 × 10⁵ cells), and nuclear matrix (equivalent to ∼7 × 10⁶ cells) fractions were analyzed as described in Materials and Methods. Shown are immunoblots of preimmune serum for anti-HP 4.1 Ab (A), anti-HP 4.1 Ab (B), anti-24 kD Ab (C), anti-10 kD Ab (D), anti-NuMA Ab (E), anti–Na⁺-K⁺-ATPase α1 Ab (F), antidynein mAb (G), and anti–γ-tubulin Ab (H). The blot in I was immunoblotted with antipericentrin Ab and was loaded with 50 μg proteins from each fraction.

Figure 6

Subcellular colocalization of protein 4.1 and NuMA at various cell cycle stages of MDCK cells. MDCK cells were fixed and processed for immunofluorescence with protein 4.1R and NuMA antibodies as described in Materials and Methods. Shown are cells in interphase (A1–A3), mitotic (B1–B3 and C1–C3), and late mitotic stages (D1–D3). Green represents anti-HP 4.1 epitopes, red represents anti-NuMA epitopes, and yellow indicates the colocalization of protein 4.1R and NuMA.

Figure 7

Coimmunoprecipitation of 4.1R and NuMA from MDCK cell nuclear extracts. MDCK nuclear extracts were prepared as described in Materials and Methods and were subjected to immunoprecipitation using different antibodies. The immunoprecipitates were analyzed by immunoblotting using anti-HP 4.1 Ab (A), anti-NuMA mAb (B), antiactin mAb (C), or antispectrin mAb (D). In A and B, lanes 1–5 were loaded with one-fourth of the immunoprecipitates of anti-HP 4.1 Ab (lane 1), preimmune serum (lane 2), anti-NuMA mAb (lane 3), anti-p53 mAb (lane 4), and mouse IgG (lane 5). Lanes 6 and 7 were loaded with one-ninth of the supernatant fractions of anti-HP 4.1 Ab and anti-NuMA mAb immunoprecipitates, respectively. For C and D, lanes 1–5 were the same as in A, and lane 6 was loaded with 40 μg of cell lysate. Arrows indicate the position of actin in C and that of α- and β-spectrin in D.

Figure 8

Immunoprecipitaion from mitotic HeLa extracts reveals that 4.1R associates with NuMA, cytoplasmic dynein, and dynactin. Mitotic HeLa nuclear extracts were prepared as described in Materials and Methods, and were subjected to immunoprecipitation using different antibodies. The immunoprecipitates were analyzed by immunoblotting using anti-HP 4.1 Ab (A and F), anti-24 kD Ab (B), anti-NuMA mAb C and G, antidynein mAb (D), or anti-p150^glued mAb (E). In A–C, lanes 1–7 were loaded with one fourth of the immunoprecipitates of anti-HP 4.1 Ab (lane 1), preimmune serum (lane 2), anti-NuMA mAb (lane 3), anti-p53 mAb (lane 4), antidynein mAb (lane 5), antidynactin mAb (lane 6), and mouse IgG (lane 7), respectively. Lane 8 was loaded with 40 μg of mitotic HeLa lysate. The anti-24 kD Ab recognized the 135-kD 4.1R isoform in the immunoprecipitates and is indicated by an arrow. The blot in D was probed with antidynein mAb and lanes 1–7 were the same as A. The blot shown in E is the same as C except that it was stripped and reprobed with antidynactin mAb. Lanes 1–4 of the blot in F were loaded with one-fourth of the immunoprecipitates of anti-HP 4.1 Ab (lane 1), preimmune serum (lane 2), anti-NuMA mAb (lane 3), and mouse IgG (lane 4), respectively. Lanes 5 and 6 were loaded with one-ninth of the supernatant fractions of anti-HP 4.1 Ab and anti-NuMA mAb immunoprecipitates, respectively. The blot shown in (G) is the same as in (F) except that it was stripped and reprobed with anti-NuMA mAb. H is a silver-stained gel of molecular mass markers (lane 1), immunoprecipitates of preimmune serum (lane 2), and anti-HP 4.1 Ab (lane 3). The migration position of 4.1R (135-kD isoform), NuMA, dynein, and dynactin are indicated in the figure. Shown in I is the alignment of NuMA residues essential for its binding to 4.1R in human and Xenopus (available in GenBank/ EMBL/DDBJ under accession number Y07624). The identical residues shared by the two proteins are shaded and the conserved amino acid changes are boxed. Numbers represent the position of respective amino acids. The peptide segment of NuMA that interacts with 4.1R is highly conserved across the species.

Figure 8

Immunoprecipitaion from mitotic HeLa extracts reveals that 4.1R associates with NuMA, cytoplasmic dynein, and dynactin. Mitotic HeLa nuclear extracts were prepared as described in Materials and Methods, and were subjected to immunoprecipitation using different antibodies. The immunoprecipitates were analyzed by immunoblotting using anti-HP 4.1 Ab (A and F), anti-24 kD Ab (B), anti-NuMA mAb C and G, antidynein mAb (D), or anti-p150^glued mAb (E). In A–C, lanes 1–7 were loaded with one fourth of the immunoprecipitates of anti-HP 4.1 Ab (lane 1), preimmune serum (lane 2), anti-NuMA mAb (lane 3), anti-p53 mAb (lane 4), antidynein mAb (lane 5), antidynactin mAb (lane 6), and mouse IgG (lane 7), respectively. Lane 8 was loaded with 40 μg of mitotic HeLa lysate. The anti-24 kD Ab recognized the 135-kD 4.1R isoform in the immunoprecipitates and is indicated by an arrow. The blot in D was probed with antidynein mAb and lanes 1–7 were the same as A. The blot shown in E is the same as C except that it was stripped and reprobed with antidynactin mAb. Lanes 1–4 of the blot in F were loaded with one-fourth of the immunoprecipitates of anti-HP 4.1 Ab (lane 1), preimmune serum (lane 2), anti-NuMA mAb (lane 3), and mouse IgG (lane 4), respectively. Lanes 5 and 6 were loaded with one-ninth of the supernatant fractions of anti-HP 4.1 Ab and anti-NuMA mAb immunoprecipitates, respectively. The blot shown in (G) is the same as in (F) except that it was stripped and reprobed with anti-NuMA mAb. H is a silver-stained gel of molecular mass markers (lane 1), immunoprecipitates of preimmune serum (lane 2), and anti-HP 4.1 Ab (lane 3). The migration position of 4.1R (135-kD isoform), NuMA, dynein, and dynactin are indicated in the figure. Shown in I is the alignment of NuMA residues essential for its binding to 4.1R in human and Xenopus (available in GenBank/ EMBL/DDBJ under accession number Y07624). The identical residues shared by the two proteins are shaded and the conserved amino acid changes are boxed. Numbers represent the position of respective amino acids. The peptide segment of NuMA that interacts with 4.1R is highly conserved across the species.

Figure 9

Alteration in the localization of NuMA in 135⁺⁺kD/ GFP 4.1R-transfected HeLa cells. HeLa cells transfected with pEGFP or 135⁺⁺kD/GFP were fixed and processed for immunofluorescence with NuMA antibody and visualized for the localization of GFP (green) and NuMA (red). Shown are cells transfected with pEGFP (A1–A3) or 135⁺⁺kD/GFP (B1–B3 and C1–C3). Green represents fluorescence of GFP, red represents anti-NuMA epitopes, and yellow indicates the colocalization of GFP and NuMA.