4.1R proteins associate with interphase microtubules in human T cells: a 4.1R constitutive region is involved in tubulin binding.
Red blood cell protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of functional roles and partners of specific nonerythroid 4.1R isoforms, we analyzed 4.1R in human T cells and found that endogenous 4.1R was distributed to the microtubule network. Transfection experiments of T cell 4.1R cDNAs in conjunction with confocal microscopy analysis revealed the colocalization of exogenous 4.1R isoforms with the tubulin skeleton. Biochemical analyses using Taxol (paclitaxel)-polymerized microtubules from stably transfected T cells confirmed the association of the exogenous 4.1R proteins with microtubules. Consistent with this, endogenous 4.1R immunoreactive proteins were also detected in the microtubule-containing fraction. In vitro binding assays using glutathione S-transferase-4.1R fusion proteins showed that a constitutive domain of the 4.1R molecule, one that is therefore present in all 4.1R isoforms, is responsible for the association with tubulin. A 22-amino acid sequence comprised in this domain and containing heptad repeats of leucine residues was essential for tubulin binding. Furthermore, ectopic expression of 4.1R in COS-7 cells provoked microtubule disorganization. Our results suggest an involvement of 4.1R in interphase microtubule architecture and support the hypothesis that some 4.1R functional activities are cell type-regulated.
Pérez-Ferreiro
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CM
Centro de Biologia Molecular "Severo Ochoa," Departamento de Biologia Molecular, Facultad de Ciencias, Universidad Autónoma de Madrid, E-28049 Madrid, Spain.
Luque
C M
CM
Correas
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I
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D009479:Neuropeptides; D017239:Paclitaxel / Q000494:pharmacology; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D009479:Neuropeptides; D020033:Protein Isoforms; D011506:Proteins; D011994:Recombinant Proteins; D014404:Tubulin; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D005982:Glutathione Transferase; D017239:Paclitaxel