Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.
A novel microtubule-associated protein (MAP) of M(r) 115,000 has been identified by screening of a HeLa cell cDNA expression library with an anti-serum raised against microtubule-binding proteins from HeLa cells. Monoclonal and affinity-purified polyclonal antibodies were generated for the further characterization of this MAP. It is different from the microtubule-binding proteins of similar molecular weights, characterized so far, by its nucleotide-insensitive binding to microtubules and different sedimentation behavior. Since it is predominantly expressed in cells of epithelial origin (Caco-2, HeLa, MDCK), and rare (human skin, A72) or not detectable (Vero) in fibroblastic cells, we name it E-MAP-115 (epithelial MAP of 115 kD). In HeLa cells, E-MAP-115 is preferentially associated with subdomains or subsets of perinuclear microtubules. In Caco-2 cells, labeling for E-MAP-115 increases when they polarize and form blisters. The molecular characterization of E-MAP-115 reveals that it is a novel protein with no significant homologies to other known proteins. The secondary structure predicted from its sequence indicates two domains connected by a putative hinge region rich in proline and alanine (PAPA region). E-MAP-115 has two highly charged regions with predicted alpha-helical structure, one basic with a pI of 10.9 in the NH2-terminal domain and one neutral with a pI of 7.6 immediately following the PAPA region in the acidic COOH-terminal half of the molecule. A novel microtubule-binding site has been localized to the basic alpha-helical region in the NH2-terminal domain using in vitro microtubule-binding assays and expression of mutant polypeptides in vivo. Overexpression of this domain of E-MAP-115 by transfection of fibroblasts lacking significant levels of this protein with its cDNA renders microtubules stable to nocodazole. We conclude that E-MAP-115 is a microtubule-stabilizing protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells.
Masson
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European Molecular Biology Laboratory, Heidelberg, Germany.
Kreis
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D000409:Alanine / Q000032:analysis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000235:genetics; D004285:Dogs; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000166:cytology; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000235:genetics; D008870:Microtubules / Q000737:chemistry / Q000648:ultrastructure; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D011233:Precipitin Tests; D011392:Proline / Q000032:analysis; D017422:Sequence Analysis, DNA; D014162:Transfection; D014709:Vero Cells
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D008869:Microtubule-Associated Proteins; D004247:DNA; D011392:Proline; D000409:Alanine; D015739:Nocodazole