Characterization of the microtubule-binding domain of microtubule-associated protein 1A and its effects on microtubule dynamics.
To determine how MAP1a interacts with microtubules we expressed several 6myc-tagged MAP1a fragments in P19 EC and HeLa cells. Confocal immunofluorescence microscopy showed that the fragment consisting of amino acids (aa) 1-281 of MAP1a did not bind while the fragment consisting of aa 1-630 did, indicating that the region of MAP1a between aa 281 and 630 contains a microtubule-binding domain. Deletion of the basic repeats from aa 336-540 did not result in loss of microtubule binding, suggesting that the regions flanking the basic repeats can bind MAP1a to microtubules. These observations were confirmed using an in vitro microtubule binding assay. The levels of acetylation and detyrosination of polymerized microtubules were assessed by quantitative dot blotting in cells expressing MAP1a fragments or MAP2c. Compared with untransfected cells, the polymerized tubulin in cells expressing full-length MAP1a was more acetylated and detyrosinated, but these increases were smaller than those seen in cells expressing MAP2c. Consistent with this, the microtubules in MAP2c expressing cells were more resistant to colchicine than those in cells overexpressing MAP1a. These data implicate aa 281-336 and/or 540-630 of MAP1a in microtubule binding and suggest that MAP1a is less able to stabilize microtubules than MAP2c.
Vaillant
A R
AR
Department of Biology, University of Ottawa, Ottawa K1N 6N5, Canada.
Müller
R
R
Langkopf
A
A
Brown
D L
DL
D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D017931:DNA Primers; C093429:MAP1A protein, human; C490547:MAP1A protein, mouse; C489788:MAP2 protein, human; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014404:Tubulin