Characterization of microtubule-associated protein MAP1B: phosphorylation state, light chains, and binding to microtubules.
We have recently described a procedure for the purification of microtubule associated protein 1B (MAP1B) from calf brain [Pedrotti, B., & Islam K. (1995) Cell Motil. Cytoskeleton 30, 301-309], and this study further characterizes the purified protein and its interaction with microtubules. We show that purified MAP1B (1) is thermostable; (2) is mainly phosphorylated at the casein kinase II (CKII) sites but only partially phosphorylated at the proline-directed protein kinase (PDPK) sites; (3) both the CKII and PDPK sites can be dephosphorylated by alkaline phosphatase; and (4) dephosphorylation results in an increased mobility on SDS-PAGE gels. The ability of MAP1B to interact with microtubules was also examined and shows that (1) phosphorylated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-P), alkaline phosphatase-treated (1B-AP), and heat-treated (1B-HT) MAP1B bind to taxol-stabilized microtubules; (2) 1 mol of 1B-P, 1B-AP, or 1B-HT each binds about 13-14 tubulin dimers; (3) light chain interaction with MAP1B heavy chain is not affected by AP- or heat-treatment; (4) MAP1B can be displaced from taxol-stabilized microtubules by titration with salt; (5) higher salt concentrations are required to displace 1B-AP compared with 1B-P from taxol-stabilized microtubules; and (6) MAP2 is able to displace both 1B-P and 1B-AP from taxol-stabilized microtubules. The role of phosphorylation in regulating MAP1B interaction with microtubules and light chains is discussed.
Pedrotti
B
B
Department of Biology, University of Milan, Italy.
Ulloa
L
L
Avila
J
J
Islam
K
K
D000469:Alkaline Phosphatase; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D010750:Phosphoproteins; C085284:microtubule-associated protein 1B; D000469:Alkaline Phosphatase