CHARACTERIZATION OF MICROTUBULE-ASSOCIATED PROTEIN MAP1B - PHOSPHORYLATION STATE, LIGHT-CHAINS, AND BINDING TO MICROTUBULES

We have recently described a procedure for the purification of microtu bule associated protein 1B (MAP1B) from calf brain [Pedrotti, B., & Is lam, K. (1995) Cell Motil. Cytoskeleton 30, 301-309], and this study f urther characterizes the purified protein and its interaction with mic rotubules, We show that purified MAP1B (1) is thermostable; (2) is mai nly phosphorylated at the casein kinase II (CKII) sites but only parti ally phosphorylated at the proline-directed protein kinase (PDPK) site s, (3) both the CKII and PDPK sites can be dephosphorylated by alkalin e phosphatase; and (4) dephosphorylation results in an increased mobil ity on SDS-PAGE gels. The ability of MAP1B to interact with microtubul es was also examined and shows that (1) phosphorylated (1B-P), alkalin e phosphatase-treated (1B-AP), and heat-treated (1B-P), alkaline phosp hatase-treated (1B-AP), and heat-treated (1B-HT) MAP1B bind to taxol-s tabilized 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 hea vy chain is not affected by AP- or heat-treatment; (4) MAP1B can be di splaced from taxol-stabilized microtubules by titration with salt; (5) higher salt concentrations are required to displace 1B-AP compared wi th 1B-P from taxol-stabilized microtubules; and (6) MAP2 is able to di splace both 1B-P and 1B-AP from taxol-stabilized microtubules. The rol e of phosphorylation in regulating MAP1B interaction with microtubules and light chains is discussed.