LOCALIZATION OF MICROTUBULE-ASSOCIATED PROTEIN 1B PHOSPHORYLATION SITES RECOGNIZED BY MONOCLONAL-ANTIBODY SMI-31

MAP 1B is a microtubule-associated phosphoprotein that is expressed ea rly in neurons and plays a role in axon growth. MAP 1B has two types o f phospho-isoforms, one of which is developmentally down-regulated aft er neuronal maturation and one of which persists into adulthood, Becau se phosphorylation regulates MAP 1B binding activity, characterisation of the phosphorylation sites and identification of the corresponding kinases/phosphatases are important goals. We have characterised the de velopmentally down-regulated phosphorylation sites recognised by monoc lonal antibody (mAb) SMI-31. We purified MAP 1B from neonatal rat brai n and mapped the mAb SMI-31 sites to specific MAP 1B fragments after c hemical cleavage, We then developed an in vitro kinase assay by using a high-speed spin supernatant from neonatal rat brain in the presence of ATP and recombinant proteins encoding selective regions of the MAP 1B molecule. Phosphorylation of the recombinant protein was detected o n western blots using mAb SMI-31. This analysis showed that mAb SMI-31 recognises two recombinant proteins corresponding to residues 1,109-1 ,360 and 1,836-2,076 of rat MAP 1B after in vitro phosphorylation, The former phosphorylation site was further defined in the in vitro kinas e assay by inhibition with peptides and antibodies from candidate regi ons of the MAP 1B sequence. This approach identified a region of 20 am ino acids, from 1,244 to 1,264, characterised by a high concentration of serines immediately upstream of prolines, indicating that the kinas e responsible is a proline-directed serine kinase.