Localisation of microtubule-associated protein 1B phosphorylation sites recognised by monoclonal antibody SMI-31

Abstract

MAP 1B is a microtubule-associated phosphoprotein that is expressed early in neurons and plays a role in axon growth. MAP 1B has two types of phosphoisoforms, one of which is developmentally down-regulated after neuronal maturation and one of which persists into adulthood. Because phosphorylation regulates MAP lB binding activity, characterisation of the phosphorylation sites and identification of the corresponding kinases/phosphatases are important goals. We have characterised the developmentally down-regulated phosphorylation sites recognised by monoclonal antibody (mAb) SMI31. We purified MAP lB from neonatal rat brain and mapped the mAb SMI-31 sites to specific MAP lB fragments after chemical 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 1 B molecule. Phosphorylation of the recombinant protein was detected on 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 1 B after in vitro phosphorylation. The former phosphorylation site was further defined in the in vitro kinase assay by inhibition with peptides and antibodies from candidate regions of the MAP lB sequence. This approach identified a region of 20 amino acids, from 1,244 to 1,264, characterised by a high concentration of serines immediately upstream of prolines, indicating that the kinase responsible is a proline-directed serine kinase.