Extracellular phoshorylation of MAP1B and its role in synapse formation

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Synapse formation between cultured rat cortical neurons is inhibited by the continuous application of K-252b, an ecto-protein kinase inhibitor, which cannot permeate the cell membrane. Application of K-252b also inhibited the phosphorylation of the extracellular domains of membrane proteins by ecto-protein kinase. Therefore, the phosphorylation of these membrane proteins may play important roles in synapse formation. To identify membrane proteins which may be involved in synapse formation, [gamma-^<33>P] ATP was applied to cultured cells for brief periods to phosphorylate their extracellular domains. The phosphorylated proteins were separated by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. Some of these bands were immediately phosphorylated, and this phosphorylation was suppressed by addition of K-252b to the medium. We examined the partial amino acid sequences of these substrates. Phosphorylated bands were cut from the gel and digested with lysyl endopeptidase. Peptide fragments were separated by capillary HPLC and analyzed by mass spectrometry. The band with the highest molecular weight, whose phosphorylation was strongly inhibited by K-252b, was identified as microtubule-associated protein (MAP) 1b. These results suggest the phosphorylation of extracellular domains of MAP1b is involved in synapse formation between cortical neurons.