Figure 1.

Mnb phosphorylates Synaptojanin at S1029 in vitro. A, Protein sequence alignment of partial Drosophila Synj-1 PRD domain with mouse and human Synj-1 PRD domain (Clustal Omega). Phosphorylation of Ser1029 by Mnb precedes a Proline residue, consistent with Mnb being a proline-directed kinase (highlighted in yellow). Gray shaded region represents the peptide fragment identified from MS. B, C, Validation that Mnb phosphorylates Synj at Ser1029 using the indicated antibodies. HA-tagged Synj^wt, Synj^S1029A, and Synj^S1029E were expressed in flies, immunoprecipitated using HA-agarose beads, and incubated with or without purified Mnb kinase following alkaline phosphatase (AP) treatment. Representative blots are shown, and results were confirmed in at least 3 independent experiments. Mutation at S1029 abolished the increased phosphorylation by Mnb.