Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids

Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PRC). Dephosphorylation is r equired for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization, An in vitro phospholipid binding ass ay was established that prevents lipid vesiculation and dynamin lipid inser tion into the lipid. Dynamin I bound the phospholipid in a concentration-de pendent and saturable manner, with an apparent affinity of 230 +/- 51 nM. O ptimal binding occurred with mixtures of phosphatidylserine and phosphatidy lcholine of 1:3 with little binding to phosphatidylcholine or phosphatidyls erine alone. Phospholipid binding was abolished after dynamin I phosphoryla tion by PKC and was restored after dephosphorylation by calcineurin. Matrix -assisted laser desorption/ionization-time of flight mass spectrometry reve aled the phosphorylation site in PKC alpha-phosphorylated dynamin I as a si ngle site at Ser-795, located near a binding site for the SH3 domain of p85 , the regulatory subunit of phosphatidylinositol 3-kinase. However, phospho rylation had no effect on dynamin binding to a bacterially expressed p85-SH 3 domain. Thus, phosphorylation of dynamin I on Ser-795 prevents its associ ation with phospholipid, providing a basis for the cytosolic localization o f the minor pool of phospho dynamin I that mediates synaptic vesicle retrie val in nerve terminals.