Phosphorylation of Dynamin I on Ser-795 by Protein Kinase C Blocks Its Association with Phospholipids

Abstract

Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 ؎ 51 nM. Optimal binding occurred with mixtures of phosphatidylserine and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone. Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed the phosphorylation site in PKC␣-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus, phosphorylation of dynamin I on Ser-795 prevents its association with phospholipid, providing a basis for the cytosolic localization of the minor pool of phospho-dynamin I that mediates synaptic vesicle retrieval in nerve terminals.

Relevance of Dopamine Signals Anchoring Dynamin-2 to the Plasma Membrane during Na+,K+-ATPase Endocytosis

“…Experiments demonstrating the interaction of p85␣ subunit of PI 3-kinase with dynamin have been performed in vitro (39,42). Our data further indicate that such interaction also occurs in intact cells and is enhanced in response to GPCR signals.…”

“…Most studies have indicated that dyn1 is a substrate for PKC. Phosphorylation of dyn1 occurs at Ser 795 , and this effect also blocks its association to phospholipids (39). Using specific dyn2 and phosphoserine antibodies, our results suggest that dyn2 is also a good phosphosubstrate, most likely for PKC.…”

“…Phosphorylated dyn2 is known to interact with many proteins as part of an "endocytic network," among them the PI 3-kinase p85␣ subunit (39), and such interaction has been shown to occur in vitro (42), but no reports have demonstrated to date its existence in intact cells. Because DA favors the interaction of PI 3-kinase with the Na ϩ ,K ϩ -ATPase ␣-subunit (14), we next examined whether an active PI 3-kinase (that interacts with Na ϩ ,K ϩ -ATPase molecules) might represent a possible anchor signal by interacting in vivo with dyn2.…”

“…Dopamine Dephosphorylates Dynamin-2 during Na ϩ ,K ϩ -ATPase Endocytosis by Activating PP2A-The state of dynamin phosphorylation/dephosphorylation is considered to be of importance for its recruitment to membrane (27,39). Therefore, we initially established whether DA increases protein phosphatase activity, and whether this effect was associated with inhibition of Na ϩ ,K ϩ -ATPase activity and endocytosis of molecules.…”

Dynamin Participates in Focal Extracellular Matrix Degradation by Invasive Cells

“…For example, a candidate in mediating Dyn2 function at the EDS might be cortactin, a tyrosine-phosphorylated cortical actin-binding protein proposed to play a role in tumor cell invasion (reviewed in Weed and Parsons, 2001), and also known to 1) mediate the regulatory function of dynamin on actin in membrane ruffling (McNiven et al, 2000b), and 2) be localized to, and required for, ECM degradation at invadopodia (Bowden et al, 1999). Interestingly, dynamin is also the target of multiple regulatory inputs such as protein kinase C (Powell et al, 2000), phosphoinositides (Lee et al, 1999;Vallis et al, 1999;Muhlberg and Schmid, 2000), and calcineurin (Liu et al, 1994;Slepnev and De Camilli, 2000). Thus, dynamin and its interacting partners are possible molecular targets for the development of antiinvasive agents.…”