DYNAMIN-I IS A CA2-SENSITIVE PHOSPHOLIPID-BINDING PROTEIN WITH VERY HIGH-AFFINITY FOR PROTEIN-KINASE-C()

Depolarization-induced Ca2+ influx into rat brain synaptosomes induces dephosphorylation of dephosphin, a 96-94-kDa protein kinase C (PKC) s ubstrate recently identified as dynamin I, a protein associated with e ndocytosis, We characterized purified dynamin I to better understand r egulation of its phosphorylation in nerve terminals, Purified dynamin I possessed a very high affinity for PKC but did not fit Michaelis-Men ten kinetics. It had an optimum phosphorylation rate of 1.42 +/- 0.02 mu mol/mg/min and a concentration giving half-maximal activity (S-0.5) of 0.14 +/- 0.02 mu M, the highest affinity reported for a PKC substr ate protein. Concentrations of dynamin greater than 0.5 mu M inhibited phosphorylation. The stoichiometry was 1.5, indicating more than one phosphorylation site. Dynamin was predominantly associated with the br ain particulate fraction under conditions of low ionic strength, and t his prevented its phosphorylation by PKC until released by moderate in creases in ionic strength (Na+, K+, and Mg2+) or by GTP or ATP. In int act synaptosomes the largest dynamin pool was associated with the part iculate fraction, while a smaller pool was cytosolic or extracted with 150 mM NaCl and contained all the phosphorylated protein. Purified dy namin also bound to phospholipid-coated controlled-pore glass beads, b ut poorly in the presence of NaCl, Mg2+, GTP, or ATP. Ca2+ induced a r eversible translocation from the cytosol to the particulate fraction ( 50% at 183 mu M Ca2+) in brain homogenates, and the purified protein a lso underwent Ca2+-sensitive translocation to phospholipid coated cont rolled-pore glass beads. We conclude that dynamin I is a nerve termina l Ca2+-sensitive phospholipid-binding protein with very high sub strat e affinity for PKC. We propose that phosphorylation by PKC occurs in t he nerve terminal soluble compartment and that Ca2+ may mediate its bi nding to the particulate fraction, thereby blocking the PKC phosphoryl ation sites. These properties may contribute to the lack of PKC phosph orylation during depolarization, despite the presence of activated PKC .