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Depolarization-induced Ca2+ influx into rat brain synaptosomes induces dephosphorylation of dephosphin, a 96-94-kDa protein kinase C (PKC) substrate recently identified as dynamin I, a protein associated with endocytosis. We characterized purified dynamin I to better understand regulation of its phosphorylation in nerve terminals. Purified dynamin I possessed a very high affinity for PKC but did not fit Michaelis-Menten kinetics. It had an optimum phosphorylation rate of 1.42 +/- 0.02 mumol/mg/min and a concentration giving half-maximal activity (S0.5) of 0.14 +/- 0.02 microM, the highest affinity reported for a PKC substrate protein. Concentrations of dynamin greater than 0.5 microM inhibited phosphorylation. The stoichiometry was 1.5, indicating more than one phosphorylation site. Dynamin was predominantly associated with the brain particulate fraction under conditions of low ionic strength, and this prevented its phosphorylation by PKC until released by moderate increases in ionic strength (Na+, K+, and Mg2+) or by GTP or ATP. In intact synaptosomes the largest dynamin pool was associated with the particulate fraction, while a smaller pool was cytosolic or extracted with 150 nM NaCl and contained all the phosphorylated protein. Purified dynamin also bound to phospholipid-coated controlled-pore glass beads, but poorly in the presence of NaCl, Mg2+, GTP, or ATP. Ca2+ induced a reversible translocation from the cytosol to the particulate fraction (50% at 183 microM Ca2+) in brain homogenates, and the purified protein also underwent Ca(2+)-sensitive translocation to phospholipid-coated controlled-pore glass beads. We conclude that dynamin I is a nerve terminal Ca(2+)-sensitive phospholipid-binding protein with very high substrate affinity for PKC. We propose that phosphorylation by PKC occurs in the nerve terminal soluble compartment and that Ca2+ may mediate its binding to the particulate fraction, thereby blocking the PKC phosphorylation sites. These properties may contribute to the lack of PKC phosphorylation during depolarization, despite the presence of activated PKC.

Type

Journal article

Journal

The Journal of biological chemistry

Publication Date

08/1994

Volume

269

Pages

21043 - 21050

Addresses

Endocrine Unit, John Hunter Hospital, Newcastle, New South Wales, Australia.

Keywords

Synaptosomes, Animals, Rats, Rats, Sprague-Dawley, Calcium, GTP Phosphohydrolases, Dynamins, Dynamin I, Protein Kinase C, Phospholipids, Precipitin Tests, Electrophoresis, Polyacrylamide Gel, Amino Acid Sequence, Protein Binding, Phosphorylation, Kinetics, Osmolar Concentration, Molecular Sequence Data