J. Biol. Chem., Vol. 263, Issue 33, 17209-17212, 11, 1988

Autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals

FS Gorelick, JK Wang, Y Lai, AC Nairn and P Greengard
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.

The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2- 5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				B. P. Harvey, S. S. Banga, and H. L. Ozer Regulation of the Multifunctional Ca2+/Calmodulin-dependent Protein Kinase II by the PP2C Phosphatase PPM1F in Fibroblasts J. Biol. Chem., June 4, 2004; 279(23): 24889 - 24898. [Abstract] [Full Text] [PDF]

				S. C. Sansom, R. Ma, P. K. Carmines, and D. A. Hall Regulation of Ca2+-activated K+ channels by multifunctional Ca2+/calmodulin-dependent protein kinase Am J Physiol Renal Physiol, August 1, 2000; 279(2): F283 - F288. [Abstract] [Full Text] [PDF]

				L. Lou, T. Zhou, P. Wang, and G. Pei Modulation of Ca2+/Calmodulin-Dependent Protein Kinase II Activity by Acute and Chronic Morphine Administration in Rat Hippocampus: Differential Regulation of alpha  and beta  Isoforms Mol. Pharmacol., March 1, 1999; 55(3): 557 - 563. [Abstract] [Full Text]

				M. A. Tehrani, M. C. Mumby, and C. Kamibayashi Identification of a Novel Protein Phosphatase 2A Regulatory Subunit Highly Expressed in Muscle J. Biol. Chem., March 1, 1996; 271(9): 5164 - 5170. [Abstract] [Full Text] [PDF]

				H Bading, D. Ginty, and M. Greenberg Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways Science, April 9, 1993; 260(5105): 181 - 186. [Abstract] [PDF]