Polypeptides of Mr 50,000 and 60,000 in isolated synaptic junctions have been compared to polypeptides of corresponding molecular weight in Ca2+/calmodulin-dependent protein kinase II. The polypeptides of corresponding molecular weight from the two preparations were shown by several criteria to be indistinguishable. These criteria included 12'I-labeled tryptic/chymotryptic peptide patterns, 32P-labeled proteolytic peptide maps, and crossreactivity on immunoblots using polyclonal and monoclonal antibodies. Furthermore, studies examining the phosphorylation of substrate proteins, by the endogenous synaptic junction kinase and by Ca2+/calmodulin-dependent protein kinase II, indicated that the two enzymes have similar substrate specificities. Since the Mr 50,000 polypeptide present in synaptic junctions is known to be the major postsynaptic density protein, the present results indicate that the major postsynaptic density protein is a component of Ca2+/calmodulin-dependent protein kinase II.A number of Ca2+/calmodulin-dependent protein kinases have recently been identified in neuronal tissue (for review, see ref. 1). One of these is a kinase purified from rat brain (2) that phosphorylates synapsin I, a synaptic vesicle-associated neuronal protein (3,4). This kinase, designated Ca2+/calmodulin-dependent protein kinase II (calmodulin kinase II), has recently been purified to near homogeneity (5, 6) and exhibits a major Mr 50,000 polypeptide and a less prominent Mr 60,000 polypeptide when analyzed by NaDodSO4/polyacrylamide gel electrophoresis. These Mr 50,000 and 60,000 polypeptides coelute with the peak of kinase activity through a variety of purification procedures, are themselves phosphorylated in a Ca2+/calmodulin-dependent manner, and are calmodulin-binding proteins (5,6).Grab et al. (7) found a calmodulin-sensitive protein kinase in a postsynaptic density (PSD) preparation isolated from canine cerebral cortex. An endogenous Mr 50,000 polypeptide is both a substrate for this kinase (7) and a calmodulin-binding protein (8). This Mr 50,000 polypeptide is the most abundant PSD protein and has been designated the major PSD protein (mPSDp) (9-11). This PSD preparation also contains a substrate protein (7) and a calmodulin-binding protein (8) in the Mr 60,000 range. Substrate proteins (12) and calmodulinbinding proteins (13,14) of Mr 50,000 and 60,000 have also been observed in PSD and synaptic junction (SJ) preparations isolated from rat forebrain. The SJ Mr 50,000 polypeptide is identical to the mPSDp (11,14). Therefore, either SJ or PSD preparations can be used as a source for the mPSDp.The similarities between the Mr 50,000 and 60,000 polypeptides in calmodulin kinase II and those of corresponding molecular weight in SJ preparations prompted us to examine their possible relationship. A preliminary account of this work has been reported (15). METHODSSubcellular Fractionation. Whole forebrains were used to isolate SJ fractions by the iodonitrotetrazolium violet/Triton X-100 method as described (11). Care was t...

A calcium/calmodulin-dependent protein kinase, which phosphorylates a synaptic vesicle-associated protein designated Synapsin I, has been shown to be present in both soluble and particulate fractions of rat brain homogenates. In the present study, the particulate activity was solubilized by washing with a low ionic strength solution, and the enzymes from the two fractions were partially purified by ion exchange chromatography and calmodulin-Sepharose affinity chromatography. By each of several criteria, the partially purified enzymes from the two sources were indistinguishable. These criteria included specificity for various substrate proteins, concentration dependence of activation by calcium and calmodulin, pH dependence, and apparent affinities for the substrates Synapsin I and ATP. The mild conditions that released the particulate enzyme indicated that it was not tightly bound to the membrane and suggested that it may exist in a dynamic equilibrium between soluble and particulate-bound states.The partially purified enzyme preparations from both the soluble and particulate fractions contained three proteins that were phosphorylated in the presence of calcium and calmodulin, a 50-kilodalton (Kd) protein and two proteins in the 60-Kd region. When compared by phosphopeptide mapping and two-dimensional gel electrophoresis, the proteins were indistinguishable from three proteins of corresponding molecular weights that were shown by Schulman and Greengard (Schulman, H., and P. Greengard (1978) Nature 271: 478-479) to be prominent substrates for calcium/ calmodulin-dependent protein kinase in a crude particulate preparation from rat brain. The 50-Kd substrate was the major Coomassie blue staining protein in both partially purified enzyme preparations. The peak of this protein coincided with that of enzyme activity during DEAE-cellulose and calmodulin-Sepharose chromatography. These results suggest that the 50-Kd phosphoprotein may be an autophosphorylatable subunit of the Synapsin I Kinase or may exist in a complex with it.

Protein phosphorylation is involved in the regulation of a wide variety of physiological processes in the nervous system. Studies in which purified protein kinases or kinase inhibitors have been microinjected into defined cells while a specific response is monitored have demonstrated that protein phosphorylation is both necessary and sufficient to mediate responses of excitable cells to extracellular signals. The precise molecular mechanisms involved in neuronal signal transduction processes can be further elucidated by identification and characterization of the substrate proteins for the various protein kinases. The roles of three such substrate proteins in signal transduction are described in this article: 1) synapsin I, whose phosphorylation increases neurotransmitter release and thereby modulates synaptic transmission presynaptically; 2) the nicotinic acetylcholine receptor, whose phosphorylation increases its rate of desensitization and thereby modulates synaptic transmission postsynaptically; and 3) DARPP-32, whose phosphorylation converts it to a protein phosphatase inhibitor and which thereby may mediate interactions between dopamine and other neurotransmitter systems. The characterization of the large number of additional phosphoproteins that have been found in the nervous system should elucidate many additional molecular mechanisms involved in signal transduction in neurons.

The molecular events that control synaptic vesicle availability in chemical synapticjunctions have not been fully clarified. Among the protein molecules specifically located in presynaptic terminals, synapsin I and calcium/calmodulindependent protein kinase H (CaM kinase II) have been shown to modulate evoked transmitter release in the squid giant synapse. In the present study, analysis of synaptic noise in this chemical junction was used to determine whether these proteins also play a role in the control of spontaneous and enhanced spontaneous transmitter release. Injections of dephosphorylated synapsin I into the presynaptic terminal reduced the rate of spontaneous and enhanced quantal release, whereas injection of phosphorylated synapsin I did not modify such release. By contrast CaM kinase II injection increased enhanced miniature release without affecting spontaneous miniature frequency. These results support the view that dephosphorylated synapsin I "cages" synaptic vesicles while CaM kinase II, by phosphorylating synapsin I, "decages" these organelles and increases their availability for release without affecting the release mechanism itself. Synapsin I, a neuron-specific protein present in nerve terminals (1), has recently been shown to be a powerful modulator of neurotransmitter release (2). This protein is known to adhere to synaptic vesicles and may cross-link them to the cytoskeleton at or near the active zone in presynaptic terminals (3). Synapsin I is a substrate for calcium/calmodulindependent protein kinase II (CaM kinase II) (4). The affinity of synapsin I for synaptic vesicles (5) and for actin (3) is reduced after phosphorylation by CaM kinase II. The physiological role of synapsin I and CaM kinase II has been studied in the squid giant synapse (2) and in the goldfish Mauthner cell (6). The results from these studies indicated that transmitter release is inhibited if dephosphorylated synapsin I is injected into the presynaptic terminal, but that it is not affected by injection of phosphorylated synapsin 1 (2, 6). Our results further indicated that CaM kinase II injections induced a significant increase of transmitter release (2).Since voltage clamp studies showed that the amplitude of the presynaptic calcium current was not modified by the injection of any of these proteins (2), it was proposed that the phosphorylation of synapsin I by CaM kinase II allows the vesicles to be freed from a bound or "caged" state and to become available for release (2, 7-9). Consistent with this hypothesis, recent video microscopic observations indicate that organelle movements in the squid axoplasm are markedly reduced after the addition of dephosphorylated synapsin I, but not after administration of phosphorylated synapsin I (10).A corollary of this hypothesis is that spontaneous and enhanced quantal transmitter release may be subjected to the same type of regulation by synapsin I and CaM kinase II. In this report, noise analysis was used to demonstrate that, depending on its state of phosphorylation, s...

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