Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Abstract

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity . As seen by autoradiographic analysis of Coomassie Blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [y-32 P]ATP and Mg t+ alone. Addition of Ca t+ alone to the native PSD preparation had little or no effect on phosphorylation . However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 Mr. Similar results were also obtained in sonicated or freeze-thawed native PSD preparations by addition of Ca t+ alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions . The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulinactivatable phosphorylation, but not of the Mgt'-dependent activity, by the Ca" chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca 21 . In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [Y-32 P]ATP, Mgt ' and Ca 21, one could not induce a Ca t+ -stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins . Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 Mr PSD protein is the one that is phosphorylated and not the 51,000 Mr component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 Mr protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg t+ -dependent kinase(s) . Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins l a and lb, the cyclic AMP-dependent protein kinase, which definitely does phosphorylate proteins l a and Ib and is present in the PSD, does not phosphorylate the 51,000 and 62,000 Mr proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins .In the previous paper we have shown that the postsynaptic density (PSD) contains a cryptic calmodulin-activatable cyclic nucleotide phosphodiesterase activity (29). A further examination of the possible function of the calmodulin in the PSD was prompted by the demonstration by several groups of the existence of calmodulin-dependent protein kinases . Calmodulin has been shown to be a subunit of the Ca"-dependent myosin light chain kinase (14,15,30,41,59,60,62,64) as well as activating muscle (12, 53) and plat...

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

“…It has been proposed that they are primarily structural, serving to anchor membrane or cytosolic proteins in the region of the postsynaptic membrane (21). The fraction is enriched in cyclic nucleotide phosphodiesterase activity (22,23), calmodulin (24), and both-cAMP- (25)(26)(27) and calmodulin-dependent protein kinase activities (27)(28)(29). Immunocytochemical evidence suggests that a calmodulin-dependent protein phosphatase, calcineurin, may also be located in PSDs in situ (30,31).…”

“…The major component of PSD fractions, maldng up 10-30% of the total protein, is a 50-kilodalton (kDa) protein of unknown function termed the "major PSD protein" (mPSDp) (18). This protein binds calmodulin (32,33) and is also a substrate for a calmodulin-dependent protein kinase (28,32).…”

“…We will refer to it as synapsin I kinase to distinguish it from other calmodulin-dependent protein kinases. It is found in both soluble and particulate fractions of brain homogenates (37) (34,(37)(38)(39), where they account for a major portion of the endogenous "substrates" for calmodulin-dependent protein kinase in the 50-to 60-kDa region of NaDodSO4/polyacrylamide gels (37).Proteins of the same molecular weights are phosphorylated by a calmodulin-dependent protein kinase in purified PSD preparations (28,29 …”

“…Proteins of the same molecular weights are phosphorylated by a calmodulin-dependent protein kinase in purified PSD preparations (28,29 …”

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin

“…Ca2+-and calmodulin-dependent protein kinase systems associated with the particulate fraction of brain have been studied in several laboratories: Ca2+-and calmodulin-dependent phosphorylation of A4, 51000 and 62000 membranous proteins by an intrinsic enzyme system [ 18,191; a particulate enzyme system catalyzing the Ca2+-and calmodulin-dependent phosphorylation of protein I, a specific synaptic protein composed of M, 80000 and 86000 polypeptide chains [20]; a Ca2+-and calmodulin-dependent protein kinase system associated with the synaptic vesicle that specifically phosphorylates the vesicle LY-and P-tubulins [21]; Ca2+-and calmodulin-dependent phosphorylation of M, 51000 and 62000 proteins in postsynaptic densities [22]; using Torpedo californica electroplax membranes, an intrinsic Ca'+-and calmodulin-dependent protein kinase system for the specific phosphorylation of acetylcholine receptor subunit proteins (Mr 65000, 58000 and 50000) [23]. However, there have been no investigations reported on the solubilization and partial purification of Ca2+-and calmodulin-dependent protein kinases from brain membranes.…”