Robert J. DeLorenzo

Identification of the Major Postsynaptic Density Protein as Homologous with the Major Calmodulin-Binding Subunit of a Calmodulin-Dependent Protein Kinase

Abstract: The major postsynaptic density protein (mPSDp), comprising >50% of postsynaptic density (PSD) protein, is an endogenous substrate for calmodulin‐dependent phosphorylation as well as a calmodulin‐binding protein in PSD preparations. The results in this investigation indicate that mPSDp is highly homologous with the major calmodulin‐binding subunit (p) of tubulin‐associated calmodulin‐dependent kinase (TACK), and that PSD fractions also contain a protein homologous with the o‐subunit of TACK. Homologies between mPSDp and a 63,000 dalton PSD protein and the p‐ and ó‐subunits of TACK were established by the following criteria: (1) identical apparent molecular weights: (2) identical calmodulin‐binding properties; (3) manifestation of Ca2+ ‐calmodulin‐stimulated autophosphorylation; (4)identical isoelectric points; (5) identical calmodulin binding and autophosphorylation patterns on two‐dimensional gels; (6) homologous two‐dimensional tryptic peptide maps; and (7) similar phosphoamino acid‐specific phosphorylation of tubulin. The results suggest that mPSDp is a calmodulin‐binding protein involved in modulating protein kinase activity in the postsynaptic density and that a tubulin kinase system homologous with TACK exists in a membrane‐bound form in the PSD.

Genetic control of two electrophoretic variants of glucosephosphate isomerase in the mouse (Mus musculus)

The autosomal variation and the genetic control of GPI has been determined by a comparison of electrophoretic patterns of F1 and backcross progeny of three inbred strains of mice. The locus controlling the production of GPI in the mouse has been designated Gpi-1. Two alleles at this locus have been described and designated Gpi-1a and Gpi-1b, which represent, respectively, the slow and fast electrophoretic forms. Twenty-seven inbred strains of mice have been classified for these two alleles. The absence of close linkage of Gpi-1 to seven other genetic loci has been determined. It has been demonstrated that the polymorphism of Gpi-1 is widely distributed in feral mice. GPI was expressed in vitro and in four types of malignant tumors.

Calcium/Ganglioside‐Dependent Protein Kinase Activity in Rat Brain Membrane

Abstract: The effects of gangliosides on phosphorylation were studied in rat brain membrane. Gangliosides stimulated phosphorylation only in the presence of Ca2+ with major phosphoproteins of 45,000, 50,000, 60,000, and 80,000 daltons and high‐molecular‐weight species. In addition, gangliosides inhibited the phosphorylation of three proteins with molecular weights of 15,000, 20,000, and 78,000 daltons. The two low‐molecular‐weight proteins comigrated with rat myelin basic proteins. Ganglioside stimulation was dependent on the formation of a Ca2+ ganglioside complex since the calcium salt of gangliosides stimulated phosphorylation maximally. Disialo and trisialo gangliosides were more potent stimulators of kinase activity than the monosialo GM1. GD1a was the most potent activator tested. Asialo‐GM1, cerebroside, sialic acid, neuraminyllactose, sulfatide, and the acidic phospholipids phosphatidylserine and phosphatidylinositol did not stimulate kinase activity. The Ca2+ ‐dependent, ganglioside‐stimulated phosphorylation was qualitatively similar to the pattern for calmodulin‐dependent phosphorylation. However, while calmodulin‐dependent kinase activity was inhibited with an IC50 of 10 μM trifluoperazine, ganglioside‐stimulated kinase was inhibited with an IC50 of 200 μM trifluoperazine. These results indicate that gangliosides have complex effects on membrane‐associated kinase activities and suggest that Ca2+ ‐ganglioside complexes are potent stimulators of membrane kinase activity.

The calmodulin hypothesis of neurotransmission

Ca2+ plays a major role in neurotransmission and synaptic modulation. Evidence is presented to support the calmodulin hypothesis of neurotransmission developed in this laboratory stating that calmodulin, a major Ca2+ binding protein in brain, mediates the effects of Ca2+ on neurotransmission. Calmodulin was isolated from highly enriched preparations of synaptic vesicles and nerve terminal cytoplasm. Ca2+ and calmodulin were shown to regulate several synaptic processes in isolated and intact preparations, including endogenous synaptic Ca2+-calmodulin protein kinase activity, neurotransmitter release, and synaptic vesicle and synaptic membrane interactions. Ca2+ and calmodulin were shown to activate a synaptic tubulin kinase system which was shown to be a distinct enzyme system from the cyclic AMP protein kinase. Ca2+ and calmodulin stimulated phosphorylation of tubulin altered the properties of tubulin, forming insoluble tubulin fibrils. Evidence for the role of Ca2+-calmodulin kinase activity, especially the calmodulin-tubulin kinase, in neurotransmission are presented. The effects of several neuroactive drugs on the synaptic calmodulin system are presented. The results support the hypothesis that calmodulin mediates many of calciums actions at the synapse, and that the effects of Ca2+ on synaptic protein phosphorylation, especially synaptic tubulin, may provide a biochemical mechanism for converting the Ca2+ signal into a motor force in the process of neurotransmission.

ROLE OF CALMODULIN IN NEUROTRANSMITTER RELEASE AND SYNAPTIC FUNCTION

An understanding of the molecular mechanisms underlying calcium-dependent neurotransmitter release ;and other calcium-modulated synaptic functions would greatly enhance our knowledge of synaptic transmission and the action of specific neuropharmacologic agents and possibly provide new insights into human disease processes involving synaptic modulation. Although the role of calcium in synaptic function h a been of great interest, little is known about the molecular mechanisms of calcium in stimulating neurotransmitter release or its other physiological functions in the nerve terminal. It is generally accepted that many of calciums effects on living tissue are mediated by heat-stable, calcium binding regulator proteins. Calmodulin is a major calcium receptor protein in brain and other tissues that has been well characterized and shown to affect several important enzyme systems. Thus, my laboratory has been investigating the possible role of calmodulin in modulating the effects of calcium or neurotransmitter releases and synaptic f u n c t i ~ n . ~ ~ In this report additional evidence is presented to further establish the role of calmodulin in neurotransmitter release and synaptic function. Calmodulin is isolated from synaptosome cytosol and highly enriched synaptic vesicle preparations. Calmodulin and calcium are demonstrated to stimulate the release of acetylcholine and norepinephrine from synaptic vesicles while simultaneously activating synaptic vesicle protein phosphorylation. Depolarization-dependent calcium uptake into synaptosomes is shown to simultaneously stimulate calmodulin modulated vesicle protein phosphorylation and release of acetylcholine and norepinephrine. Calmodulin is also shown to modulate the effects of Ca2+ on protein phosphorylation in preparations of synaptic junctional complexes and postsynaptic densities. In an attempt to investigate the mechanism of calcium and calmodulin in stimulating the release of transmitter from vesicles, a morphological study was undertaken to determine if synaptic vesicles change shape or configuration in the presence of calcium and calmodulin. The results suggest that calcium and calmodulin initiate synaptic vesicle and synaptic membrane interactions that may play a role in mediating the effects of these agents on synaptic function. The results presented in this report provide evidence that calmodulin mediates several synaptic events and are consistent with and expand the initial hypothesis from this laboratory that calmodulin plays a major role in modulating the effects of Caz+ on synaptic function and neurotransmitter

Calcium dependent neurotransmitter release and protein phosphorylation in synaptic vesicles

A highly purified preparation of synaptic vesicles was prepared to study the relationship between calcium-dependent neurotransmitter release and protein phosphorylation. Calcium ions simultaneously produced significant increases in both the endogenous release of norepinephrine from the synaptic vesicles and the endogenous incorporation of [32p] phosphate into specific synaptic vesicle proteins. The results are compatible with the hypothesis that the action of calcium on the phosphorylation of specific synaptic vesicle proteins is the molecular mechanism mediating some of the effects of calcium on neurotransmitter release and synaptic vesicle function.

Glutamate oxalate transaminase (GOT) genetics in Mus musculus: Linkage, polymorphism, and phenotypes of the Got-2 and Got-1 loci

A comparison of electrophoretic patterns of F1 and backcross progeny of two inbred strains of mice has revealed a new autosomal variant of the mitochondrial form of GOT. The loci controlling the production of the soluble and mitochondrial forms of GOT have been designated Got-1 and Got-2, respectively. The two alleles of the Got-2 locus have been designated Got-2a and Got-2b, which represent the slow- and fast-migrating electrophoretic forms. Twenty-seven inbred strains of mice have been classified for Got-2a and Got-2b. It has been demonstrated that the polymorphism of Got-2 is widely distributed in feral mice. Got-2 was shown to be linked to Es-1, and evidence is also presented for linkage between Got-2 and Es-2, Es-5, and oligosyndactyly (Os). The absence of linkage of Got-2 to seven other loci has also been demonstrated. GOT was expressed in vitro in cell lines derived from human and mouse tissues.

Calcium-dependent phosphorylation of specific synaptosomal fraction proteins: Possible role of phosphoproteins in mediating neurotransmitter release

Abstract Calcium ions caused a marked increase in the level of endogenous phosphorylation of specific proteins from synaptosomal fractions prepared from rat cerebral cortex. The levels of phosphorylation of these specific proteins were dependent upon the presence of calcium and regulated by small changes in the concenrration of calcium ions. The effect of calcium was independent of ATP concentration over a wide range of concentrations. The results are compatible with the hypothesis that some of the effects of calcium on synaptic transmission might be mediated by the effect of calcium on the phosphorylation of specific synaptosomal proteins.

Phosphorylation of Microtubule-Associated Protein 2 at Distinct Sites by Calmodulin-Dependent and Cyclic-AMP-Dependent Kinases

Abstract: Microtubule‐associated protein 2 (MAP2) is an excellent substrate for both cyclic‐AMP (cAMP)‐dependent and Ca2+/calmodulin‐dependent kinases. A recently purified cytosolic Ca2+/calmodulin‐dependent kinase (now designated CaM kinase II) phosphorylates MAP2 as a major substrate. We now report that microtubule‐associated cAMP‐dependent and calmodulin‐dependent protein kinases phosphorylate MAP2 on separate sites. Tryptic phosphopeptide digestion and two‐dimensional phosphopeptide mapping revealed 11 major peptides phosphorylated by microtubule‐associated cAMP‐dependent kinase and five major peptide species phosphorylated by calmodulin‐dependent kinase. All 11 of the cAMP‐dependently phosphorylated peptides were phosphorylated on serine residues, whereas four of five major peptides phosphorylated by the calmodulin‐dependent kinase were phosphorylated on threonine. Only one peptide spot phosphorylated by both kinases was indistinguishable by both migration and phosphoamino acid site. The results indicate that cAMP‐dependent and calmodulin‐dependent kinases may regulate microtubule and cytoskeletal dynamics by phosphorylation of MAP2 at distinct sites.

Kindling induces a long-lasting change in the activity of a hippocampal membrane calmodulin-dependent protein kinase system

Septal kindling has been shown to produce a long-lasting decrease in endogenous calcium/calmodulin-dependent phosphorylation of hippocampal synaptic plasma membrane proteins, including two major bands of approximately 50,000 and 60,000 Daltons. These two proteins differ from the B-50 protein and tubulin, as evidenced by differences in migration in SDS-PAGE gels and by lack of cross-immunoreactivity with specific antibodies. Identity of these two proteins with the rho and sigma subunits of purified calmodulin-dependent kinase (CaM Kinase II) is suggested by similar migration in SDS-PAGE and two-dimensional gels, by similar calmodulin binding in two-dimensional gels, and similar 125I-peptide mapping of the 50,000 Dalton protein. These results demonstrate that septal kindling is associated with changes in the activity of a major Ca2+/calmodulin-dependent kinase system in hippocampal synaptic plasma membrane. This long-lasting modulation of kinase activity may provide a molecular insight into some aspects of neuronal plasticity.