The N-methyl-D-aspartate (NMDA) receptor contributes to synaptic plasticity in the central nervous system and is both serine-threonine and tyrosine phosphorylated. In CA1 pyramidal neurons of the hippocampus, activators of protein kinase C (PKC) as well as the G-protein-coupled receptor ligands muscarine and lysophosphatidic acid enhanced NMDA-evoked currents. Unexpectedly, this effect was blocked by inhibitors of tyrosine kinases, including a Src required sequence and an antibody selective for Src itself. In neurons from mice lacking c-Src, PKC-dependent upregulation was absent. Thus, G-protein-coupled receptors can regulate NMDA receptor function indirectly through a PKC-dependent activation of the non-receptor tyrosine kinase (Src) signaling cascade.

Abstract. Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825. To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADE We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADE Immunoprecipitation of both isoforrns from extracts of cells prelabeled with [32p]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADE pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from ~,,18% to 150% of the amount of unphosphorylated ADE pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre-and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.

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