Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein ␤␥ subunit complex (G␤␥) that has been recently shown to catalyze the formation of the G␤␥ dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for G␤␥ dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the G␤␥-assembly process. The results reported here delineate a mechanism for G␤␥ assembly in which a stable ternary complex is formed between PhLP, the nascent G␤ subunit, and CCT that does not include G␥. PhLP phosphorylation permits the release of a PhLP⅐G␤ intermediate from CCT, allowing G␥ to associate with G␤ in this intermediate complex. Subsequent interaction of G␤␥ with membranes releases PhLP for another round of assembly.Eukaryotic cells employ heterotrimeric G proteins to transduce a wide variety of hormonal, neuronal, and sensory signals that control numerous physiological processes. As a result, malfunctions in G protein pathways contribute to many diseases (1-3), and therapeutic agents targeting G protein-coupled receptors represent the single largest class of current pharmaceuticals (4). There are three fundamental steps in the propagation of a G protein-mediated signal. First, a ligand binds a receptor, resulting in a change in the packing of the seven transmembrane ␣-helices found in all G protein-coupled receptors. Second, the activated receptor catalyzes exchange of GDP for GTP on the ␣ subunit of a heterotrimeric G protein (G␣) 2 on the intracellular surface of the receptor. GTP binding causes G␣ to dissociate from the G protein ␤␥ subunit complex (G␤␥). Third, the G␣⅐GTP and G␤␥ complexes control the activity of effector enzymes and ion channels that regulate the intracellular concentration of second messengers (cyclic nucleotides, inositol phosphates, and Ca 2ϩ ) and the plasma membrane electrical potential (mainly via K ϩ channels). Changes in these properties in turn orchestrate the cellular response to the stimulus (5).Phosducin-like protein (PhLP) is a member of the phosducin gene family (6 -8) that is believed to participate in G protein signaling by virtue of its ability to bind the G␤␥ dimer with high affinity (9 -11). Many in vitro and overexpression experiments have shown that PhLP binding to G␤␥ blocks its ability to interact with G␣ or effectors (9,10,(12)(13)(14). From these experiments, it was suggested that the physiological role of PhLP was to down-regulate G protein signaling by sequestering G␤␥. However, the results of several recent studies have seriously challenged this model. Specifically, disruption of the PhLP1 gene in the chestnut blight fungus Cryphonectria parasitica (15) and in the soil amoeba Dictyostelium discoideum (7) yielded the same phenotype as the disruption of the G␤ gene. Moreover, PhLP deletion blocked G protein signaling in Dictyosteliu...

Depolarization of synaptosomes is known to cause a calcium‐dependent increase in the phosphorylation of a number of proteins. It was the aim of this study to determine which protein kinases are activated on depolarization by analyzing the incorporation of 32P1 into synaptosomal phosphoproteins and phosphopeptides. The following well‐characterized phosphoproteins were chosen for study: phosphoprotein “87K,” synapsin Ia and Ib, phosphoproteins IIIa and IIIb, the catalytic subunits of calmodulin kinase II, and the B‐50 protein. Each was initially identified as a phosphoprotein in lysed synaptosomes after incubation with [γ‐32P]ATP. Mobility on two‐dimensional polyacrylamide gels and phosphorylation by specific protein kinases were the primary criteria used for identification. A technique was developed that allowed simultaneous analysis of the phosphopeptides derived from all of these proteins. Phosphopeptides were characterized in lysed synaptosomes after activating cyclic AMP‐, calmodulin‐, and phospholipid‐stimulated protein kinases in the presence of [γ‐32P]ATP. Phosphoproteins labelled in intact synaptosomes after incubation with 32Pi were then compared with those seen after ATP‐labelling of lysed synaptosomes. As expected from previous work, phosphoprotein “87K,” and synapsin Ia and Ib were‐labelled, but for the first time, phosphoproteins IIIa, IIIb, and the B‐50 protein were identified as being labelled in intact synaptosomes; the calmodulin kinase II subunits were hardly phosphorylated. From a comparison of the phosphopeptide profiles it was found that cyclic AMP‐, calmodulin‐, and phospholipidstimulated protein kinases are all active in intact synaptosomes and their activity is dependent on extrasynaptosomal calcium. The activation of cyclic AMP‐stimulated protein kinases in intact synaptosomes was confirmed by the addition of dibutryl cyclic AMP and theophylline which specifically increased the labelling of phosphopeptides in synapsin Ia and Ib and in phosphoproteins IIIa and IIIb. On depolarization of intact synaptosomes, a number of phosphopeptides showed increased labelling and the pattern suggested that cyclic AMP‐, calmodulin‐, and phospholipid‐stimulated protein kinases were all activated. No new peptides were phosphorylated, suggesting that depolarization simply increased the activity of already active protein kinases and that there was no depolarization‐specific increase in protein phosphorylation.