Protein tyrosine phosphorylation in purified synaptic vesicles from rat forebrain has been studied in the presence of Mn2" and orthovanadate. High levels of endogenous protein tyrosine phosphorylation were observed. Four major phosphoproteins, with apparent molecular masses of 105, 94, 38, and 30 kDa, were shown to contain phosphotyrosine. The 38-kDa phosphoprotein was identified as synaptophysin (p38), a well-characterized integral membrane protein of synaptic vesicles. The three other phosphotyrosinecontaining proteins distributed in the same manner as synaptophysin in all subcelular fractions. Like synaptophysin, the two high molecular weight phosphotrosine proteins (105 and 94 kDa) were found to be glycoproteins by lectin chromatography. Tyrosine phosphorylation of synaptophysin was an intravesicular reaction and reached 50% of maximal level within 3 min. Triton X-100, a nonionic detergent, inhibited tyrosine phosphorylation of endogenous protein substrates but not the phosphorylation ofan exogenous substrate, poly(Gluse,-Tyrn0). Tyrosine phosphorylation of synaptophysin was also demonstrated in synaptosomes, indicating that tyrosine phosphorylation of synaptic vesicle proteins occurs in intact nerve terminals.There is now a great deal of evidence that protein phosphorylation is involved in the regulation of neuronal function (for reviews, see refs. 1 and 2). Serine/threonine phosphorylation of proteins in synaptic vesicles appears to play an important regulatory role' in nerve terminals. It has been demonstrated that serine phosphorylation of synapsin I, a synaptic vesicle-specific protein, is regulated by depolarizing reagents and by neurotransmitters (1). Phosphorylation of synapsin I by calcium/calmodulin-dependent protein kinase II appears to facilitate'neurotransmitter release (3).Phosphorylation of proteins on tyrosine residues has generally been thought to be associated with the regulation of cell growth and transformation (4). The intrinsic tyrosine kinase activities of certain hormone receptors and oncogene protein products have been shown to be necessary for their biological activities (5-7). However, the presence of high levels of tyrosine kinase activity in adult brain (8-11) and other nonproliferative tissues (8, 12, 13) were incubated at 30'C for 15 min. The reaction was terminated by adding 100 1.L of a "stop-solution" containing 12% (vol/vol) NaDodSO4, 20o (vol/vol) glycerol, 0.25 M TrisHCl (pH 7), and 0.01% bromophenol blue, followed by boiling for'3 min. The phosphorylated proteins were analyzed by NaDodSO4/PAGE (10% acrylamide) (15). After staining and destaining, the gels were dried, and the phosphoproteins were visualized by autoradiography.Alkali treatment of gels was carried out essentially as described (16). The destained gel was shaken in 250 ml of 1 M KOH at room temperature for 15 min, then in 500 ml of 1 M KOH at 60'C for 2 hr. and finally in 800 ml of destaining solution at room temperature for 1 hr. The dried gel was autoradiographed.Phosphoamino Acid Analysis. To det...

The amino acid sequences surrounding three major phosphorylation sites in rat and bovine synapsin I have been determined by employing automated gas-phase sequencing and manual Edman degradation of purified phosphopeptide fragments. Site 1 is a serine residue phosphorylated by cAMP-dependent protein kinase and by calcium/cahuodulindependent protein kinase I. The sequence around site 1 was derived from tryptic/chymotryptic phosphopeptides and overlapping cyanogen bromide cleavage fragments. This sequence, identical in rat and bovine synapsin I, is Asn-Tyr-Leu-ArgArg-Arg-Leu-Ser(P)-Asp-Ser-Asn-Phe-Met. Site 1 is located at the NH2 terminus of the protein, within the collagenaseresistant head region. Sites 2 and 3 are serine residues phosphorylated by calcium/calmodulin-dependent protein kinase II. The sequences surrounding bovine site 2 and site 3 were derived from tryptic phosphopeptides and overlapping fragments generated by cleavage with chymotrypsin, collagenase, and endoproteinase Lys-C. The sequence around bovine site 2 is Thr-Arg-Gln-Thr-Ser(P)-Val-Ser-Gly-Gln-Ala-ProPro-Lys, and the sequence around bovine site 3 is Thr-ArgGln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Met-Pro-Arg. Sites 2 and 3 are located within the COOH-terminal, collagenase-sensitive tail region of the molecule, separated by 36 amino acids. The sequences surrounding rat site 2 and site 3 were derived from tryptic phosphopeptides. The sequence around rat site 2 is Gln-Ala-Ser(P)-Ile-Ser-Gly-Pro-Ala-Pro-Pro-Lys, and the sequence around rat site 3 is Gln-Ala-Ser(P)-Gln-Ala-Gly-ProGly-Pro-Arg. Thus, the sequences surrounding the four sites that are phosphorylated by calcium/calmodulin-dependent protein kinase II, namely sites 2 and 3 in rat and bovine synapsin I, exhibit a high degree of homology.

The catalytically active, tyrosyl-phosphorylated form of insulin receptor kinase was isolated from human placenta by a procedure which exploits the propensity for the intact alpha 2 beta 2 form of insulin receptor to undergo insulin-promoted autophosphorylation at tyrosyl residues and concomitant activation as a tyrosyl kinase. Purification of tyrosyl-phosphorylated insulin receptor was effected by adsorption on and elution (with a hapten) from a column of O-phosphotyrosyl-binding antibody immobilized on protein A-Sepharose (Ab-protein A). The starting material for the purification process was protein which had been solubilized from placental membranes and purified by chromatography on immobilized wheat germ agglutinin. After chromatography on Ab-protein A to remove preexisting O-phosphotyrosyl-containing proteins, the fraction which did not adsorb to the Ab-protein A column was incubated with insulin and briefly treated with ATP so as to maximize selective autophosphorylation of insulin receptor. This material was then subjected to chromatography on Ab-protein A. Although the amount of the intact alpha 2 beta 2 form of insulin receptor present in the starting material was only a small fraction of the protein (approximately 0.2%) and only approximately 20% of the insulin-binding forms of the receptor present, it was eluted (with 10 mM p-nitrophenyl phosphate) from the column in greater than or equal to 80% purity. Chromatography on Ab-protein A appears to have an advantage over the alternative affinity chromatographic procedures which utilize immobilized insulin or antiinsulin receptor antibody to adsorb insulin receptor, since these procedures do not resolve the intact alpha 2 beta 2 form of insulin receptor from the nicked insulin-binding forms of the receptor which do not undergo insulin promoted autophosphorylation.

Using antiphosphotyrosine antibodies, we have investigated protein phosphorylation in mouse brain during development in intact animals and in reaggregated cerebral cultures. Under basal conditions, in vivo and in vitro, the levels of two main phosphoproteins, of Mr 120,000 and 180,000 (pp180), increased with development, reaching a maximum in the early postnatal period and decreasing thereafter. In adult forebrain, pp180 was still highly phosphorylated, but it was not detected in cerebellum or in peripheral tissues. In reaggregated cortical cultures, epidermal growth factor (EGF), type I insulin-like growth factor (IGF-I), and insulin enhanced protein tyrosine phosphorylation of several proteins, which were specific for EGF or IGF-I/insulin. In highly enriched neuronal or astrocytic monolayer cultures, some proteins phosphorylated in basal conditions, or in response to EGF and IGF-I, were found in both types of culture, whereas others appeared cell type specific. In addition, in each cell type, some proteins were phosphorylated under the action of both growth factors. These results indicate that tyrosine protein phosphorylation is maximal in mouse brain during development and is regulated by growth factors in neurons as well as in astrocytes.

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