Protein tyrosine phosphorylation in synaptic vesicles.

Abstract

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...

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity

“…The tyrosine kinase activity of USV was higher than that of STSV (Fig. 6), as evaluated by 32 P incorporation into either poly(Glu-80,Tyr-20) or endogenous vesicleassociated tyrosine kinase substrates such as synaptophysin and p29͞synaptogyrin (4,5). Phosphorylation of vesicleassociated proteins was not increased further by the addition of exogenous purified c-Src.…”

“…It seemed possible that domain D or other proline-rich regions in synapsin I might interact with other SH3 domain-containing proteins within the nerve terminal and that these interactions might have a physiological role in presynaptic function. One such candidate, the SH3 domain-containing nonreceptor tyrosine kinase c-Src, is expressed at high levels in postmitotic neurons and is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity (3)(4)(5)(6). Using purified components in vitro, we now report that a domain Dmediated interaction of synapsin I with the SH3 domain of c-Src results in a 5-fold activation of the catalytic activity of c-Src.…”

Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons

“…What are the potential mechanisms that preferentially favor the formation and͞or maintenance of syp-positive wildtype synapses over the mutant synapses? Syp is phosphorylated by both Ser͞Thr and Tyr kinases (17)(18)(19), and it is one of the major phosphotyrosine-containing proteins in the mature nerve terminal (20). Thus, it is possible that syp is a downstream target of a second messenger system which, when phosphorylated, promotes synapse stabilization by acting on other synaptic proteins required for maintaining the synaptic architecture.…”

Hypoxic Response of Synaptosomal Proteins in Term Guinea Pig Fetuses

“…Several proteins endowed with tyrosine kinase activity have been localized in synaptosomes, for example, pp60 c-src (Hirano et al, 1988;Pang et al, 1988), insulin receptor (e.g., Raizada et al, 1988), epidermal growth factor receptor (Faúndez et al, 1992), and proline-rich tyrosine kinase 2/cell adhesion kinase ␤ (Siciliano et al, 1996). Synaptophysin, a major protein in synaptosomes, is a well characterized tyrosine kinase substrate (Pang et al, 1988;Barnekow et al, 1990). Some tyrosine kinases have been proven to be associated with synaptosomal membrane glycoproteins (Hanissian and Sahyoun, 1992).…”

“…Analysis of subcellular fractions is a convenient approach to characterizing low-abundance proteins, which are likely to escape detection in wholecell proteome studies (Herbert et al, 1997). Several proteins endowed with tyrosine kinase activity have been localized in synaptosomes, for example, pp60 c-src (Hirano et al, 1988;Pang et al, 1988), insulin receptor (e.g., Raizada et al, 1988), epidermal growth factor receptor (Faúndez et al, 1992), and proline-rich tyrosine kinase 2/cell adhesion kinase ␤ (Siciliano et al, 1996). Synaptophysin, a major protein in synaptosomes, is a well characterized tyrosine kinase substrate (Pang et al, 1988;Barnekow et al, 1990).…”