Phosphorylation Characteristics of Brain Clathrin-Coated Vesicle Endogenous Proteins

Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin-coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl-dependent enzymes in the following order of potency: Zn2+ greater than Hg2+ greater than Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodinitrobenzene, phenacyl bromide, and N-ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from dis-assembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin-coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components.     

Calmodulin Affinity for Brain Coated Vesicle Proteins

A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of 125I-labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin-C. Scatchard analysis revealed one high-affinity, low-capacity binding site, KD = 3.9 +/- 0.6 nM, Bmax = 16.3 +/- 2.4 pmol/mg, and one low-affinity, high-capacity binding site, KD = 102 +/- 15.0 nM, Bmax = 151 +/- 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 microgram calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X-100-treated extract of coated vesicles was passed through a Sepharose 4B-calmodulin affinity column, polypeptides with Mrs (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca2+-dependent manner. A 30,000 Mr protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca2+-Mg2+]-ATPase activity as well as Ca2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 Mr calmodulin binding proteins represent the [Ca2+-Mg2+]-ATPase complex, the other coated vesicle calmodulin binding site.     

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.     

Organization and dynamics of lipids in bovine brain coated and uncoated vesicles

Three characteristics have been demonstrated by the chemical analysis of bovine brain coated vesicles following removal of the coat proteins: a high protein content, a high cholesterol/lipid ratio and a high percentage of phosphatidylethanolamine amongst the phospholipids.The study of lipid bilayer organization and dynamics has been performed using the fluorescent probes pyrene and parinaric acid (cis and trans). This has allowed the study of both lateral mobility and rotational motion in the lipid bilayer of the coated and uncoated vesicles.Lateral mobility in the fluid phase of the lipid is slightly reduced by the presence of the clathrin coat, as indicated by the lower diffusion coefficient of pyrene in coated compared with uncoated vesicles.At all temperatures from 6° to 30°C, solid-phase domains, probed by trans parinaric acid, coexist with fluid-phase domains in the lipid bilayer. The temperature dependence of the parinaric acid lifetimes and of their amplitudes strongly suggests that the solid phase domains decrease in size with temperature, both in coated and uncoated vesicles.However, the difference in the value of the anisotropy at long times (r ), between coated and uncoated vesicles (a difference which is more pronounced for cis than for trans parinaric acid), indicates that the presence of the clathrin coat introduces disorder in the surrounding lipids, thus suggesting a possible role of the clathrin in the formation of the pits on the plasma membrane.Abbreviations CVs coated vesicles - UVs uncoated vesicles - TLC thin layer chromatography - DMSO dimethylsulfoxide - DPPC dipalmitoylphosphatidylcholine - cis Pna cis parinaric acid - (9,11,13,15-cis-trans-trans-cis) octadecatetraenoic acid - Trans Pna Trans parinaric acid - (9,11,13,15-all-trans) octadecatetraenoic acid     

Organization of Brain Synaptic Vesicle Proteins

Abstract: The topographical arrangement of proteins and glycoproteins of mouse brain synaptic vesicles was studied with trypsin and galactose oxidase, reagents known to be impermeable with respect to other membranes. Incubation of vesicles with trypsin at a concentration of 1 μg/ml extensively degraded seven polypeptides of molecular weights (M.W.) (×10^-3) 125, 107, 95, 83, 70, 60, and 36; higher concentrations degraded two additional species of 75,000 and 46,000 M.W., while leaving unaffected polypeptides of M.W. 66,000, 55,000, 33,000, 26,000, 22,000, 19,000, and 16,000. All of the trypsin-sensitive species of greater than 70,000 M.W. stained positively with the periodic acid-Schiff reagent; several other glycoproteins, all of M.W. less than 70,000, were identified, and all of these were insensitive to trypsin. Galactose oxidase-NaB³H₄ treatment of synaptic vesicles heavily and exclusively labeled material of greater than 70,000 M.W. All of the polypeptides studied were sensitive to each reagent when the synaptic vesicles were first treated with detergents. Extraction of vesicles with 0.05 M-NaOH partially or completely removed a wide variety of polypeptides, including most of those in the M.W. range 46,000–83,000; none of the glycoproteins was solubilized. Essentially the opposite results were obtained when the vesicles were extracted with 0.5% Triton X-100. Most of the vesicle's species were insensitive to several bisimidate cross-linking reagents. These results suggest that: (a) The polypeptides of M.W. 125K, 107K, 95K, 83K, 75K, 70K, 60K, 46K, and 36K are externally oriented in the vesicle, whereas those of 66K, 55K, 33K, 26K, 22K, 19K, and 16K are internally oriented; (b) the vesicles contain two classes of glycoproteins, one consisting of high-molecular-weight, externally oriented species that are rich in galactose, and the other consisting of low-molecular-weight, internally oriented species of relatively low galactose content; (c) the vesicles contain a large class of nonglycosylated species that are relatively loosely attached to the membrane; and (d) most of the vesicles' polypeptides are probably freely mobile in the membrane. The organization of synaptic vesicle proteins is compared with that of the proteins of synaptosomal plasma membrane, with which the vesicle is believed to fuse.     

Comparison of a 52-kDa Phosphoprotein from Synaptic Plasma Membranes Related to Long-Term Potentiation and the Major Coated Vesicle Phosphoprotein

In the in vitro hippocampal slice preparation a short tetanus induces long-term potentiation (LTP) and an increase in the post hoc phosphorylation of a 52-kDa protein in synaptosomal plasma membranes (SPM) prepared from these slices. This 52-kDa SPM phosphoprotein closely resembles the predominant phosphoprotein in coated vesicles, pp50, with respect to the insensitivity of its phosphorylation to Ca2+/calmodulin and cyclic AMP. This resemblance prompted us to compare in rat brain the 52-kDa SPM protein with pp50 in isolated coated vesicles. Both proteins appear to be very similar on basis of the following criteria: relative molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, phospho-amino acid content, and isoelectric point. Since coated vesicles are thought to be involved in receptor-mediated endocytosis and membrane recycling, our data suggest that LTP-correlated changes in 52-kDa phosphorylation may reflect increased coated vesicle activity.     

The isolation of coated vesicles from protoplasts of soybean

Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant clathrin, had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.NRCC No. 23142     

Vacuole biogenesis and protein transport to the plant vacuole: A comparison with the yeast vacuole and the mammalian lysosome

Summary The vacuole is often termed the lytic compartment of the plant cell. The yeast cell also possesses a vacuole containing acid hydrolases. In animal cells these enzymes are localized in the lysosome. Recent research suggests that there is good reason to regard these organelles as homologous in terms of protein transport. Although sorting motifs for the recognition of vacuolar proteins within the endomembrane system differ between the three organelles, there is an underlying similarity in targeting determinants in the cytoplasmic tails of Golgi-based receptors. In all three cases these determinants appear to interact with adaptins of clathrin-coated vesicles which ferry their cargo first of all to an endosomal compartment. The situation in sorting and targeting of plant vacuolar proteins is complicated by the fact that storage and lytic vacuoles may exist together in the same cell. The origin of these two types of vacuole is also a matter of some uncertanity.Abbrevations AP assembly protein - ALP alkaline phosphatase - ARF adenosine diphosphate ribosylation factor - BiP immunoglobulin binding protein - CCV clathrin coated vesicle - CPY carboxypeptidase-Y - DPAP dipeptidyl aminopeptidase - ER endoplasmic reticulum - GApp Golgi apparatus - LAMPs lysosomal associated membrane protein(s) - LAP lysosomal acid phosphatase - LIMPs lysosomal integral membrane protein(s) - MPRs mannosyl 6-phosphate receptors - MVB multivesicular bodies - NSF N-ethylmaleimide sensitive fusion (protein) - PAT phosphinotricine acetyltransferase - PB protein body - PHA phytohemagglutinin - PM plasma membrane - PSV protein storage vacuole - SNAPs soluble NSF attachment protein(s) - SNAREs SNAP receptor(s) - TGN trans Golgi network - TIP tonoplast integral protein - VPS vacuolar protein sorting - ZIO zinc iodide/osmium     

Calmodulin Inhibition of Brain Membrane Phosphorylation

Abstract: Calmodulin has been found to inhibit the phosphorylation of rat brain membrane proteins of molecular weight 14,900–18,900 in a dose-dependent manner. This phenomenon was seen under conditions in which calmodulin simultaneously produced a stimulatory effect on the phosphorylation of proteins of molecular weight 51,000 and above. This inhibition required calcium, but was not sensitive to cyclic AMP or increasing ATP concentration and was not due to activation of a phosphatase. These results suggest either that calmodulin induces its inhibitory effects on phosphorylation by an indirect mechanism via a presently unknown pathway, or that in addition to the kinase stimulated by calmodulin, there exists another distinct kinase which is inhibited by calmodulin.     

Characterization of Cyclic AMP-Dependent Phosphorylation of Neuronal Membrane Proteins

We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.     

Effect of Oral Administration of Tri-o-cresyl Phosphate on In Vitro Phosphorylation of Membrane and Cytosolic Proteins from Chicken Brain

Abstract: The effects of a single oral dose of 750 mg/kg tri- o -cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were assessed in male adult chickens following the development of delayed neurotoxicity. Phosphorylation of crude synaptosomal (P₂) membrane and synaptosomal cytosolic proteins was assayed in vitro by using [γ-³²P]ATP as phosphate donor. Following resolution of brain proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, specific protein phosphorylation was detected by autoradiography and quantified by microdensitometry. TOCP administration enhanced the phosphorylation of both cytosolic (M_r 65,000 and 55,000) and membrane (20,000) proteins by as much as 146% and 200%, respectively.     

Phosphorylation of Superior Cervical Ganglion Proteins During Regeneration

The incorporation of radioactive phosphate into proteins of both normal and regenerating ganglia of the sympathetic nervous system of the rat is reported. The incorporation reactions were carried out in vitro by incubating homogenates of excised ganglia with [gamma-32P]ATP under various conditions. It was found that incorporation of phosphate into proteins of regenerating ganglia in the molecular mass range 10,000-100,000 daltons increased up to 40% over incorporation into proteins from control ganglia during the first 3 days following injury and returned to control levels after 14 days. Analysis of the proteins by two-dimensional electrophoresis revealed that only few, i.e., less than 20, became radioactively labelled in homogenates of superior cervical ganglia in the presence of Ca2+, and even fewer in the presence of cyclic AMP. Furthermore, all these proteins fell within a narrow pI range of 4-6. The growth-associated protein, variously designated GAP-43, B-50, F-1, and pp46, has an enhanced level of expression and phosphorylation in regenerating ganglia compared with controls at day 3. Injury also caused consistently higher levels of incorporation into two other proteins with molecular masses at positions 55,000 and 85,000 and pI values of 5.1 and 4.5, respectively; the former protein most probably is beta-tubulin. The fact that both proteins are found in the 15,000 g pellet after the tissue has been solubilized in 0.5% nonionic detergent indicates that they may indeed by components of filament assemblies. Thus, the results suggest that protein phosphorylation is a mechanism involved in cytoskeletal function in regenerating nerve.     

Phosphorylation of Microtubule Proteins in Rat Brain at Different Developmental Stages: Comparison with That Found in Neuronal Cultures

The phosphorylation of rat brain microtubule protein on intracranial injection of labeled phosphate has been analyzed. The major microtubule protein components phosphorylated in vivo in rat brain are the high-molecular-weight microtubule-associated proteins (MAPs) MAP-1A, MAP-1B, and MAP-2. A slight phospholabeling of beta-tubulin, which corresponds to the phosphorylation of a minor neuronal beta-tubulin isotype, is also observed. Whereas MAP-1B, MAP-2, and beta-tubulin are phosphorylated in the brain of 5-day-old rat pups, when most neurons of the CNS are extending processes, MAP-1A phosphorylation is observed only after neuronal maturation takes place. The phosphorylation of MAP-1A, MAP-1B, and beta-tubulin may be due mainly to casein kinase II or a related enzyme, whereas MAP-2 appears to be modified by other enzymes such as the cyclic AMP-dependent protein kinase (protein kinase A) and the calcium/phospholipid-dependent protein kinase (protein kinase C). Microtubule protein phosphorylation has also been studied in neuronal cultures. In differentiated neuroblastoma cells, only MAP-1B and beta-tubulin are phosphorylated in a manner coupled to neurite outgrowth. In primary cultures of fetal rat brain neurons, the pattern of microtubule protein phosphorylation resembles that found in vivo in rat pup brain. As phosphorylated MAP-1A and MAP-1B are present mainly on assembled microtubules, whereas the phosphorylation of MAP-2 decreases its interaction with microtubules, a role can be suggested for the phosphorylation of these proteins in the regulation of microtubule assembly and disassembly during neuronal development.     

GPS 5.0: An Update on the Prediction of Kinase-specific Phosphorylation Sites in Proteins

In eukaryotes, protein phosphorylation is specifically catalyzed by numerous protein kinases(PKs), faithfully orchestrates various biological processes, and reversibly determines cellular dynamics and plasticity. Here we report an updated algorithm of Group-based Prediction System(GPS) 5.0 to improve the performance for predicting kinase-specific phosphorylation sites(p-sites). Two novel methods, position weight determination(PWD) and scoring matrix optimization(SMO), were developed. Compared with other existing tools, GPS 5.0 exhibits a highly competitive accuracy. Besides serine/threonine or tyrosine kinases, GPS 5.0 also supports the prediction of dual-specificity kinase-specific p-sites. In the classical module of GPS 5.0, 617 individual predictors were constructed for predicting p-sites of 479 human PKs. To extend the application of GPS5.0, a species-specific module was implemented to predict kinase-specific p-sites for 44,795 PKs in161 eukaryotes. The online service and local packages of GPS 5.0 are freely available for academic research at http://gps.biocuckoo.cn.     

Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly

Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg²⁺, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES–1 mM EGTA–1 mM MgCl₂–0.02% NaN₃, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg²⁺ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl₂ prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl₂ raised to 10 mM, reassembly occurs. These results suggest that Mg²⁺ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg²⁺ (10 μM–10 mM) increases reassembly whereas chelation of Mg²⁺ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl₂ do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.     

Phosphorylation of Bcl-2 and Bax Proteins during Hypoxia in Newborn Piglets

Studies indicate that phosphorylated Bcl-2 cannot form a heterodimer with Bax and thus may lose its antiapoptotic potential. The present study tests the hypothesis that graded hypoxia in cerebral tissue induces the phosphorylation of Bcl-2, thus altering the heterodimerization of Bcl-2 with Bax and subsequently leading to apoptosis. Anesthetized, ventilated newborn piglets were assigned to a normoxic and a graded hypoxic group. Cerebral cortical neuronal nuclei were isolated and immunoprecipitated; immune complexes were separated and reacted with Bcl-2 and Bax specific antibodies. The results show an increased level of serine/tyrosine phosphorylated Bcl-2 in nuclear membranes of hypoxic animals. The level of phosphorylated Bcl-2 protein increased linearly with decrease in tissue PCr. The level of phosphorylated Bax in the neuronal nuclear membranes was independent of cerebral tissue PCr. The data shows that during hypoxia, there is increased phosphorylation of Bcl-2, which may prevent its heterodimerization with Bax and lead to increased proapoptotic activity due to excess Bax in the hypoxic brain. Further increased phosphorylation of Bcl-2 may alter the Bcl-2/Bax-dependent antioxidant, lipid peroxidation and pore forming activity, as well as the regulation of intranuclear Ca²⁺ and caspase activation pathways. We speculate that increased phosphorylation of Bcl-2 in neuronal nuclear membranes is a potential mechanism of programmed cell death activation in the hypoxic brain.     

Phosphorylation of Adrenal Medulla Cell Proteins in Conjunction with Stimulation of Catecholamine Secretion

Abstract: Enhanced phosphorylation of two specific protein bands accompanied catecholamine secretion from cultured bovine adrenal medulla cells stimulated by different secretagogues. Cells preincubated with ³²P_i were treated with nicotine, veratridine, Ionomycin, or barium. Each of these secretagogues stimulated the phosphorylation of two protein bands with apparent molecular weights of 60,000 and 95,000. Phosphorylation of the 60,000 M.W. protein band was two- to threefold higher than that of the 95,000 M.W. band on stimulation with nicotine, veratridine, or barium, but Ionomycin stimulated phosphorylation of each protein band to the same extent. In general, the increase in phosphorylation was most rapid during the first minute of stimulation and occurred prior to detectable secretion. Phosphorylation reached a relatively constant level within 5 min after onset of stimulation at a time when catecholamine release was still proceeding at a rapid rate. Nicotine-stimulated phosphorylation and catecholamine secretion were calcium-dependent and blocked by d -tubocurarine, whereas tetrodotoxin inhibited veratridine-stimulated secretion and phosphorylation. We conclude that catecholamine secretion and protein phosphorylation occur under similar conditions and that Ca²⁺-dependent incorporation of phosphate into specific proteins may be a link in stimulus-secretion coupling.     

Phosphorylation of Brain Synaptic and Coated Vesicle Proteins by Endogenous Ca2+/Calmodulin- and cAMP-Dependent Protein Kinases

Phosphorylation of brain synaptic and coated vesicle proteins was stimulated by Ca2+ and calmodulin. As determined by 5-15% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), molecular weights (Mr) of the major phosphorylated proteins were 55,000 and 53,000 in synaptic vesicles and 175,000 and 55,000 in coated vesicles. In synaptic vesicles, phosphorylation was inhibited by affinity-purified antibodies raised against a 30,000 Mr protein doublet endogenous to synaptic and coated vesicles. When this doublet, along with clathrin, was extracted from coated vesicles, phosphorylation did not take place, implying that the protein doublet may be closely associated with Ca2+/calmodulin-dependent protein kinase. Affinity-purified antibodies, raised against clathrin used as a control antibody, failed to inhibit Ca2+/calmodulin-dependent phosphorylation in either synaptic or coated vesicles. Immunoelectron cytochemistry revealed that this protein doublet was present in axon terminal synaptic and coated vesicles. Synaptic vesicles also displayed cAMP-dependent kinase activity; coated vesicles did not. The molecular weights of phosphorylated synaptic vesicle proteins in the presence of Mg2+ and cAMP were: 175,000, 100,000, 80,000, 57,000, 55,000, 53,000, 40,000, and 30,000. Based on the different phosphorylation patterns observed in synaptic and coated vesicles, we propose that brain vesicle protein kinase activities may be involved in the regulation of exocytosis and in retrieval of synaptic membrane in presynaptic axon terminals.     

Effect of Digestion with Phospholipase A2 on Endogenous Protein Phosphorylation in Particulate Fractions from Rat Brain Synaptosomes

The endogenous phosphorylation of synapsin 1 in cyclic AMP-containing media was greatly decreased by digestion of synaptic vesicles and synaptosomal membranes with phospholipase A2, suggesting that the system is functionally dependent on the membrane structure. Treatment of the synaptic vesicle fraction with phospholipase A2 also caused a small but significant inhibition of the Ca2+/calmodulin-dependent phosphorylation of the same protein. The Ca2+/calmodulin-dependent phosphorylation of other major acceptors, and the basal phosphorylation of a 52-kD acceptor enriched in the vesicle fraction, remained unchanged after cleavage of the membrane phospholipids with phospholipase A2. The significance of the selective effect of phospholipase A2 treatment on endogenous membrane phosphorylation is discussed.     

Vestibular Compensation Affects Endogenous Phosphorylation of Frog Brain Proteins

The effect of unilateral labyrinthectomy followed by the process of vestibular compensation on the incorporation of radioactive phosphate into frog brain proteins was investigated. Phosphoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. The present data show that unilateral labyrinthectomy affects the incorporation of 32P into various frog brain proteins. In particular, the phosphorylation of a 20-kDa protein appeared enhanced during early stages of vestibular compensation (4-12 days). This 20-kDa protein was shown to be immunologically related to myelin basic protein and its phosphorylation was regulated by an endogenous calcium/calmodulin-dependent protein kinase. These data might indicate that in addition to neuronal components, components of glial origin are also involved in biochemical events that lead to functional recovery after neuronal lesions.