Phosphorylation Of Synaptic Membrane Proteins Research Articles

Calcium-stimulated protein phosphorylation in synaptic membranes.

Synaptic membranes from rat brain contain several calcium-requiring protein kinase (PK) activities with different substrate specificities: (a) an activity (CaH-PK) effective at high concentrations of Ca2+ ion in the absence of Mg2+ (active on class F substrates); (b) a (Ca + Mg)-PK activity that is mediated by Ca2+ ion in the presence of Mg2+ (active on class B substrates); (c) (Ca-CaM)-PK activities that exhibit simultaneous requirements for both Ca2+ ion and CaM (for class C and D substrates). Also described are three activities (d-f) that do not require Ca2+ ion: (d) a Mg-PK activity in which the presence of Ca2+ causes the inhibition of phosphorylation (active on class A substrates); (e) an activity affecting a diverse group of substrates (class E substrates), the phosphorylation of which occurs in the presence of Mg2+ ion alone (Mg-PK activity) and is unaffected by the addition of Ca2+ ion and CaM, the substrates of which show different responses to several types of inhibitors; and, finally, (f) the previously well characterized cAMP-dependent PK activities. Several of the substrates of these kinases have been identified in a fairly unambiguous manner: among them are P43 (class A), as the alpha subunit of pyruvate dehydrogenase; P54 (class B), as the presynaptic protein B50; and the doublet P75-P80, as proteins IA and IB of Ueda and Greengard. The most interesting activity is that requiring both Ca2+ and CaM. The half-maximal stimulation (K0.5) for Ca2+ in the presence of CaM was found to be 1.0 microM Ca2+F in untreated membranes. There is little change in this value on prior EGTA extraction of the membranes, which removes the bulk of its Ca2+ and reduces its residual CaM by greater than or equal to 50%. The apparent K0.5 for CaM in the presence of excess Ca2+ ion was found to equal 0.4 microgram per reaction mixture (8 micrograms/ml) or 1.35 micrograms per reaction mixture (27 micrograms/ml), for the untreated and EGTA-treated membranes, respectively.

The role of calmodulin in opioid-induced changes in the phosphorylation of rat striatal synaptic membrane proteins.

Chronic morphine treatment of rats decreased the level of phosphorylation of synaptic membrane proteins of the striatum assayed in vitro. Although the patterns of phosphorylated proteins separated on SDS-gel electrophoresis from morphine-tolerant rats resembled patterns produced by lowering Ca2+ levels in the assay, supplementation of the protein kinase assay with Ca2+ and its binding protein, calmodulin, did not restore full kinase activity. The addition of methadone or etorphine to the protein kinase in vitro however, was able to block the Ca2+-calmodulin stimulation of phosphorylation in both synaptic membranes and intact synaptosomes. These data suggest that opioids produce an irreversible (or slowly reversible) defect in the Ca2+-dependent protein kinase system of striatal membranes.

Phosphorylation of synaptic membranes.

Abstract: In vivo and in vitro phosphorylation of synaptic membrane proteins were compared. In vivo phosphorylation was carried out by injecting rats intraventricularly with 1 mCi of 32Pi‐orthophosphate. After a 40‐min isotope incorporation period, rats were decapitated and synaptic membranes isolated. In vitro phosphorylation was accomplished by incubating unlabeled synaptic membranes, isolated from noninjected rats, with 5 μM‐[γ‐32P]ATP. In vivo and in vitro32P‐labeled synaptic membranes were then fractionated electrophoretically on an SDS‐polyacrylamide (7.5%) slab gel. The Coomassie blue protein patterns for in vivo and in vitro32P‐labeled synaptic membranes were identical. In contrast, autoradiographs of these gels showed striking differences in the pattern of phosphate incorporation. Cyclic‐AMP (10 μM) maximally stimulated) the in vitro phosphate labeling of two protein bands (80,000 and 55,000 apparent molecular weight) which were not substantially labeled by the in vivo procedure.

Long duration phosphorylation of synaptic membrane proteins

It has been proposed that the phosphorylation of membrane proteins is the mechanism by which postsynaptic membrane permeability is controlled at catecholaminergic synapses. Part of the evidence in favour of this interpretation is the rapid turnover of protein-bound phosphate observed in isolated synaptosomal plasma membranes in vitro. In the present study the rate of incorporation of phosphate from [γ- 32P]adenosine 5′-triphosphate into the protein of synaptic membranes isolated from rat brain was found to be dependent upon the concentration of adenosine triphosphate. At 10 μ m, maximum phosphate incorporation occurred after 30 s but at 1 μ m the maximum was not reached until 5 min. The net amount of phosphate incorporated also rose with increasing adenosine 5'-triphosphate concentration, being lowest at 10 μ m and rising to reach a maximum at between 1 and 5 m m adenosine triphosphate. When the phosphorylation of individual synaptic membrane proteins was followed by gel electrophoresis and autoradiography even more pronounced differences were found between the two concentrations of adenosine triphosphate: at 10 μ m there was a rapid phosphorylation and a striking stimulation by cyclic adenosine monophosphate whereas at 1 m m the time-course of turnover was prolonged and stimulation by cyclic adenosine monophosphate was more modest. At both concentrations of adenosine triphosphate the phosphorylation of synaptic membrane proteins was not completely reversible in vitro. In both cases more than 50% of the incorporated phosphate was still bound to membrane protein after dephosphorylation had ceased. It seems probable that results obtained with micromolar concentrations of adenosine triphosphate are spurious, the phosphorylation process being prematurely terminated by the consumption of the substrate by non-kinase membrane adenosine 5′-triphosphatase activity. The longer time-course found with adequate levels of adenosine triphosphate (millimolar concentrations) is not incompatible with a transmission-dependent role for synaptic membrane phosphorylation. However, it does suggest that the phenomenon may be part of a more general metabolic response to afferent stimulation than has hitherto been proposed.

Complexity of cyclic AMP‐dependent phosphoproteins in membranes from brain tissue containing synapses

FEBS LettersVolume 75, Issue 1-2 p. 61-64 Full-length articleFree Access Complexity of cyclic AMP-dependent phosphoproteins in membranes from brain tissue containing synapses M. Reddington, Corresponding Author M. Reddington Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGDr M. Reddington, Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGSearch for more papers by this authorE. Mehl, E. Mehl Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGSearch for more papers by this author M. Reddington, Corresponding Author M. Reddington Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGDr M. Reddington, Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGSearch for more papers by this authorE. Mehl, E. Mehl Max-Planck-Institut für Psychiatrie, Neurochemische Abteilung, Kraepelinstr. 2, D-8000 München 40, FRGSearch for more papers by this author First published: March 15, 1977 https://doi.org/10.1016/0014-5793(77)80053-7Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 F.E. Bloom, Rev. Physiol. Biochem. Pharmacol., 74, (1975), 1– 103. 2 M. Reddington, R. Rodnight, M. Williams, Biochem. J., 132, (1973), 475– 482. 3 M. Williams, R. Rodnight, Biochem. J., 154, (1976), 163– 170. 4 M. Williams, Brain Res., 109, (1976), 190– 195. 5 I. Levitan, C. Madsen, S. Barondes, J. Neurobiol., 5, (1974), 511– 525. 6 M. Weller, R. Rodnight, Biochem. J., 124, (1971), 406– 511. 7 M. Weller, R. Rodnight, Biochem. J., 132, (1973), 483– 492. 8 T. Ueda, H. Maeno, P. Greengard, J. Biol. Chem., 248, (1973), 8295– 8305. 9 A. Routtenberg, Y.H. Ehrlich, Brain Res., 92, (1975), 415– 430. 10 M. Weller, I. Morgan, Biochim. Biophys. Acta, 436, (1976), 675– 685. 11 P. Dunkley, H. Holmes, R. Rodnight, Biochem. J., 157, (1976), 661– 666. 12 U.K. Laemmli, Nature, 227, (1970), 680– 685. 13 E.M. Reimann, D.A. Walsh, E.G. Krebs, J. Biol. Chem., 246, (1971), 1986– 1995. 14 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 275. 15 E. Miyamoto, J. Neurochem., 26, (1976), 573– 577. 16 C.S. Rubin, J. Biol. Chem., 250, (1975), 9044– 9052. 17 O.M. Pinkett, R.L. Pertman, Biochim. Biophys. Acta, 372, (1974), 379– 387. 18 J. Schultz, B. Hamprecht, J.W. Daly, Proc. Natl. Acad. Sci. USA, 436, (1972), 1266– 1270. 19 Blas A. De, H.R. Mahler, Biochem. Biophys. Res. Commun., 72, (1976), 24– 32. 20 M. Weller, I. Morgan, Biochim. Biophys. Acta, 391, (1976), 162– 169. Citing Literature Volume75, Issue1-2March 15, 1977Pages 61-64 ReferencesRelatedInformation

A possible role of the phosphorylation of synaptic membrane proteins in the control of calcium ion permeability

Incubation of synaptosomes under conditions which result in complete phosphorylation of membrane bound accepter proteins does not affect the permeability to Na + or K + as measured by a spectrophotometric method. This technique was not, however, sensitive enough to determine permeability to Ca 2+ which was thus estimated using 45Ca 2+. It was found that although phosphorylation did not affect the equilibrium binding of 45Ca it did lower the rate of both Ca 2+ uptake and efflux. The most likely interpretation of these results is that phosphorylation of proteins in the synaptic membrane lowers the permeability of the membrane to Ca 2+. This could have a role in the regulation of synaptic transmission.

Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Partition of substrates and protein kinase activities with triton X-100

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase activity catalysing the phosphorylation of endogenous substrates. Extraction of membrane fragments with Triton X-100 solubilized less than 20% of the kinase activity and left the major part of the endogenous substrates in the insoluble fraction.