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• W2045392503 abstract "Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 ± 51 nm. Optimal binding occurred with mixtures of phosphatidylserine and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone. Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed the phosphorylation site in PKCα-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus, phosphorylation of dynamin I on Ser-795 prevents its association with phospholipid, providing a basis for the cytosolic localization of the minor pool of phospho-dynamin I that mediates synaptic vesicle retrieval in nerve terminals. Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 ± 51 nm. Optimal binding occurred with mixtures of phosphatidylserine and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone. Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed the phosphorylation site in PKCα-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus, phosphorylation of dynamin I on Ser-795 prevents its association with phospholipid, providing a basis for the cytosolic localization of the minor pool of phospho-dynamin I that mediates synaptic vesicle retrieval in nerve terminals. protein kinase C matrix-assisted laser desorption/ionization mass spectrometry time of flight phosphatidylserine phosphatidylcholine phosphatidylinositol glutathioneS-transferase bovine serum albumin polyacrylamide gel electrophoresis adenosine 5′-O-(thiotriphosphate) adenosine 5′-(β,γ-imino)triphosphate immobilized metal affinity chromatography casein kinase II adenosine 5′-O-(thiotriphosphate) guanosine 5′-(β,γ-imino)triphosphate Dynamin I is a GTPase enzyme required for the retrieval of synaptic vesicles after exocytosis. It functions in endocytosis by stimulated assembly as a helix around the neck of invaginating synaptic vesicles (1.Koenig J.H. Ikeda K. J. Neurosci. 1989; 9: 3844-3860Crossref PubMed Google Scholar, 2.Stowell M.H. Marks B. Wigge P. McMahon H.T. Nat. Cell Biol. 1999; 1: 27-32Crossref PubMed Scopus (310) Google Scholar). Dynamin can self-assemble as a series of rings in the absence of guanine nucleotide (3.Hinshaw J.E. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (654) Google Scholar), and helices form around phospholipid vesicles in vitro (4.Sweitzer S.M. Hinshaw J.E. Cell. 1998; 93: 1021-1029Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar) or around the neck of invaginating synaptic vesicles (5.Takei K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (650) Google Scholar). The helices behave like a nanospring, with GTP hydrolysis producing an increase in the helix pitch, suggesting that endocytosis might occur by a nucleotide-dependent conformational change in dynamin that cleaves vesicles from the plasma membrane (2.Stowell M.H. Marks B. Wigge P. McMahon H.T. Nat. Cell Biol. 1999; 1: 27-32Crossref PubMed Scopus (310) Google Scholar). Dynamin I is also a phosphoprotein found in intact nerve terminals where it is apparently phosphorylated by PKC1 (6.Robinson P.J. J. Biol. Chem. 1992; 267: 21637-21644Abstract Full Text PDF PubMed Google Scholar). It is rapidly dephosphorylated by calcineurin on stimulation of endocytosis by depolarization and calcium influx (7.Liu J.P. Sim A.T.R. Robinson P.J. Science. 1994; 265: 970-973Crossref PubMed Scopus (183) Google Scholar) and blocking dephosphorylation prevents endocytosis in nerve terminals (8.Marks B. McMahon H.T. Curr. Biol. 1998; 8: 740-749Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). It remains dephosphorylated during endocytosis of most vesicles and is rephosphorylated while endocytosis is completing (9.Robinson P.J. Liu J.P. Powell K.A. Fykse E.M. Südhof T.C. Trends Neurosci. 1994; 17: 348-353Abstract Full Text PDF PubMed Scopus (115) Google Scholar). Therefore the phosphorylation of dynamin is not likely to play a role during endocytosis but is probably a priming step prior to endocytosis.Many of the proteins essential for endocytosis are constitutively phosphorylated in nerve terminals at rest and are coordinately dephosphorylated by calcineurin upon a burst of exocytosis and endocytosis. These “dephosphins” include dynamin I, amphiphysins I and II, synaptojanin, epsin and Eps15 (8.Marks B. McMahon H.T. Curr. Biol. 1998; 8: 740-749Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 10.Robinson P.J. Sontag J.-M. Liu J.P. Fykse E.M. Slaughter C. McMahon H.T. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar, 11.McPherson P.S. Takei K. Schmid S.L. De Camilli P. J. Biol. Chem. 1994; 269: 30132-30139Abstract Full Text PDF PubMed Google Scholar, 12.McPherson P.S. Garcia E.P. Slepnev V.I. David C. Zhang X.M. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar, 13.Bauerfeind R. Takei K. De Camilli P. J. Biol. Chem. 1997; 272: 30984-30992Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 14.Chen H. Slepnev V.I. Di Fiore P.P. De Camilli P. J. Biol. Chem. 1999; 274: 3257-3260Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In vitrodephosphorylation of rat brain extracts promotes the assembly of dynamin I, synaptojanin, amphiphysin, clathrin, and AP-2 into complexes (15.Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. DeCamilli P. Science. 1998; 281: 821-824Crossref PubMed Scopus (271) Google Scholar). Thus, phosphorylation appears to regulate the association and dissociation cycle of the endocytic machinery. This suggests that the role of dynamin phosphorylation may be to either prevent inappropriate protein-protein interactions (15.Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. DeCamilli P. Science. 1998; 281: 821-824Crossref PubMed Scopus (271) Google Scholar) or to keep dynamin away from sites of exocytosis until required for endocytosis (6.Robinson P.J. J. Biol. Chem. 1992; 267: 21637-21644Abstract Full Text PDF PubMed Google Scholar).The precise role of the phosphorylation of dynamin has not been fully determined, but several biochemical correlates have been reported.In vitro phosphorylation by cPKC (the Ca2+-dependent PKC family comprising PKCα, -β, and -γ) stimulates the GTPase activity of dynamin I (10.Robinson P.J. Sontag J.-M. Liu J.P. Fykse E.M. Slaughter C. McMahon H.T. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar), promotes the formation of disulfide bonds and its assembly into a tetramer, and increases low affinity Ca2+ binding (16.Liu J.P. Zhang Q.-X. Baldwin G. Robinson P.J. J. Neurochem. 1996; 66: 2074-2081Crossref PubMed Scopus (37) Google Scholar). Phosphorylation by a cyclin-dependent protein kinase, Cdc2, prevents dynamin association with microtubules in vitro(17.Hosoya H. Komatsu S. Shimizu T. Inagaki M. Ikegami M. Yazaki K. Biochem. Biophys. Res. Commun. 1994; 202: 1127-1133Crossref PubMed Scopus (22) Google Scholar). Phosphorylation of dynamin I by an unidentified cytosolic kinase activity inhibits its binding to amphiphysin (15.Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. DeCamilli P. Science. 1998; 281: 821-824Crossref PubMed Scopus (271) Google Scholar), although phospho-dynamin from intact nerve terminals is capable of binding to the SH3 domains of amphiphysins I and II (8.Marks B. McMahon H.T. Curr. Biol. 1998; 8: 740-749Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In none of these examples was the phosphorylation site identified, and it might differ for each protein kinase.Dynamin I is known to be phosphorylated exclusively on serine on a site within the C terminus by cPKC in vitro and by endogenous protein kinases in intact nerve terminals (10.Robinson P.J. Sontag J.-M. Liu J.P. Fykse E.M. Slaughter C. McMahon H.T. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar, 18.Robinson P.J. FEBS Lett. 1991; 282: 388-392Crossref PubMed Scopus (19) Google Scholar, 19.Liu J.P. Powell K.A. Südhof T.C. Robinson P.J. J. Biol. Chem. 1994; 269: 21043-21050Abstract Full Text PDF PubMed Google Scholar). Phospho-dynamin I has a subcellular localization restricted to the cytosolic fraction of intact nerve terminals, despite that greater than 90% of the dynamin I is membrane-associated (19.Liu J.P. Powell K.A. Südhof T.C. Robinson P.J. J. Biol. Chem. 1994; 269: 21043-21050Abstract Full Text PDF PubMed Google Scholar). Similarly, the larger dynamin I pool that is associated with brain membranes or cytoskeleton cannot be phosphorylated in vitro by PKC until dynamin is extracted from the membranes (19.Liu J.P. Powell K.A. Südhof T.C. Robinson P.J. J. Biol. Chem. 1994; 269: 21043-21050Abstract Full Text PDF PubMed Google Scholar). These data suggest that phosphorylation of dynamin I may be required to generate or maintain a cytosolic pool of tetrameric phospho-dynamin which associates with proteins required to target dynamin to sites of endocytosis following a dephosphorylation stimulus. There are examples where one consequence of phosphorylation of other peripheral membrane proteins such as myristoylated alanine-rich C kinase substrate, synapsin I, and spectrin is an inhibition of their binding to phospholipid and consequent release into the cytosol (20.Wang J.K. Walaas S.I. Sihra T.S. Aderem A.A. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2253-2256Crossref PubMed Scopus (157) Google Scholar, 21.Vorotnikov A.V. Bogatcheva N.V. Gusev N.B. Biochem. J. 1992; 284: 911-916Crossref PubMed Scopus (14) Google Scholar, 22.Kim J. Shishido T. Jiang X. Aderem A. McLaughlin S. J. Biol. Chem. 1994; 269: 28214-28219Abstract Full Text PDF PubMed Google Scholar, 23.Fowler V.M. Adam E.J. J. Cell Biol. 1992; 119: 1559-1572Crossref PubMed Scopus (61) Google Scholar, 24.Tarelli F.T. Bossi M. Fesce R. Greengard P. Valtorta F. Neuron. 1992; 9: 1143-1153Abstract Full Text PDF PubMed Scopus (81) Google Scholar). However, in the case of dynamin I this concept has not been directly tested. Therefore our aims were to examine the consequences of phosphorylation by PKC on phospholipid binding by dynamin I and to determine the phosphorylation site sequence.RESULTSTo measure phospholipid-binding of dynamin I a plate-based assay was developed. The use of lipid-coated plastic or glass beads prevents insertion of dynamin into a lipid bilayer (19.Liu J.P. Powell K.A. Südhof T.C. Robinson P.J. J. Biol. Chem. 1994; 269: 21043-21050Abstract Full Text PDF PubMed Google Scholar, 25.Ando Y. Imamura S. Hong Y.M. Owada M.K. Kakunaga T. Kannagi R. J. Biol. Chem. 1989; 264: 6948-6955Abstract Full Text PDF PubMed Google Scholar) and prevents dynamin from assembling as helices or vesiculating the phospholipid (4.Sweitzer S.M. Hinshaw J.E. Cell. 1998; 93: 1021-1029Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar), which would otherwise confound interpretation of phospholipid association. In background experiments to develop the method, the relative affinity of dynamin I for different compositions of phospholipid was assessed (Fig.1 A). Coating solutions were prepared from mixtures of two phospholipids with a fixed total concentration of phospholipid but with varying ratios. Dynamin I binding was greatest to a mixture of PS and PC and was reduced in mixtures of PI and PC or PS and PI. Dynamin I did not bind well to plates coated with any of the three individual phospholipids (data not shown). The highest binding level was achieved with a PS/PC ratio of 1:3. These conditions also best reflect the in vivosituation in regards to membrane lipid composition in cells and were used for all other experiments. In previous studies dynamin was found to dissociate from lipid-coated beads or rat brain membranes with NaCl (19.Liu J.P. Powell K.A. Südhof T.C. Robinson P.J. J. Biol. Chem. 1994; 269: 21043-21050Abstract Full Text PDF PubMed Google Scholar). We found that NaCl also efficiently blocked dynamin I association with phospholipid in the plate assay (Fig. 1 B). To demonstrate that binding was specific, we increased the detergent concentration in the assay to 1% Tween 80, which almost abolished dynamin binding (Fig. 1 C). As dynamin I concentrations were increased, it bound to the phospholipid-coated plates in a linear and saturable manner (Fig. 1 C). The calculated apparent affinity of dynamin for the phospholipid mixture using double-reciprocal plots was 230 ± 51 nm (n = 3).To determine if the plate binding assay sensitively detects alterations in the conformational status of dynamin, we next explored the effect of adenine and guanine nucleotides on dynamin binding to phospholipids (Fig. 2). Metal ions such as Mg2+ (Fig. 2 A) and Mn2+ (data not shown) had no effect on dynamin binding to phospholipid, whereas Ca2+ had variable effects at concentrations above 200 μm (data not shown). Mg2+ was included in equimolar concentrations with each guanine or adenine nucleotide. GTP potently reduced dynamin association with phospholipid (Fig.2 A). Other nucleotides also reduced binding but with greatly reduced efficacy. Concentrations producing 50% inhibition of binding (IC50) were 0.30 mm for GTP, and 2.5, 4.3, and 14 mm for GDP, GMP, and cGMP respectively. This suggests specificity in the action of GTP, as 0.3 mm is close to the physiological concentration of GTP in cells (30.Otero A.D. Biochem. Pharmacol. 1990; 39: 1399-1404Crossref PubMed Scopus (102) Google Scholar). To determine whether GTP binding or GTP hydrolysis was responsible for the reduced binding, two non-hydrolyzable GTP analogues were used, and both potently reduced the ability of dynamin I to bind to phospholipid (Fig. 2 B). GTPγS had a very similar response to GTP, and GMP-PNP was less potent. IC50 values for the analogues were 0.35 mm (Mg/GTPγS and 0.9 mm (Mg/GMP-PNP). This demonstrates that the effect of nucleotides on dynamin I binding to phospholipid is a consequence of GTP binding, rather than GTP hydrolysis. Qualitatively similar results were obtained with ATP and its analogues, except that the potency of all adenine nucleotides was much less than guanine nucleotide analogues (Fig. 2, C andD). The IC50 values were (in mm) ATP 2.0, ADP 5.1, AMP 9.5, ATPγS 2.9, and AMP-PNP 4.8.Figure 2Effect of guanine or adenine nucleotides on dynamin I binding to phospholipid. A, the effect of guanine nucleotides. The phospholipid-binding plate assay was performed in wells coated with PS/PC at 1:3 and in the presence of 0.8 μg/well dynamin I, with the addition of increasing concentrations of Mg2+ alone (filled circles) or Mg2+with GTP (open circles), GDP (open squares), GMP (open triangles), or cGMP (open diamonds). All nucleotides were included in the presence of equimolar Mg2+concentrations. B, the effect of non-hydrolyzable analogues of GTP. Concentration-response curves for GTP (circles), GTPγS (squares), and GMP-PNP (triangles).C, the effect of adenine nucleotides. Concentration-response curves for ATP (open circles), ADP (open squares), AMP (open triangles), or GTP (filled circles). D, the effect of non-hydrolyzable analogues of ATP. Concentration-response curves for ATP (circles), ATPγS (squares), and AMP-PNP (triangles). In all panels values plotted have had the blank reading (no dynamin I) subtracted and are means of three experiments, error barsindicate S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The effect of phosphorylation by cPKC on the ability of dynamin I to bind to phospholipid was then determined in the phospholipid plate assay. Binding of non-phosphorylated dynamin I to phospholipid-coated plates increased in a linear manner, whereas phosphorylated dynamin I essentially failed to bind (Fig. 3). Some minor binding was achieved with the phosphorylated preparation as the concentration of dynamin was increased, the amount of which varied between different preparations of purified dynamin I. This variability might result from different amounts of phospho-dynamin contaminating each preparation of the protein. Phosphorylation of dynamin I was confirmed by either inclusion of [γ-32P]ATP in some experiments and autoradiography or by the ability of phosphorylated dynamin I to form tetramers in non-reducing SDS gels (16.Liu J.P. Zhang Q.-X. Baldwin G. Robinson P.J. J. Neurochem. 1996; 66: 2074-2081Crossref PubMed Scopus (37) Google Scholar) (data not shown). Addition of sodium chloride abolished binding of non-phosphorylated dynamin I to phospholipid but had no effect on the lack of binding by phosphorylated dynamin I (data not shown). To confirm that the in vitro effect of cPKC is mimicked in intact neurons by the endogenous dynamin I kinase, phospho-dynamin I was purified from rat brain. This form of dynamin eluted much earlier in a salt gradient from the Q-Sepharose column (data not shown) and was confirmed to be phosphorylated by its assembly as a disulfide-linked tetramer (16.Liu J.P. Zhang Q.-X. Baldwin G. Robinson P.J. J. Neurochem. 1996; 66: 2074-2081Crossref PubMed Scopus (37) Google Scholar) and elevated intrinsic GTPase activity (10.Robinson P.J. Sontag J.-M. Liu J.P. Fykse E.M. Slaughter C. McMahon H.T. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar). This preparation of in vivo phospho-dynamin I also completely failed to associate with phospholipid in this assay (data not shown).Figure 3Phosphorylation of dynamin I (Dyn I) by PKC abolishes its ability to bind to phospholipid in the phospholipid-binding plate assay. Purified dynamin I was phosphorylated in vitro using cPKC and its appropriate substrates and co-factors and was repurified. A concentration-response curve was generated using either phosphorylated dynamin I (squares) or dynamin I from the same purification that had not been phosphorylated but had been repurified in parallel with the phosphorylated protein (circles). Results have had the blank (no dynamin I) subtracted and are from three experiments anderror bars indicate S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To verify that phosphorylation abolishes phospholipid binding by dynamin I, we employed a distinct phospholipid binding assay that also does not allow dynamin insertion into bilayers nor vesiculation of the lipid. Dynamin I strongly associates with phospholipid-coated CPG beads in a centrifugation-based assay (19.Liu J.P. Powell K.A. Südhof T.C. Robinson P.J. J. Biol. Chem. 1994; 269: 21043-21050Abstract Full Text PDF PubMed Google Scholar). To test the effect of phosphorylation, dynamin I was phosphorylated in vitro by recombinant PKCα (Fig. 4) or cPKC (data not shown), repurified, and incubated with the phospholipid-coated CPG beads. Phosphorylated dynamin showed reduced binding to phospholipids (lanes 3 and 4). As a control, dynamin was also phosphorylated by CK2, which is known not to affect its GTPase activity (10.Robinson P.J. Sontag J.-M. Liu J.P. Fykse E.M. Slaughter C. McMahon H.T. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar). CK2-phosphorylation did not affect its ability to associate with phospholipid (lanes 5 and 6).Figure 4Binding of dynamin I to phospholipid-coated controlled-pore glass beads is reduced by phosphorylation.Purified dynamin I (1.0 μg) was phosphorylated with cPKC (lanes 3 and 4) or CK2 (lanes 5 and 6) and repurified. Non-phosphorylated (lanes 1 and2) and phosphorylated dynamin I were mixed with phospholipid-coated CPG beads for 30 min. Samples were separated into supernatant (S) or pellet (P) fractions by brief centrifugation and applied to SDS-PAGE (7.5–15% gradient acrylamide gels), and the protein was stained with Coomassie Blue. The same results were obtained in a second, independent experiment.Ctrl, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the ability of dynamin I to bind phospholipid could be restored by its dephosphorylation, a preparation of dynamin I was phosphorylated in the presence of [γ-32P]ATPin vitro and then dephosphorylated by addition of the purified protein phosphatase, calcineurin. The dynamin phosphorylation status was monitored by SDS-PAGE and autoradiography (Fig.5, A and B). Dynamin I was efficiently dephosphorylated by 0.75 μg of calcineurin (lane 3). Phosphorylated dynamin I was then incubated with 0.15 μg or 0.75 μg of calcineurin in the presence of calmodulin, Mn2+, and Ca2+ (activators of calcineurin). Neither divalent cation affected the binding of dynamin I to phospholipid in this assay system (Fig. 5 C). Increasing the concentration of calcineurin correspondingly increased dynamin I binding to the phospholipid. Controls showed that calcineurin and its activators had no effect on non-phosphorylated dynamin I binding to phospholipid (Fig. 5 C).Figure 5Dephosphorylation of dynamin I (Dyn I) restores phospholipid binding. Dynamin I was phosphorylated in vitro with cPKC and [γ-32P]ATP and was repurified. Dephosphorylation with calcineurin plus calmodulin (CaM), Ca2+, and Mn2+ was performed at 30 °C for 30 min, and samples were applied to SDS-PAGE for Coomassie staining (B) autoradiography (A) or were used in the phospholipid-binding plate assay (C). A, autoradiograph andB, Coomassie blue-stained gel demonstrating the dephosphorylation of dynamin. Dynamin I without phosphorylation (lane 1 and 6) or after cPKC phosphorylation (lanes 2–5). Non-phosphorylated dynamin I prior to phosphorylation (lane 1), phosphorylated dynamin I (lane 2), dephosphorylation of phosphorylated dynamin I with calcineurin and calmodulin (CaM, lane 3), phosphorylated dynamin I with calcineurin but no calmodulin (lane 4), phosphorylated dynamin I with calmodulin, Ca2+, and Mn2+, but no calcineurin (lane 5), and non-phosphorylated dynamin I with calcineurin and calmodulin (lane 6). C, dynamin or PKC-phosphorylated dynamin I (2.0 μg per well) were added to phospholipid-coated wells and incubated with the phosphatase calcineurin (0.15 or 0.75 μg) plus its activators (calmodulin, calcium, and manganese) for 30 min at 30 °C. Results have had the blank reading (no dynamin I) subtracted and are the means of three experiments ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The identification of the PKC phosphorylation site in dynamin I responsible for the loss of phospholipid binding was performed using high mass accuracy MALDI-TOF mass spectrometry. Three different approaches were used as follows: (i) identification of the phosphopeptide in the peptide mass map of the whole peptide mixture derived by tryptic digestion of dynamin I, before and after alkaline phosphatase treatment; (ii) identification of the phosphopeptides after affinity purification using an IMAC column, and (iii) identification of the phosphopeptides by MALDI MS of the radioactive fraction after high pressure liquid chromatography separation. The tryptic peptides of dynamin I and phosphorylated dynamin I were analyzed by MALDI-TOF MS, and the observed peptides were identified based on their predicted masses determined from the known sequence. Comparison of the peptide mass map of phosphorylated dynamin I and dynamin I revealed a single peptide of mass-over-charge ratio (m/z) of 1175.74 Da that was unique to dynamin I and was not found in phosphorylated dynamin I (data not shown). However, the corresponding phosphorylated peptide predicted at an m/z 1255.75 Da was not observed in the spectrum of the tryptic digest of the phosphorylated sample, probably due to suppression effects frequently observed in MALDI-TOF MS. Another peptide signal at m/z 1256.69 Da could be correlated to a peptide derived from trypsin (amino acids 4–14) (Fig.6 A). Treatment of the mixture with alkaline phosphatase resulted in a very weak peak atm/z 1175.74, indicating that the phosphorylated peptide might have been present (data not shown). To demonstrate this, the phosphopeptides in the tryptic digest of phosphorylated dynamin I were selectively purified by immobilized metal affinity chromatography (IMAC) using Fe3+-charged nitrilotriacetic acid resin and were desalted prior to MALDI-TOF MS (Fig. 6 B). A major mass peak at m/z 1255.74 Da was found, accompanied by a characteristic metastable fragment ion, diagnostic of the loss of H3PO4 by post-source decay in the first field free region in the MALDI (Fig. 6 B). A minor peak atm/z 1411.86 Da was also observed, with its accompanying fragment ion, corresponding to alternative tryptic cleavage after Arg-694 instead of Arg-695. To confirm further that these peptides were indeed phosphorylated, they were dephosphorylated with alkaline phosphatase directly on the MALDI target, resulting in a mass shift of −80 Da to m/z 1175.75 Da concomitant with the disappearance of the metastable species (Fig. 6 C). This mass corresponds to the dynamin 785–796 fragment APAVPPARPGSR, which contains a single serine at Ser-795. In the experiment shown in Fig. 6 B, them/z 1411.86-Da peptide (B) did not appear atm/z 1331.86 Da (C) probably due to its low abundance. However, a weak signal corresponding to this peptide was detected in two additional experiments (data not shown). Furthermore, a radioactively labeled peak detected by high pressure liquid chromatography was found by MALDI-TOF MS to represent them/z 1255.74-Da peptide, accompanied by the metastable fragment ion signal. This peak shifted to m/z 1175.74 Da after alkaline phosphatase treatment. Since we previously demonstrated that dynamin I is exclusively phosphorylated on serine residues by PKC (10.Robinson P.J. Sontag J.-M. Liu J.P. Fykse E.M. Slaughter C. McMahon H.T. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar, 18.Robinson P.J. FEBS Lett. 1991; 282: 388-392Crossref PubMed Scopus (19) Google Scholar, 19.Liu J.P. Powell K.A. Südhof T.C. Robinson P.J. J. Biol. Chem. 1994; 269: 21043-21050Abstract Full Text PDF PubMed Google Scholar), this result confirms Ser-795 as the PKCα phosphorylation site in dynamin I.Figure 6Dynamin I (dyn I) phosphorylation site by PKC α determined by MALDI-MS. A, dynamin I was phosphorylated with recombinant PKCα, digested with trypsin, and the digest subjected to MALDI-TOF MS analysis. The mass of each peak is indicated in Da.B, the same digest was subjected to micro-purification on IMAC followed by R3 to purify the phosphopeptides. Only two peaks (1255.79 and 1411.86 Da) and peaks corresponding to their metastable loss of H3PO4 were detected. Both masses represented two cleavage variants derived from the same sequence stretch (the amino acid sequence is shown), separated only by the mass of a single arginine residue. C, the samples fromB were incubated on the sample plate (on-target) with alkaline phosphatase and subjected to MALDI-TOF MS again. A single mass was detected, corresponding to the non-phosphorylated peptide shown. Results are from 1 of 3 experiments with qualitatively the same results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A synthetic peptide encompassing the Ser-795 PKC phosphorylation site (Dyn788–803, VPPARPGSRGPAPGPP) was not appreciably phosphorylated by PKC in an in vitro kinase assay (data not shown), nor was another peptide encompassing the adjacent serines in dynamin770–784 (AGRRSPTSSPTPQRR) phosphorylated by PKC in vitro. This result contrasts with the high affinity phosphorylation of dynamin previously reported for the" @default.
• W2045392503 created "2016-06-24" @default.
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• W2045392503 date "2000-04-01" @default.
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• W2045392503 title "Phosphorylation of Dynamin I on Ser-795 by Protein Kinase C Blocks Its Association with Phospholipids" @default.
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