the in vivo phosphorylation sites of rat brain dynamin i* · protein kinases and new...

The in vivo Phosphorylation Sites of Rat Brain Dynamin I* Mark E. Graham‡, Victor Anggono‡, Nicolai Bache§, Martin R. Larsen§, George E. Craft‡

and Phillip J. Robinson‡

‡ Cell Signalling Unit, Children’s Medical Research Institute, Locked Bag 23,

Wentworthville, NSW 2145, Australia. § Department of Biochemistry and Molecular Biology, Campusvej 55, University of

Southern Denmark, Odense, Denmark

Running Title: In vivo phosphorylation sites in rat dynamin I Address correspondence to: Phillip J. Robinson, Cell Signalling Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia, Tel. +61-2-9687-2800; Fax. +61-2-9687-2120; E-mail: [email protected].

Dynamin I (dynI) is phosphorylated in synaptosomes at Ser-774 and Ser-778 by cdk5 to regulate recruitment of syndapin I for synaptic vesicle endocytosis (SVE), and in PC12 cells on Ser-857. Hierarchical phosphorylation of Ser-774 precedes phosphorylation of Ser-778. In contrast, Thr-780 phosphorylation by cdk5 has been reported as the sole site (Tomizawa et al., J. Cell Biol. 163, 813-824). To resolve the discrepancy and to better understand the biological roles of dynI phosphorylation we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phospho-amino acid analysis, exclusively phospho-serine residues were found. Thr-780 phosphorylation was not detectable. Mutation of Ser-774, Ser-778 and Thr-780 confirmed that Thr-780 phosphorylation is restricted to in vitro conditions. Mass spectrometry of 32P-labelled phosphopeptides separated by 2D mapping revealed 7 in vivo phosphorylation sites: Ser-774, Ser-778, Ser-822, Ser-851, Ser-857, Ser-512, and Ser-347. Quantification of 32P-radiation in each phosphopeptide showed that Ser-774 and Ser-778 were the major sites (up to 69% of the total), followed by Ser-851 and Ser-857 (12%), and Ser-853 (2%). Phosphorylation of Ser-851 and Ser-857 was restricted to the long tail splice variant dynIxa and was not hierarchical. Co-purified, 32P-labelled dynIII was phosphorylated at Ser-759, Ser-763 and

Ser-853. Ser-853 is homologous to Ser-851 in dynIxa. The results identify all major and several minor phosphorylation sites in dynI and provide the first measure of their relative abundance and relative responses to depolarization. The multiple phospho-sites suggest subtle regulation of SVE by new protein kinases and new protein-protein interactions. The homologous dynI and dynIII phosphorylation indicates a high mechanistic similarity. The results suggest a unique role for the long splice variants of dynI and dynIII in nerve terminals.

Synaptic vesicle endocytosis (SVE)1 is triggered by a coordinated dephosphorylation of a group of at least eight proteins called the dephosphins (1). The dephosphins are constitutively phosphorylated in nerve terminals and their collective rephosphorylation after SVE is necessary for maintaining synaptic vesicle recycling and thus synaptic transmission. One such dephosphin is dynamin I (dynI), a large GTPase enzyme which is crucial for the fission stage of SVE (2). During SVE, dynI is dephosphorylated by the calcium-dependent phosphatase calcineurin (3) and is subsequently rephosphorylated by cyclin-dependent kinase 5 (cdk5) on Ser-774 and Ser-778 (4;5). These phosphorylation sites, located in the proline-rich domain (PRD), are thought to regulate the interaction with the src-3 homology (SH3) domain

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Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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containing proteins involved in SVE. A long list of SH3 domain containing proteins has been shown to bind the dynI PRD in vitro. Recently, we have identified syndapin I (sdpnI) as the phosphorylation-regulated dynI partner in vivo and that its interaction with dynI is crucial to SVE (6).

Apart from phosphorylation of Ser-774 and Ser-778 by cdk5, there have been a number of reports on other dynI phosphorylation sites and their potential protein kinases, both in vivo and in vitro. DynI is phosphorylated on Ser-857 by minibrain kinase/Dyrk1A in vitro and the phosphorylation regulates binding of dynI to amphiphysin I (amphI) and Grb2 (7). This phosphorylation was shown to occur in PC12 cells and was responsive to depolarising stimuli, strongly suggesting a physiological relevance. It has also been reported that dynI is phosphorylated at Thr-780 by cdk5 which apparently regulates its binding to amphI (8). This is in conflict with our previous in vivo phosphorylation site analysis of dynI, which did not detect phosphorylation at this site (5). Other studies have suggested that there may be other kinases capable of phosphorylating dynI in vitro, but the in vivo relevance of these events has not been established (9-12). DynI and dynII are also substrates for the tyrosine kinase c-src in non-neuronal cells on Tyr-231 and Tyr-597 (13;14). The two sites are highly conserved between all three dynamin genes. However, the tyrosine phosphorylation of dynI has only been reported in transfected non-neuronal cells where it is not known to be normally expressed (13), while that of dynII occurs under endogenous conditions (13;14). This suggests that in vivo phosphorylation of these tyrosine phospho-sites in dynI remains an open question.

In this study, we sought to determine whether there were other in vivo dynI phosphorylation sites, in addition to Ser-774, Ser-778 and Ser-857, that may prove to be functionally important for SVE. We established a method that ensures maximum purification of 32P-labelled dynI so that none of the relevant phosphorylation sites were missed. We show that dynI was exclusively phosphorylated on serine residues by 32P-phospho-amino acid analysis. We identified two new dynI phosphorylation sites at Ser-347 and Ser-512 and two new stimulation-dependent dynI phosphorylation sites at Ser-822 and Ser-851. We also showed that phosphorylation of Thr-780 by

cdk5 was restricted to in vitro conditions, did not occur in vivo (within detection limits) and it did not regulate binding of amphI.

EXPERIMETNAL PROCEDURES

DNA constructs and protein expression – The dynI PRD (rat, amino acids 746-864) was amplified from the GFP-tagged dynamin and subcloned into pGEX4T-1 as described previously (6). DynI point mutants were generated using the QuickChange site-directed mutagenesis kit (Stratagene), and were confirmed by DNA sequencing. GST-amphI SH3 domain was from Pietro de Camilli (Yale, New Haven, CT). GST-sdpnI SH3 domain was from Markus Plomann (University of Cologne, Germany). GST-endophilin I (endoI) SH3 domain was from Peter McPherson (McGill, Canada). GST-cdk5 and GST-p25 was from Jerry Wang (Hong Kong University, Hong Kong) and Li-Huei Tsai (Massachusetts Institute of Technology, Cambridge, MA), respectively. All GST fusion proteins were expressed in Escherichia coli and purified using glutathione-sepharose beads (Amersham Biosciences) according to the manufacturers’ instructions.

Synaptosomal dynI purification – Crude (P2) synaptosomes were prepared from rat brain and labelled with 32P as described previously (10). Briefly, synaptosomes were incubated with 0.75 mCi/ml [γ-32P]-Pi for 1 h at 37°C. The synaptosomes were briefly depolarized with 41 mM KCl and immediately lysed as described previously (4). DynI was purified from synaptosome lysates using the GST-amphI SH3 domain bound to glutathione sepharose beads as described previously (4). GST-endoI SH3 domain and GST-sdpnI SH3 domain were also used as bait to pull-down dynI from synaptosome lysates, as described previously (6). Beads were washed extensively, eluted in 2x SDS sample buffer and resolved on 7.5-15% gradient SDS gels.

Tryptic digestion and phosphopeptide enrichment – DynI gel bands, each containing approximately 2 μg of purified protein, were excised and diced from colloidal Coomassie Blue stained SDS gels. Four untreated and four KCl treated 32P-labelled dynI bands were used in each two dimensional (2D) map. Twenty unlabelled dynI bands were TiO2-enriched (see below) and

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added to the untreated sample for 2D mapping. Two dynI bands were used in the nanoLC-MSMS experiments (see below). The bands were destained in three 1 ml washes of 25 mM ammonium bicarbonate in a 50% acetonitrile solution with 1 h of vortexing between washes. The gel pieces were digested in 25 mM ammonium bicarbonate aqueous solution containing 12.5 ng/μl trypsin at 37°C overnight. The digest solution was made up to 50% acetonitrile and the tryptic peptides were extracted after 15 min of vortexing. A second extraction was obtained with 50% acetonitrile solution after 15 min of vortexing. A final extraction was obtained with 80% acetonitrile, 5% formic acid solution after 15 min of vortexing. The combined extract was dried down to 2.5 μl.

Phosphopeptides from a tryptic digest of non-radioactive dynI were enriched using TiO2 as described earlier (15). Briefly, the tryptic digest was concentrated into 5 μl and added to 25 μl of loading solution (300 mg/μl dihydroxybenzoic acid in 0.1% TFA 80% acetonitrile or 5% TFA in 80% acetonitrile in the absence of dihydroxybenzoic acid). The sample was loaded onto a GELoader tip (Eppendorf), converted to a microcolumn, packed with TiO2 and washed twice with loading solution and then twice with a wash solution 0.1% TFA 80% acetonitrile, without the presence of dihydroxybenzoic acid. The sample was eluted in 20% ammonium hydroxide 20% acetonitrile solution and immediately dried.

Phospho-amino acids analysis and 2D phosphopeptide mapping The 32P-labelled dynI tryptic digest was analysed by phospho-amino acid analysis as described previously (4;10). 2D phosphopeptide mapping was done on 20 x 20 cm cellulose plates (Merck) by electrophoresis at pH 4.7 (16) followed by ascending chromatography in 35% 1-butanol, 20% pyridine, 7.5% acetic acid, 2.5% acetonitrile and 35% water. The radioactive spots were detected by quantitative phosphorimaging (Storm 860, Amersham Biosciences), scraped from the plate and extracted from the cellulose as described (16), except that the extraction was done in 5% formic acid solution. The phosphopeptides were concentrated using C18 or graphite microcolumns and detected by matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS,

Voyager DE-PRO, Applied Biosystems) as described previously (5).

A theoretical 2D phosphopeptide map was generated firstly by determining the theoretical electrophoretic mobility by predicting the charge at pH 4.7 and dividing by the mass of the phosphopeptide as described by Meisenhelder et al. (16). The mobility in the second dimension, which is based on relative hydrophobicity, was estimated from the reversed phase HPLC index (17) as calculated using GPMAW v7.01 (Lighthouse Data, Denmark). The theoretical HPLC index was decreased by two units for each additional phosphate group to account for the observed decreased mobility of multiply phosphorylated peptides (Fig. 2B-C).

Nano-liquid chromatography mass spectrometry – An 8 μl aliquot (estimated at 0.5-1.5 pmol) of TiO2-enriched dynI phosphopeptides was loaded onto the nano-HPLC system (LC Packings Ultimate HPLC system, Dionex, Netherlands) with a 75 μm inside diameter (i.d.) pre-column of C18 reversed phase material (ReproSil-Pur 120 C18-AQ, 3 μm beads, Dr Maisch, Germany) in 5 mins. It was then eluted through a 50 μm i.d. C18 column of the same material at 100 nl/min. The gradient was from 100% phase A (0.1% formic acid in water) during loading, then increasing to 10% phase B (90% acetonitrile, 0.1% formic acid and 9.9% water) in three minutes, then to 50% phase B in twenty-eight minutes, then to 60% phase B in three minutes and finally to 100% phase B in one minute. The eluate was sprayed through a 10 μm i.d. distal coated SilicaTip (New Objective, USA) into a QSTAR XL quadrupole-TOF (QqTOF) MS (Applied Biosystems, USA) or a QTOF Ultima MS (Micromass/Waters, UK) using 1900 V on the tip. For the detection of phosphopeptides of a known molecular mass the precursor ion selection was fixed at the specific m/z of the phosphopeptide in its most abundant charge state with a wide m/z setting (2-3 units) using consecutive two second scans. Information-dependent data acquisition was done by using a 1 second survey scan from which the three most abundant doubly, triply or quadruply charged peptides were selected for product ion scans (2 seconds). The data for the phospho-dynI343-364 was from experiments using stable isotope labelling,

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the full details of which will be published elsewhere. As a consequence, the N-terminus of a phospho-dynI343-364 was labelled with iTRAQ reagent (Fig. 4C). In this instance, the TiO2–enriched digest was first treated with iTRAQ 116 reagent (Applied Biosystems, USA) according to the manufacturer’s instructions before nanoLC-MSMS analysis. All phosphopeptides reported in this study were detected in at least three independent experiments.

In vitro phosphorylation – GST-dynI PRD (1 µg) immobilized on glutathione-sepharose beads was phosphorylated by recombinant cdk5/p25 in a total volume of 40 µl containing 30 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM MgCl2, 0.05% Tween-20 and 40 µM [γ-32P]ATP (6.9 x 106 cpm/nmol) at 37°C. Phosphorylation proceeded for 10 min and reactions were terminated by addition of 3x SDS sample buffer and boiling. Samples were analysed by SDS-PAGE and phosphoproteins were detected by autoradiography. A single band each of in vitro phosphorylated GST-dynI PRD wild type and double mutant Ser to Ala were digested as above and enriched for phosphopeptides using Fe3+-IMAC as described previously (5). The phospho-box phosphopeptides were sequenced by nano-LCMSMS by fixing the precursor ion selection at a specific m/z as described above.

Pull-down experiments – A rat brain or synaptosomal extract was prepared as described previously (6). Various GST-dynI PRD or GST-amphI SH3 recombinant proteins were then incubated with the same amounts of tissue lysate at 4°C for 1 h. All pull-downs were done in the presence of 150 mM NaCl unless stated otherwise. Beads were washed extensively, eluted in 2x SDS sample buffer and analysed by SDS-PAGE.

Antibodies and Western blots – The anti-amphiphysin I monoclonal antibody was from Pietro de Camilli. The anti-dynI antibodies and phospho-specific antibodies to phospho-Ser-774 and phospho-Ser-778 in dynI were reported previously (4). Immunoprecipitations from rat brain synaptosomes were performed as described previously (10). Protein samples were separated by SDS-PAGE on 10% or 12% acrylamide gels and transferred to nitrocellulose membrane. Western blots were analysed by enhanced chemiluminescence method using the SuperSignal

West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

RESULTS

Quantitative extraction of dynI by GST-amphI SH3 domain - To catalogue all in vivo phosphorylation sites in dynI from rat brain synaptosomes, we used a method capable of extracting and purifying all dynI from synaptosome lysates, as judged by SDS-PAGE analysis and autoradiography. We have previously reported that the SH3 domain of amphI quantitatively extracts synaptosomal dynI independently of its phosphorylation status (6). However, another study has proposed that the interaction of GST-amphI SH3 with dynI is phospho-dependent (8). Here, we compared the efficiency of GST-fusion proteins of amphI, endoI and sdpnI SH3 domains to quantitatively extract dynI by three sequential pull-downs. Initial pull-downs with GST-amphI SH3 and GST-sdpnI SH3 recovered a large amount of endogenous dynI protein (Fig. 1A) and 32P-labelled phospho-dynI (Fig. 1B), whereas GST-endoI SH3 bound less dynI. A second pull-down of the same extracts with GST-amphI SH3 was performed to capture residual dynI missed in the first pull-down. There was less than 2% residual dynI protein and phospho-dynI following amphI or sdpnI in the first pull-down (Fig. 1A and B). In contrast, there was significant dynI and phospho-dynI captured following endoI SH3 in the first pull-down (Fig. 1A and B, middle panels). This is in agreement with a previous report that endoI SH3 is partly sensitive to dynI phosphorylation in vitro (6), and shows that amphI SH3 can indeed capture this small pool. A third sequential pull-down with sdpnI SH3 or endoI SH3 domains recovered no residual dynI protein or phospho-dynI after two previous extractions with amphI SH3, endoI SH3 or sdpnI SH3 (Fig. 1A and B). Therefore the SH3 domain of amphI and sdpnI quantitatively extracts synaptosomal dynI from synaptosomes.

In an independent experiment, we performed the similar GST-amphI SH3 triple pull-downs using synaptosome extracts and blotted with anti-dynI antibodies. Two rounds of GST-amphI SH3 pull-down was enough to quantitatively extract all synaptosomal dynI because no trace of unbound dynI was found in the

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second or third pull-down (Fig. 1C). We thus used this system of double GST-amphI SH3 pull-downs in all subsequent experiments to purify all synaptosomal dynI, regardless of its in vivo phosphorylation status.

DynI is phosphorylated exclusively on serine residues - DynI phosphorylation on Thr-780 has been controversial (18). It was not detected in a previous in vivo study (5) but was claimed to be found after in vitro phosphorylation with cdk5 (8). A simple way to address whether a protein is phosphorylated on serine, threonine or tyrosine residues is by phospho-amino acid analysis after 32P-labelling. There may be some phosphorylation sites that are not labelled with 32P, however in dynI these sites are unlikely to be relevant to SVE as they do not turnover 32P in 1 hour of labelling. The first published report of phospho-amino acid analysis on synaptosomal dynI showed only serine was phosphorylated (19). The source of dynI in that experiment was bands cut from an SDS gel of a synaptosome lysate, which may be contaminated by another underlying protein with similar size. The same result was reported when dynI was purified by a single pull-down with the GST-amphI SH3. Phosphorylation at threonine or tyrosine was absent (4). In this study, we performed phospho-amino acid analysis using dynI purified by GST-amphI-SH3 pull-downs from 32P-labelled synaptosomes and again found only serine was phosphorylated (Fig. 2A). We conclude that synaptosomal dynI is exclusively phosphorylated on serine residues with no detectable 32P-labelling on threonine or tyrosine residues.

Separation and detection of in vivo 32P-labelled dynI phosphopeptides - A tryptic digest of dynI purified from 32P-labelled synaptosomes was subjected to 2D phosphopeptide mapping (Fig. 2B). Similarly, a tryptic digest of dynI from synaptosomes depolarised with 41 mM KCl for 10 s was also analysed in parallel (Fig. 2C). The 32P-labelled phosphopeptides were detected by autoradiography. We observed 15 clearly distinct spots on the 2D map (A to O). The majority of the radiation was in spots A to D (Fig. 2B). These spots have previously been shown to contain overlapping tryptic phosphopeptides (including tryptic cleavages and missed cleavages) from the dynI phospho-box (772RRSPTSSPTPQRR784) (6).

Spots C and D were previously shown to contain almost exclusively phospho-Ser-774, and spots A and B were shown to contain doubly-phosphorylated peptides that are equal parts phosphorylated at Ser-774 and Ser-778 (5).

Tandem MS identification of phosphorylation sites in dynI phosphopeptides - In order to account for all the 32P that is distributed among the various dynI phosphorylation sites, the phosphopeptides contained within each spot on the 2D map were extracted from the cellulose and analysed by MALDI-MS and tandem MS. Spots A-D contained the dynI phospho-box peptides (772RRSPTSSPTPQRR784, data not shown) that we identified previously (6). Spots E to H had low relative radiation levels and were analysed by MALDI-TOF MS, but no phosphopeptides were detectable (Fig. 2B). Similarly, no phosphopeptides were detected in spots J, M and N which had even lower levels of total radiation (approximately 2%, 1% and 1% respectively, Fig. 2B).

Mono- and di-phosphopeptides matching the tail of the long splice variant dynIxa, 847SGQASPRSPESPRPPFDL864, were found in spots K and I, respectively. The mono-phosphorylated peptide in spot K was determined to be dynIxa847-864, where either Ser-851 or Ser-857 is phosphorylated (data not shown). Neither phosphorylation site was grossly dominant in this mixture of two phosphopeptides of equal molecular mass. The di-phosphorylated peptide in spot I was determined to be dynIxa847-864 where both Ser-851 and Ser-857 are phosphorylated (Fig. 3A). Phospho-Ser-851 was detected between y13 and y14 and also as a dehydroalanine between b4 and b5. Phospho-Ser-857 was revealed as the only possible position for the second phosphorylation site in the sequence 855PES857 between the b8/y10 and b11/y7 ions. In contrast to the two sites in the dynI phospho-box (5;6), there appears to be no hierarchy between phospho-Ser-851 and 857, suggesting independent regulation and potentially independent protein kinases.

A weak signal, that was sensitive to phosphatase treatment, was detected by MALDI-TOF MS in spot O. NanoLC-MSMS was used to sequence this peptide by selecting the quadruply charged precursor at m/z 606.3. The sequence was determined to be dynIIIxxb 849RPPPSPTRPTIIRPLESSLLD869 where Ser-853

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is phosphorylated (Fig. 3B). DynIII was previously found to be co-purified with dynI from P2 synaptosomes (5). Small y-type ions and both small and large b-type ions were detected, with little information on the middle part of the sequence. However, the position of the phosphorylation site was unequivocally determined by crucial ions such as the b2 and b5 ions, which limit the phosphorylation site to the sequence 851PPS853, of which only Ser-853 can be phosphorylated. In addition to the homologous phosphorylation of the dynI and dynIII phospho-box (5), we have now revealed that dynIIIxxb has a third in vivo phosphorylation site in a sequence location that is homologous to the dynIxa sequence containing phospho-Ser-851. As for dynI, this site is present only in the long-tailed splice variant of dynIII.

Spot L was analysed by MALDI-TOF MS and a phosphopeptide was observed, as confirmed by dephosphorylation with Antarctic phosphatase (data not shown). NanoLC-MSMS was required to sequence this phosphopeptide. The sequence was dynI 797PAPGPPPAGSALGGAPPVPSRPGASPDPFGPPPQVPSRPNR838 where Ser-822 is phosphorylated (Fig. 4A). Small b-type ions and y-type ions throughout the sequence were used to determine the phosphorylation site and the identity of the phosphopeptide. Y-type ions up to y16 and b-type ions up to b16 (and then a weak signal at b25) were not phosphorylated. Since y17 and higher ions were phosphorylated, the phosphorylation site can be unequivocally localised to Ser-822. This is the fifth proline-directed serine (i.e. SP sequence) in the dynIxa PRD that was found to be phosphorylated in vivo.

Finally, we used another independent approach to maximise detection of any relatively minor phosphorylation sites. Extraction of phosphopeptides from cellulose plates after 2D peptide mapping may not be completely efficient due to adsorption to surfaces. Therefore to maximize the number of phosphopeptides detected, without relying on 32P-labelling, we used TiO2 to enrich for all phosphopeptides from a dynI tryptic digest and analysed them using nanoLC-MSMS with information-dependent data acquisition. Two new dynI phosphorylation sites were found. Automatic selection of the precursor at m/z 712.8 produced a unique fragmentation

spectrum (Fig. 4B). This phosphopeptide matched the sequence of dynI 511TSGNQDEILVIR522 where Ser-512 is phosphorylated. The sequence was entirely described by y-type ions. The phosphorylation site was detected as the dehydroalanine residue between y11 and y10. In the same experiment, we also sequenced phospho-dynI510-522 and found that Ser-512 was phosphorylated in this slightly larger phosphopeptide (data not shown). Automatic selection of the precursor at m/z 750.0 produced another unique fragmentation spectrum (Fig. 4C). This phosphopeptide matched dynI 343RIEGSGDQIDTYELSGGARINR364 where Ser-347 is phosphorylated. Non-phosphorylated y-type ions ruled out the possibility of phosphorylation near the C-terminal half of this phosphopeptide. The phosphorylation site was deduced to be at Ser-347, before the residues producing phosphorylated (b5 to b11) fragments and after the residues producing non-phosphorylated fragments (b3). This is the first time that in vivo dynI sites have been detected outside of the PRD. It is possible that these sites were 32P-labelled, but were not detected by MALDI-TOF MS.

It can be predicted where the peptides encompassing Ser-512 and Ser-347 would appear in the 2D map in Fig. 2B, if they were 32P-labelled. We plotted the theoretical HPLC index versus the estimated electrophoretic mobility for each of the detected dynI and dynIII peptides (Fig. 5A). Only peptides with up to one missed trypsin cleavage are shown. The theoretical electrophoretic mobility correlated with the experimentally observed mobility of the sequenced phosphopeptides (Fig. 2B-C and Fig. 5A). In the second dimension, the HPLC index was a good predictor of mobility, except for the large peptide in spot L (Fig. 2B-C and Fig. 5A). This may reflect an over-emphasis on peptide molecular mass when using the HPLC index. DynI511-522 has a HPLC index greater than dynI774-

783 (spots A to D) and less than dynI847-864 (spots K and I, Fig. 5A). The phosphopeptides dynI511-522, dynI511-523 and dynI510-522 are predicted to migrate in the second dimension near the unidentified spots at E to H (Fig. 2B). Therefore, phosphorylated dynI510-523 theoretically accounts for at least two of the spots in the region of E to H. There are two unassigned spots (M and N, Fig.

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2B) above dynI847-864 (spots K and I). Spot N (Fig. 2B) might be accounted for by dynI839-864 which would be expected to be phosphorylated at Ser-851 and/or Ser-857. The theoretical mobility of dynI343-361 predicts migration to a spot above and to the left of dynI847-864 (spots K and I). Therefore, dynI343-361 theoretically accounts for spot M. However, direct evidence is lacking for Ser-347 and Ser-512 being 32P–labelled in 1 hr during synaptosome labelling or depolarisation. Moreover, the possibility of non- or semi-tryptic dynI proteolysis products and minor contaminating non-dynI phosphopeptides in the 2D map cannot be ruled out.

Distribution of the 32P between phospho-sites - We next quantified the amount of phosphate in each spot (Table 1). Quantitative analysis allowed a determination of the potential in vivo significance of each site in terms of its abundance. Note that “cold phosphorylation” (i.e. that occurring prior to 32P-labelling) was not quantifiable in this way. The 32P-labelled peptides were detected by autoradiography and the intensity of each spot was quantified and expressed as a percentage of the total radiation in all spots (Fig. 5B). Using quantitative phosphorimaging data, plus the knowledge of which phosphopeptide accounted for each spot it was possible to deduce that phospho-Ser-774 and Ser-778 were 32P-labelled in a ratio of two to one (6). We now extend this analysis to cover all phosphorylation sites. The phosphopeptides identified in each spot are shown (Fig. 5B). Spots A-C each contained about 20% of the total 32P and contained Ser-774 or Ser-774 plus Ser-778. Therefore these previously reported sites contain 69% of the total 32P in dynI. Note that a small, unknown amount of this radiation is derived from dynIII (5), but this amount was ignored in the present calculation. MALDI-MS spectra have indicated that dynIII phospho-box phosphopeptides are between five and ten-fold less abundant the homologous dynI phosphopeptides (4;5). Spot O was dynIII Ser-853 and had 2% of the total 32P (Fig. 5B). Spots K and I together accounted for 12% of the total radiation. The majority of the 32P was in spot K which was a mix of Ser-851 or Ser-857 phosphopeptides. The minority of this label was in spot I which was doubly-phosphorylated on both sites; therefore the double phosphorylation is relatively rare. Spot L had 5% of the radiation and contained Ser-822.

Spots E-H together contained a combined value of 8% of the total 32P radiation. Although likely to include Ser-512, the presence of other sites or phosphopeptides from contaminating proteins cannot be ruled out. Therefore Ser-512 must represent less (or much less) than 8% of the total dynI phosphorylation. Spots M-N contained ~2% of the total 32P. No sites were identified, but Ser-347 is predicted to migrate in this region. Assuming this to be the case, Ser-347 has less than 2% of the dynI total phosphorylation. Overall, all the sites identified to date can be ranked in decreasing order of abundance: 774, 778, 857 and 851, 822, 512 and 347.

To predict the potential biological significance of each phosphorylation site we next determined which sites were responsive to depolarisation (Fig. 5C). This provides a crude measure of the relevance of each site to depolarisation-induced SVE. The amount by which each phosphopeptide in spots A to D was decreased upon depolarization was quantified (Fig. 5B) and correlated to the phosphorylation site identified (found here and previously). The amount of 32P-labelled phospho-Ser-774 was decreased by 30% (±2% s.e.m.) and phospho-Ser-778 was decreased by 47% (±3% s.e.m.) following depolarization (Fig. 5C). Again, the small amount of radiation from co-purified dynIII phospho-Ser-769 and Ser-773 in spots A to D was ignored. Although the phosphopeptide containing the sites Ser-851 and Ser-857 were not highly 32P-labelled, the phosphorylation was reduced by 34% (range of ± 1%) following depolarization (Fig. 5C). Therefore, the two C-terminal phosphorylation sites in dynIxa are dephosphorylated upon depolarization. The phosphopeptide containing Ser-822 was significantly dephosphorylated by 56% (± 0.5% s.e.m.) following depolarization (Fig. 5B-C). No depolarisation-dependent change in the phosphorylation of Ser-512 could be measured since there was no 32P-label directly associated with this site. However, if it was part of spots E-H as we surmise, then it was depolarisation-sensitive (Fig. 5B). There was no depolarisation-dependent change in the phosphopeptide surmised to contain Ser-347.

Phosphorylation of Thr-780 on dynI is restricted to in vitro conditions - It was previously reported that dynI is phosphorylated by cdk5 only

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at Thr-780 in vitro and that all phosphorylation is blocked by mutation of Thr-780 to Ala (8). This data strongly suggests Thr-780 is the sole (cdk5-mediated) phosphorylation site in the dynI PRD and conflicts with our main findings. We previously found no evidence for Thr-780 phosphorylation in vivo (5). Mass spectrometry is not a good tool to resolve this discrepancy since it cannot be used to definitively prove the absence of a phosphorylation site. Therefore we used a site-directed mutagenesis strategy to determine how a single mutation on Thr-780 might abolish [γ-32P]ATP phosphorylation of dynI on Ser-774 or Ser-778 by cdk5 in vitro. The results contradict those of Tomizawa et al. (8). We generated a series of single, double or triple point mutations of Ser-774, Ser-778 and/or Thr-780 to Ala in GST-dynI PRD for the purpose of testing the circumstances under which phosphorylation of the phospho-box by recombinant cdk5/p25 could be prevented (Fig. 6A). Single or double mutation of Ser-774 and Ser-778 had little effect on the extent of phosphorylation by cdk5, as determined by autoradiography (Fig. 6B) and by quantitative phosphorimager analysis (Fig. 6C). A similar result was obtained with single mutation on Thr-780 (Fig. 6B-C). However, when all three sites in the phospho-box were mutated to Ala, phosphorylation was greatly reduced (32 ± 3% of Dyn1 WT, P < 0.001, Fig. 6B-C). It appears that mutation of any one of these three sites results in a compensatory increase in phosphorylation at one or more of the remaining sites. So it is only when all three sites are mutated that a significant drop in phosphorylation was detected in the PRD. However, note that 30% of the total phosphorylation remained in the triple mutant, suggesting a major contribution from other sites in the PRD. The dynI PRD construct used in these studies was based on the long splice variant dynIxa, therefore there are at least three additional sites available for phosphorylation by cdk5; Ser-822, Ser-851 and Ser-857. Some or all of these may account for the remaining phosphorylation in Fig. 6B.

Using tandem mass spectrometry, we confirmed that the phospho-box of the in vitro phosphorylated wild type GST-dynI PRD was indeed phosphorylated at Ser-774, Ser-778 and Thr-780 (Supplementary Fig. S1). As expected, in vitro phosphorylation at Thr-780 was most easily

observed by analysing the double mutant dynI PRD (Fig. 6D). The phosphorylation at Thr-780 was determined by the y3 and y4-98 ions. The relatively strong signal for the phosphorylated y5 fragment ion, owing to the favourable cleavage of an X-P bond (20), was a clear signal that the phosphorylation site on this peptide was C-terminal to Pro-779, i.e. it must only be Thr-780 since no other part of 779PTPQR783 can be phosphorylated. This y5 ion was also a strong signal in the spectra of the wild type sequences (Supplementary Fig. S1), alternately conferring and denying phosphorylation to Thr-780. Curiously, the phosphorylated y5 ion was absent from the spectrum of a similar in vitro analysis by Tomizawa et al. (8).

The results demonstrate that a single mutation in a protein that is multiply phosphorylated is insufficient to detect reduced overall in vitro phosphorylation. Whilst Thr-780 is easily detected as an in vitro phosphorylation site, it does not necessarily follow that Thr-780 is phosphorylated in vivo. Our systematic approach contradicts the overall results and conclusions of Tomizawa et al. (8).

AmphI binding is independent of dynI phosphorylation - There are several reports that amphI binding to dynI is regulated by the in vitro phosphorylation status of dynI (8;21). To extend these in vitro observations, we also generated a series of Glu mutations at Ser-774, Ser-778 and Thr-780 in GST-dynI PRD (Fig. 6A). Pull-down experiments with GST-dynI PRD wild type and GST-dynI Ala mutants (non-phosphorylatable) or Glu mutants (pseudo-phosphorylation) did not significantly alter the binding of native full-length amphI as shown by Western blot (Fig. 7A). The results were confirmed by quantitative densitometry analysis of multiple experiments (Fig. 7B).

We then performed a reverse pull-down experiment, using recombinant GST-amphI SH3 to capture native dynI in the presence of different sodium chloride concentrations in the pull-down buffer. As the salt concentration increased from 0 to 1 M, the amount of total dynI protein bound to GST-amphI SH3 decreased (Fig. 7C). However, the amount of phosphorylated dynI bound was unchanged, even at high salt concentration (Fig. 7C). This suggests that amphI SH3 binding to dynI is not regulated by dynI phosphorylation (see

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Fig. 1). The observation that GST-amphI SH3 has an apparently higher binding affinity towards phospho-dynI than non-phospho-dynI is interesting, but not likely to have any physiological relevance because it only occurred at 0.5-1M NaCl.

We have shown how the dynI PRD interacts with full-length amphI and how amphI SH3 interacts with full-length dynI. Next, we used an immunoprecipitation experiment to ask whether the association of native full-length dynI with native full-length amphI might be phosphorylation-regulated. Pretreatment of synaptosomes with Ba2+ for 1 hr was previously shown to produce massive dynI dephosphorylation (22) by chronic depolarisation (23). We prepared Ba2+-treated synaptosomes and compared them to resting (0.1 mM Ca2+) synaptosomes. DynI was immunoprecipitated from each of these preparations. DynI was massively dephosphorylated upon Ba2+-treatment, as shown by Western blot analysis with the anti-phospho-Ser-774 antibody (Fig. 7D). However, the interaction between amphI and dynI was not affected (Fig. 7D and E). From these interactions studies we conclude that amphI is not a phosphorylation-dependent partner for dynI, in vivo or in vitro.

DISCUSSION

DynI has seven phosphorylation sites - An exhaustive strategy was used to identify and rank all the phosphorylation sites in dynI. Mass spectrometry of synaptosomal 32P-labelled dynI phosphopeptides separated by 2D tryptic mapping revealed seven in vivo phosphorylation sites at Ser-774, Ser-778, Ser-822, Ser-851, Ser-857, Ser-512 and Ser-347 (Fig. 8A). Thr-780 phosphorylation was not detected. It was shown previously that a peptide containing Ser-857 was phosphorylated in vitro by Mnb/Dyrk1A (7), whereas a peptide containing Ser-851 was not. In this study, we confirmed the existence of Ser-857 as an in vivo phosphorylation site (7) and identified Ser-851 as a new in vivo phosphorylation site within the same tryptic peptide. These sites are restricted to the tail of the longer splice variant, dynIxa (Fig. 8B). We found that Ser-851 and Ser-857 were phosphorylated to a similar extent and there was no hierarchy to their

phosphorylation. It has been shown that Ser-857 phosphorylation reduced dynI binding to amphI SH3 domain (7). However, our data suggested that dynI phosphorylation does not affect binding of amphI. It may be difficult to discern the true effect of phosphorylation at Ser-857 on amphI binding from our experiments since it is <12% of the total radiation and we focused mainly on cdk5 substrates within the phospho-box.

The phosphorylation site found at Ser-512 is near the start of the pleckstrin homology domain (Fig. 8A). It is not known how much the phosphate on this site is turning-over since we were unable to correlate a radioactive spot with the sequenced phosphopeptides. Nonetheless, some of the unidentified spots were good candidates for containing phospho-Ser-512, based on peptide mobility prediction. If Ser-512 is involved in SVE, then it is most likely involved in regulating dynI interaction with phospholipids. The phosphorylation at Ser-347 is nearest to the GTPase domain and may have some influence on this domain. Along with part of the assembly domain, this domain is thought to be involved in dyn I tetramerization and higher-order self assembly (24). The lack of a crystal structure or functional role for the sequence encompassing this site precludes further comment on its function. This site is predicted to have low or no turnover of 32P in a 1 h incubation of synaptosomes and it is therefore difficult to envisage an important role for Ser-347 in SVE. Both Ser-347 (RIEGSGDQID) and Ser-512 (KKKTSGNQDE) are not situated in front of a proline residue (Table 1). Ser-512 is more clearly in a basophilic context than Ser-347, on the N-terminal side of the phosphorylation site, so they may not be phosphorylated by the same protein kinase. However, both Ser-347 and Ser-512 share some common amino acid sequences (SGxQ) followed closely by an acidic residue on the C-terminal side. Hence, we conclude that there is at least one other protein kinase, apart from cdk5 (and potentially minibrain kinase/Dyrk1A (7)), that phosphorylates dynI in vivo.

Ser-822 (RPGASPDPF) is located in front of a proline and might be phosphorylated by one of the same protein kinases that phosphorylate Ser-774, Ser-778, Ser-851 or Ser-857. Ser-822 is flanked on either side by PxxP motifs, which bind SH3 domain containing proteins. The motifs

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immediately C-terminal to Ser-822 are involved in binding grb2 and amphI (these are called Site 8 and Site 9, respectively, Fig. 8B) (25). Therefore, it is possible that this binding may be phospho-regulated by Ser-822.

New information about the hierarchical nature of Ser-774 and Ser-778 was revealed when the extent of dephosphorylation of each site was quantified. The data showed that following depolarization, phospho-774 and phospho-778 are dephosphorylated to a similar extent, perhaps phospho-778 slightly more. More importantly, the pool of doubly phosphorylated dynI (with both Ser-774 and Ser-778 phosphorylated) was dephosphorylated, in preference to the singly phosphorylated pool (with only Ser-774). This implies a greater relative importance on the removal of phospho-Ser-778. We propose that dynI phosphorylation at Ser-774 has a different regulatory role to phosphorylation at Ser-778 in SVE.

Phosphorylation at Thr-780 is an in vitro artefact - Controversially (18), it was reported that cdk5 phosphorylates dynI at Ser-774 and Ser-778 in two studies (4;5) and solely at Thr-780 in another study (8). However, we have now demonstrated that although Thr-780 is phosphorylated in vitro by cdk5 and phosphorylation mutants may be able to produce a functional effect in vivo (8), it is unlikely that dynI is phosphorylated at Thr-780 in vivo in synaptosomes within the approximately 100-fold detection limits of our methods. The most likely explanation is that phosphorylation at Thr-780 is restricted to in vitro conditions; i.e. it is simply an artefact of cdk5 phosphorylation in vitro. Three experiments supported our conclusion that Thr-780 is not phosphorylated by cdk5 in synaptosomes. Firstly, phospho-amino acid analysis of purified dynI from 32P-labelled synaptosomes revealed exclusive phosphorylation of dynI on serine residues. This experiment alone, within the limits of detection, restricts Thr-780 or any tyrosine phosphorylation to being a static phosphorylation site (i.e. not labelled with 32Pi after 1 h) in synaptosomes or to not being present in synaptosomes at all. Secondly, we were unable to detect any Thr-780 phosphorylation by exhaustive 2D phosphopeptide mapping of 32P-labelled dynI and MS analysis. This experiment, within the limits of detection, rules out the

presence of a major static pool of dynI phosphorylated on Thr-780. Any such pool must contain less than 1% of the phosphorylation in dynI. Thirdly, a single mutation of Thr-780 to Ala did not abolish cdk5 phosphorylation in the dynI-PRD, in contrast to observations reported by Tomizawa et al. (8). Like Thr-780, a single Ala mutation on Ser-774 and Ser-778, or in combination had no effect on dynI phosphorylation by cdk5 in vitro. It was only when all three sites at Ser-774, Ser-778 and Thr-780 were mutated to Ala that phosphorylation by cdk5 was significantly reduced. This suggests that there is a compensatory phosphorylation of alternative sites within the PRD when any single site is mutated. In fact, we have previously shown in phospho-amino acid analysis that phospho-threonine only appeared when dynI was phosphorylated by cdk5 in vitro, but was absent when dynI was purified from 32P-labelled synaptosomes (4). Potential differences between the two PRD splice variants are possible. The long form was used in our study (containing six SP or TP motifs, while there are only four in the short form), but the form used by Tomizawa et al. (8) was not reported (see Fig. 8). Placing further focus on in vitro phosphorylation vs in vivo phosphorylation site analysis does not appear to be constructive.

The remaining discrepancy between our study and that of Tomizawa et al. relates to the use of their claimed phospho-specific antibody to Thr-780. With this antibody it was shown that the phosphorylation of Thr-780 occurred in synaptosomes and brain slices and was stimulus-sensitive (see Fig. 7 of Tomizawa et al. (8)). While the data appears compelling at face value, it assumes there is specificity for this antibody towards phospho-Thr-780, which was not reported. Rather, that antibody was raised against a synthetic peptide that encompasses all three phospho-sites, Ser-774, Ser-778 and Thr-780, and was phosphorylated in vitro prior to immunisation of rabbits. It is not possible to prevent phosphorylation at Ser-774 or Ser-778 under these conditions, and indeed we showed that all three are phosphorylated in vitro in the PRD (Supplementary Fig S1). The site specificity of the antibody has not been tested and it is highly likely to cross-react with both phospho-serines. Therefore, the results obtained with its use cannot

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be ascribed to Thr-780, but match previous data for phosphorylation at the serines, particularly if Thr-780 is not phosphorylated in vivo.

DynIII has three phosphorylation sites - We previously found that a small amount of dynIII co-purifies during the dynamin purification from P2 synaptosomes and were able to identify the homologous phospho-box phosphorylation sites to Ser-774 and Ser-778 for dynIII (5). The amino acid sequences around these sites are highly conserved between dynI and dynIII. In this study we found a new dynIII phosphorylation site at Ser-853 that was 32P-labelled. DynIII has previously been reported to be mainly expressed in the post-synapse (26). It is unlikely that the P2 synaptosome preparation contained post-synaptic dynIII that could acquire 32P-label. Therefore, we conclude that the 32P-labelled dynIII was pre-synaptic. Ser-853 is homologous to Ser-851 of dynI. Like dynI, this site is only present in the long splice form. We note that Ser-857 from dynI is not present in dynIII (Fig. 8B). This third homologous phosphorylation site, Ser-853, suggests that dynIII may perform an analogous function to dynI in the pre-synaptic nerve terminal. Its phosphorylation on the same sites in synaptosomes suggests that the same protein kinases may be involved and that there is a physiologically significant role for Ser-853 (dynIII) and Ser-851 (dynI). The amino acid sequence of the tail of the long splice variants of dynI and dynIII are not similar except for 5 amino acids, SPxRP, that encompass the phosphorylation site motif (Fig. 8B).

It remains to be determined whether all the phosphorylation sites in dynIII have now been identified, although the results suggest that the main dynIII sites have been found. Among the 7 dynI sites, only Ser-774, 778, 851 and 347 are conserved in the sequence of dynIII. Three of them have now been identified, and while phosphorylation at Ser-347 in dynIII is possible, it would be well below detection limits in our current approaches. No sites were detected that were not homologous to dynI. However, the amino acid sequences of dynIII are sufficiently different as to indicate that different protein partners are phospho-regulated by these sites. The results suggest dynIII has a role in nerve terminals, however it must be a highly specialised role.

DynII phosphorylation? - Perhaps surprisingly, we found no evidence for phosphorylation of dynII in this study. DynII was present in the synaptosomes, as judged by western blot and mass spectrometry data (data not shown). The GST-amphI SH3 domain pull-down also extracts dynII and III (data not shown). Although we have not directly determined that the extraction of dynII or III is quantitative, it is likely to be so. In fact, our laboratory purifies dynII by pull-down on GST-amphII SH3 beads. The absence of any dynII phosphorylation sites in synaptosomes is not conclusive evidence that none are present, but is compelling because sites in dynIII were detectable at low levels. Only two of the seven dynI phospho-sites are conserved in the sequence of dynII. Ser-774 is missing in dynII, although Ser-778 and Ser-347 are conserved. Ser-822 and Ser-512 are missing in dynII, although the surrounding amino acid sequences are highly conserved. The tail of dynII is not subjected to the same alternative splicing as dynI and III, however it is highly related in sequence to the long tail of dynIII, both of which are missing Ser-857 (dynI). The main difference is that dynII specifically lacks Ser-851 (dynI, or Ser-853 in dynIII), hence it has none of the sites present in either of the other dynamins and would not be expected to be phosphorylated by a proline-directed protein kinase in this region. The absence of detection of phosphorylation of dynII at Tyr-231 and Tyr-597 (13;14) is not surprising since the synaptosomes were not stimulated with any growth factor receptors. However, these sites may be phosphorylated in other cellular compartments or after the appropriate stimulation.

A complete description of dynI in vivo phosphorylation? - We have attempted to map all the in vivo phosphorylation sites in dynI. However, no technology is yet sufficiently sensitive to allow unequivocal conclusions that all sites in any protein have been identified. Other previously reported sites were not found: Thr-780 (8), Tyr-231 and Tyr-597 in dynI (13;27) and dynII (14), or Ser-795 (28). We conclude that phosphorylation of Thr-780 and Ser-795 is restricted to in vitro conditions only. We found no evidence for phosphorylation on tyrosine, but cannot rule out several possibilities. Phosphorylation of Tyr-231 and Tyr-597 was first observed in dynI transfected into non-neuronal

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cells, thus phosphorylation occurred in an inappropriate in vivo context. Although we found no evidence for phosphorylation of dynI on Tyr, it, and other sites, cannot be ruled out completely for a variety of reasons. Firstly, other sites in dynI may be phosphorylated in different subcellular compartments or at different stages of development. Secondly, it is possible (although unlikely) that some phosphopeptides do not bind TiO2 or Fe3+-IMAC and therefore would have been missed. A number of phosphopeptides are not mass spectrometry “friendly” (i.e. do not produce sufficient signal), although the specific properties of such phosphopeptides have yet to be described. Within such limitations we conclude that any new sites discovered in the future must represent <2% of the total phosphorylation, since the majority of the 32P-label on dynI has been accounted for. The caveats on this conclusion are the possibility that a co-migrating, poorly detected phosphopeptide could have been missed in one of the spots, or the prior existence of high stoichiometry phosphorylation sites that may not

be labelled with 32P after 1 hour. The potential physiological significance of the latter hypothetical sites would be questionable in the context of SVE.

A thorough and deliberate strategy of maximized phosphoprotein capture, 32P-labelling and highly sensitive mass spectrometry has been utilized to avoid missing significant in vivo phosphorylation sites.

The discovery of four new in vivo dynI phosphorylation sites at Ser-512, Ser-822, Ser-851 and Ser-347 in addition to the three already identified sites (Ser-774, Ser-778 and Ser-857) provides a basis for further study of the phospho-regulation of dynI and SVE. It remains to be shown whether these new phosphorylation sites in the PRD regulate binding of other SH3 domain containing proteins, besides syndapin I (6), or whether they have a supplementary role in the same process of endocytosis or perhaps a major part in a mechanistically distinct mode of endocytosis.

FOOTNOTES

* We thank the large number of our colleagues who have generously provided materials for this study. This work was supported by grant from the National Health and Medical Research Council of Australia (P.J.R.) and University of Sydney Postgraduate Awards (V.A. and G.E.C.). 1 The abbreviations used are: Cdk5, cyclin-dependent kinase 5; 2D, two dimensional; ESI, electrospray ionisation; GFP, green fluorescent protein; MALDI, matrix assisted laser desorption ionization; MS, mass spectrometry; MSMS, tandem mass spectrometry; QqTOF, quadrupole-TOF hybrid; PRD, proline rich domain; SH3, src-3 homology; SVE, synaptic vesicle endocytosis; TOF, time of flight.

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FIGURE LEGENDS Fig. 1. AmphI SH3 domain quantitatively extracts dynI from nerve terminals. A, GST-amphI SH3, GST-sdpnI SH3 or GST-endoI SH3 bound to GSH-sepharose were used in pull-down experiments from 32P-labelled P2 synaptosomes lysed in Triton X-100 in the presence of 150 mM NaCl. Bound proteins were analysed by SDS-PAGE and visualised by Coomassie Blue staining. DynI (arrow) was extracted in duplicate pull-downs (top panel). GST-amphI SH3 was then used sequentially in the 2nd pull-down to recover any remaining unbound dynI in the initial extract (second panel). In the 3rd sequential pull-down of this extract GST-sdpnI SH3 or GST-endoI SH3 were used to recover any remaining unbound dynI (third panel). The amount of GST-SH3 domains used in the pull-down are shown (lower panel, which is from the same gel as in the top panel). B, Phospho-dynI was visualised by autoradiography of the same pull-down experiments. Results are representative of three independent experiments. C, GST-amphI SH3 bound to GSH sepharose was used in the triple pull-downs from synaptosome lysates. 10% each of the starting material (input), bound proteins from the 1st, 2nd and 3rd pull-downs and the unbound proteins from each pull-down were subjected to Western blotting and probed with specific anti-dynI and anti-phospho-Ser-774 antibodies (top two panels). The amount of protein loaded onto the SDS gel was visualised by Coomassie Blue staining (bottom panel). Fig. 2. Phospho-amino acid analysis and 2D tryptic phosphopeptide mapping. A, Autoradiograph of the phospho-amino acid analysis of dynI from 32P-labelled synaptosomes. DynI was purified using multiple sequential pull-downs with the GST-amphI SH3 domain. The migration of a standard mixture (std) of phospho-serine, -threonine and –tyrosine is shown alongside, as determined by ninhydrin staining. B, Autoradiograph of a 2D tryptic phosphopeptide map of purified dynI from control synaptosomes. Radioactive spots are labelled alphabetically above and to the left of each spot position. The phosphopeptides of the dynI phospho-box (772RRSPTSSPTPQRR784) are enclosed in a rectangle. These phosphopeptides were previously identified by tandem MS (5;6). C, Autoradiograph of the 2D phosphopeptide map of purified dynI from depolarised synaptosomes. Fig. 3. Phosphorylation sites in dynI and dynIII phosphopeptides detected by 2D tryptic mapping. A, Tandem mass spectrum of the phosphopeptide detected in spot I (such as in Fig. 2B). The phosphopeptide was extracted from the cellulose plate and analysed by ESI-QqTOF MS. The triply charged precursor at m/z 695.6 was selected and produced a sequence of b- and y-type ions that describe phospho-dynIxa847-864 where Ser-851 and Ser-857 are phosphorylated. Ions showing neutral loss of phosphoric acid (-98 Da) are shown. B, Tandem mass spectrum of the phosphopeptide detected in spot O (such as in Fig. 2B). A dynI tryptic digest enriched for phosphopeptides using TiO2 was analysed by nanoLC-MSMS by selecting the quadruply charged precursor at m/z 606.3. The fragment ions match the sequence of dynIIIxxb849-869 where Ser-853 is phosphorylated. The m/z range from 850 to 1150 has been multiplied by a factor of eighteen to improve clarity. Fig. 4. Phosphorylation sites from the dynI phosphopeptide in spot L and from two dynI phosphopeptides not detected in spots from 2D mapping. A, Tandem mass spectrum of the phosphopeptide detected in spot L. The quadruply charged precursor at m/z 1,010.3 was selected in a nanoLC-MSMS analysis of a dynI tryptic digest enriched for phosphopeptides using TiO2. The fragment ions match the sequence of dynI797-838 where Ser-822 is phosphorylated. The m/z range from 790 to 1,600 has been multiplied by a factor of eight to improve clarity. B, A dynI tryptic digest enriched for phosphopeptides using TiO2 was analysed by nanoLC-MS/MS using information-dependent data acquisition. The precursor at m/z 712.8 was selected for sequencing and produced a sequence which matches the sequence of dynI511-522 where Ser-512 is phosphorylated. C, As for panel b except that the precursor at m/z 750.0 was selected for sequencing and produced a sequence which matches the sequence of dynI343-364 where Ser-347 is phosphorylated. The N-terminus was labelled with ITRAQ 116 reagent affecting the mass of the b-type ions.

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Fig. 5. Quantitative analysis of dynI phosphorylation sites. A, Theoretical 2D map produced by plotting HPLC index versus predicted electrophoretic mobility. Known dynI and dynIII phosphopeptides with up to one missed trypsin cleavage are shown. Phosphopeptides that were detected from the 2D map in Figure 2B are shown with the same lettering. B, Quantitative analysis of the radiation for each spot in Figure 2B-C normalised to the total counts detected in all spots in panel b (defined as 100%). Filled bars are from synaptosomes at rest and open bars are after depolarisation for 10 s with 41 mM KCl. The phosphorylation site(s) associated with each spot are shown. The sites are as identified previously (5) or in this study. C, Quantitative analysis of the total radiation for each phosphorylation site in dynI purified from control (solid bars) and depolarised synaptosomes (open bars). Data for Ser-774, Ser-778 and Ser-822 are averages and errors (± s.e.m.) from three phosphopeptide maps. Data for Ser-851 and Ser-857 are the average of two phosphopeptide maps and the errors are the ranges for these two experiments. Note that the phosphorylation of Ser-851 and Ser-857 could not be individually apportioned, as was done for Ser-774 and Ser-778 (6), so they are grouped together. Fig. 6. In vitro phosphorylation of dynI PRD by cdk5. A, DynI consists of four distinct domains: the GTP hydrolysis domain (GTPase), a pleckstrin homology (PH) domain, an assembly domain (AD) and a proline-rich domain (PRD). Point mutations were made in phospho-box residues at the indicated positions (arrows). B, GST-dynI PRD either WT or Ala mutants were phosphorylated by recombinant cdk5/p25 in the presence of γ-32P[ATP] in vitro. An autoradiograph is shown (top panel). The protein load was visualised by Coomassie Blue staining (bottom panel). C, The amount of incorporated γ-32P[ATP] in experiments such as that in panel b was quantified by Storm phosphorimager (n = 3). Data was expressed as a percent of dynI PRD WT ± s.e.m. One-way ANOVA was applied (***, P < 0.001 against dynI-WT). D, GST-dynI PRD dmA was phosphorylated in vitro with cdk5/p25, as in lane 5 of B, was digested with trypsin, enriched for phosphopeptides using Fe3+-IMAC and the mutated phospho-box phosphopeptide was sequenced by tandem mass spectrometry. Fragmentation of the triply charged precursor at m/z 421.2 produced a spectrum which matched the sequence of dynI773-783 where Ser774 and Ser-778 are each substituted for Ala and Thr-780 is phosphorylated. The diagnostic y5

2+ ion was the most intense at a height of 6.4 counts. Fig. 7. DynI phosphorylation has no effect on amphI interaction. A, Interaction of pseudo-phosphorylated dynI PRD with amphI. GST-dynI PRD WT or Glu mutants were bound to GSH-sepharose and used in pull-down experiments from rat brain lysates. The samples were blotted with anti-amphI antibodies (top panel). The amount of GST-dynI PRDs used in the pull-downs are shown (bottom panel). B, The amount of amphI bound to GST-dynI PRD mutants was quantified by densitometric analysis of Western blots such as in panel a (n = 4). Data was expressed as a percent of dynI PRD WT ± s.e.m. One-way ANOVA was applied, but no statistically significant differences were found. C, GST-amphI SH3 binds phospho-dynI with high affinity. GST-amphI SH3 was used in pull-down experiments from P2 synaptosomes lysed in Triton X-100 in the absence or presence of 0.15, 0.5 or 1 M NaCl. DynI was detected by Coomassie Blue staining of the gel (top panel). Lysates from the same experiment were probed with antibodies to phospho-Ser-774 and phospho-Ser-778. Immunoblots are displayed (bottom two panels). Data are representative of two independent experiments. D, Interaction of phospho-dynI with amphI. Synaptosomes were incubated for 60 min in Krebs-like buffer containing 0.1 mM Ca2+ or 2.5 mM Ba2+, lysed and immunoprecipitated with anti-dynI antibodies. The complexes were subjected to Western blotting analysis with antibodies to amphI and phospho-Ser-774 (top two panels). The amount of dynI immunoprecipitated by the dynI antibodies was visualised by Ponceau staining of nitrocellulose membrane. Blots are representative of three independent experiments. E, Quantitative analysis from Western blots of the amount of amphI co-immunoprecipitated with dynI (panel D). Results are from 3 independent experiments.

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Fig. 8. In vivo phosphorylation sites in the PRD of dynI and dynIII. A, Domain structure of rat dynIxa (long splice variant) showing the location of all in vivo phosphorylation sites. All sites are serine residues. Dotted lines indicate sites present only in the long splice variant. B, An amino acid alignment of the C-terminal PRD from dynI and III long and short tail splice variants. In dynI the long variant is xa, while in dynIII it is called xb (29). The location of the phosphorylation sites identified in this and a previous study are shown with arrows. The phospho-box is indicated. Four of the 13 DynI PxxP motifs are shown in boxes (called site 1 to site 13). In dynI, sdpnI binds Site 2, endoI binds sites 2+3, grb2 binds site 8 and amphI binds site 9. Note that dynI and III share conservation of Ser-774 and Ser-778 in both splice variants. DynI Ser-851 and Ser-857 are restricted to the long splice variant, and only the former site is conserved in dynIIIxb. DynI Ser-822 is not conserved in dynIII. An additional site identified in this study at dynI Ser-347 is also conserved in dynIII (not shown), while Ser-512 in dynI is missing in dynIII.

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TABLES

Table 1. Distribution of 32P radiation on all dynI and dynIII phosphorylation sites detected from 2D tryptic phosphopeptide mapping and nanoLC-MSMS. Phosphorylation sites identified by MS from phosphopeptides extracted from the 2D map were correlated with the 32P radiation detected in each spot to determine the percentage of total radiation. Phosphorylation sites that were not extracted from the 2D map, rather by nano-LCMSMS alone, were correlated with the spots in the 2D map that match the theoretically predicted migration of the phosphopeptides (see Experimental Procedures and Fig. 5A). Note, it is likely that an unknown phosphopeptide contributed to part of this measured radiation predicted to be associated with Ser-512. Note also, that a small, but unknown amount of radiation is likely to be contributed by dynIII Ser-769 and Ser-773, reducing the proportion of radiation from dynI Ser-774 and Ser-778.

Phosphorylation Site Context of Site Measured 32P-Radiation (%)

Predicted 32P-Radiation (%)

Ser-347 KRIEGSGDQID - 1% Ser-512 NKKKTSGNQDE - ≤ 8% Ser-774 PAGRRSPTSSP ≤ 47% Ser-778 RSPTSSPTPQR ≤ 22% Ser-822 SRPGASPDPFG 5% Ser-851 RSGQASPSRPE 12% combined Ser-857 PSRPESPRPPF 12% combined 1%

DynIII Ser-853 RRPPPSPTRPT 2% Unknown spots 2%

Total 90% 10%

Reference List

1. Cousin, M. A. and Robinson, P. J. (2001) Trends Neurosci. 24, 659-665

2. Sweitzer, S. M. and Hinshaw, J. E. (1998) Cell 93, 1021-1029

3. Liu, J. P., Sim, A. T. R., and Robinson, P. J. (1994) Science 265, 970-973

4. Tan, T. C., Valova, V. A., Malladi, C. S., Graham, M. E., Berven, L. A., Jupp, O. J., Hansra, G., McClure, S. J., Sarcevic, B., Boadle, R. A., Larsen, M. R., Cousin, M. A., and Robinson, P. J. (2003) Nat.Cell Biol. 5, 701-710

5. Larsen, M. R., Graham, M. E., Robinson, P. J., and Roepstorff, P. (2004) Mol Cell Proteomics 3, 456-465

6. Anggono, V., Smillie, K. J., Graham, M. E., Valova, V. A., Cousin, M. A., and Robinson, P. J. (2006) Nat.Neurosci. 9, 752-760

7. Huang, Y., Chen-Hwang, M. C., Dolios, G., Murakami, N., Padovan, J. C., Wang, R., and Hwang, Y. W. (2004) Biochemistry 43, 10173-10185

16


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8. Tomizawa, K., Sunada, S., Lu, Y. F., Oda, Y., Kinuta, M., Ohshima, T., Saito, T., Wei, F. Y., Matsushita, M., Li, S. T., Tsutsui, K., Hisanaga, S. I., Mikoshiba, K., Takei, K., and Matsui, H. (2003) J.Cell Biol. 163, 813-824

9. Hosoya, H., Komatsu, S., Shimizu, T., Inagaki, M., Ikegami, M., and Yazaki, K. (1994) Biochem.Biophys.Res.Commun. 202, 1127-1133

10. Robinson, P. J., Sontag, J.-M., Liu, J. P., Fykse, E. M., Slaughter, C., McMahon, H. T., and Südhof, T. C. (1993) Nature 365, 163-166

11. Earnest, S., Khokhlatchev, A., Albanesi, J. P., and Barylko, B. (1996) FEBS Lett. 396, 62-66

12. Yoshimura, Y., Aoi, C., and Yamauchi, T. (2000) Brain Res.Mol.Brain Res. 81, 118-128

13. Ahn, S., Kim, J., Lucaveche, C. L., Reedy, M. C., Luttrell, L. M., Lefkowitz, R. J., and Daaka, Y. (2002) J.Biol.Chem. 277, 26642-26651

14. Shajahan, A. N., Timblin, B. K., Sandoval, R., Tiruppathi, C., Malik, A. B., and Minshall, R. D. (2004) J.Biol.Chem. 279, 20392-20400

15. Larsen, M. R., Thingholm, T. E., Jensen, O. N., Roepstorff, P., and Jorgensen, T. J. (2005) Mol Cell Proteomics 4, 873-886

16. Meisenhelder, J., Hunter, T., and Van der Geer, P. (1999) Current Protocols In Protein Science 13.9, 13.9.1-13.9.27

17. Sakamoto, Y., Kawakami, N., and Sasagawa, T. (1988) J.Chromatogr. 442, 69-79

18. Nguyen, C. and Bibb, J. A. (2003) J.Cell Biol. 163, 697-699

19. Robinson, P. J. (1991) FEBS Lett. 282, 388-392

20. Kapp, E. A., Schutz, F., Reid, G. E., Eddes, J. S., Moritz, R. L., O'Hair, R. A., Speed, T. P., and Simpson, R. J. (2003) Anal.Chem. 75, 6251-6264

21. Slepnev, V. I., Ochoa, G. C., Butler, M. H., Grabs, D., and DeCamilli, P. (1998) Science 281, 821-824

22. Robinson, P. J. and Dunkley, P. R. (1983) Neurosci.Lett. 43, 85-90

23. Sihra, T. S., Piomelli, D., and Nichols, R. A. (1993) J.Neurochem. 61, 1220-1230

24. Ramachandran, R., Surka, M., Chappie, J. S., Fowler, D. M., Foss, T. R., Song, B. D., and Schmid, S. L. (2007) EMBO J. 26, 559-566

25. Grabs, D., Slepnev, V. I., Songyang, Z., David, C., Lynch, M., Cantley, L. C., and De Camilli, P. (1997) J.Biol.Chem. 272, 13419-13425

26. Gray, N. W., Fourgeaud, L., Huang, B., Chen, J., Cao, H., Oswald, B. J., Hemar, A., and McNiven, M. A. (2003) Curr.Biol. 13, 510-515

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27. Ahn, S., Maudsley, S., Luttrell, L. M., Lefkowitz, R. J., and Daaka, Y. (1999) J.Biol.Chem. 274, 1185-1188

28. Powell, K. A., Valova, V. A., Malladi, C. S., Jensen, O. N., Larsen, M. R., and Robinson, P. J. (2000) J.Biol.Chem. 275, 11610-11617

29. Cao, H., Garcia, F., and McNiven, M. A. (1998) Mol.Biol.Cell 9, 2595-2609

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AmphI AmphI SdpnI EndoI

AmphI AmphI AmphI AmphI

SdpnI EndoI EndoI SdpnI

Coomassie Blue

1st

2nd

3rd

Pull-

Dow

n

AmphI AmphI SdpnI EndoI

AmphI AmphI AmphI AmphI

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Autoradiograph

1st

2nd

3rd

Pull-

Dow

n

A

B

Fig 1 - ME Graham et al

Pull-

Dow

n

1st

GST-SH3s

Western Blot

Coomassie Blue

Input 1st 2nd 3rd

C

43 kDa

30 kDa

116

94

67

PD: GST-AmphI-SH3

Blot: Dyn I

kDa

Unbound Bound to SH3

1st 2nd 3rd

Blot: Phospho-Dyn I-774


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pThr

pTyr

pSer

stdDynI

TLC

A

B

A B

C D

E FG H

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M N

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Electrophoresis

Control Depolarised


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B849RPPPpSPTRPTIIRPLESSLLD869

2 9 181

8 2 1

5 8

16

19

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y2

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b2

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b82+ b18

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16

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b193+

b183+

b163+

24

0

m/z500100 900 1100300 700

Abs

olut

e In

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ity (c

ount

s) 18x

Abs

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e In

tens

ity (c

ount

s)847SGQApSPSRPEpSPRPPFDL864

400

0

b3

y1

y2b2

y7

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300100 700 900500

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3 8 132

7 5 4 2 1

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15 1013

y132+

y142+

y152+

14

b172+

b132+

17

[b8-98]2+

[b11-98]2+

[y15-98]2+-98

m/z

A


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Fig 4A-B - ME Graham et al

m/z

Abs

olut

e In

tens

ity (c

ount

s)797GPAPGPPPAGSALGGAPPVPSRPGApSPDPFGPPPQVPSRPNR838

30

0

b3

y1

b2y6

2+

b4

600100 16001100

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12

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ity (c

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y1

y2 y3

y4

y5

y6

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C343RIEGpSGDQIDTYELSGGAR361

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8 45 1

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ity (c

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0

m/z100 500 900 1300

y6 y7

y9

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y12

6791112

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y3

3

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8

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Fig 4C - ME Graham et al


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Perc

enta

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50% pSer85150% pSer857

pSer822

pSer774

50% pSer77450% pSer778

pSer851pSer857mixture

B

A C D E F G H I J K L M N OB

30

20

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60

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Phosphorylation Sites

Perc

enta

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f tot

al

32P-

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l (%

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dex

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785-838797-846

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20

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-1.5 -0.5 0.5 1.5


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100 200 300 400 500 600 700 800


Coomassie Blue

AutoRad

Cdk5 - + + + + + +WT WT 774A 778A 780AdmA tmA

Phospho-Box

769 PAGRRSPTSSPTPQRRAPAVPPARPGSRGP 798

AE

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SingleMutants

DoubleMutants (dm)

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TripleMutants (tm)

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B

WT 774A 778A dmA 780A tmA0

40

80

120

Phos

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D

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C

Abs

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e In

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ity (c

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s)

m/z

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b62+ y6

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65

6 4 1358


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A

WT

774A

774E

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778E dm

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100

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Western Blot

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Western Blot

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50

100

150

Amph

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sin

Boun

d (%

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E

C

Western Blot

Coomassie Blue

NaCl (M)0 0.15 0.5 1

DynI

P-774

P-778

Coomassie Blue

GST-PRDs


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70 80 90 100 110 120| | | | | |

1 DYNIxb GAPPVPSRPGASPDP------FGPPPQVPSRPNRAPPGVPRITISDP---------------- 8512 DYNIxa GAPPVPSRPGASPDP------FGPPPQVPSRPNRAPPGVPSRSGQASPSRPESPRPPFDL--- 8643 DYNIIIxa GAPPVPFRPGPLPPFPNSSDSYGAPPQVPSRPTRAPPSVPRFGAVKEEAVEP----------- 8594 DYNIIIxb GAPPVPFRPGPLPPFPNSSDSYGAPPQVPSRPTRAPPSVPSRRPPPSPTRPTIIRPLESSLLD 869

10 20 30 40 50 60| | | | | |

1 DYNIxb PMPPPVDDSWLQVQSVPAGRRSPTSSPTPQRRAPAVPPARPG--SRGPAPGPPPAGSALG 8102 DYNIxa PMPPPVDDSWLQVQSVPAGRRSPTSSPTPQRRAPAVPPARPG--SRGPAPGPPPAGSALG 8103 DYNIIIxa PAPPPVDDSWLQHS-----RRSPPPSPTTQRRLTLSAPLPRPASSRGPAPAIPSPG-PHS 7964 DYNIIIxb PAPPPVDDSWLQHS-----RRSPPPSPTTQRRLTLSAPLPRPASSRGPAPAIPSPG-PHS 796

Site 2 Site 3Phospho-Box


Site 8 Site 9

S774 S778(T780)

S853 (DynIIIxb)

S822 S851 S857

822

778774

857851512347

GTPase PH AD PRD1 230 518 625 752 864

A

B


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Phillip J. RobinsonMark E. Graham, Victor Anggono, Nicolai Bache, Martin R. Larsen, George E. Craft and

The in vivo phosphorylation sites of rat brain dynamin I

published online March 20, 2007J. Biol. Chem.

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the in vivo phosphorylation sites of rat brain dynamin i* · protein kinases and new...

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