Differential Phosphorylation of Dynamin I Isoforms in Subcellular

Differential Phosphorylation of Dynamin I Isoforms in Subcellular Compartments Demonstrates the Hidden Complexity of Phosphoproteomes Ling-Shan Chan, Gurdip Hansra, Phillip J. Robinson, and Mark E. Graham* Cell Signalling Unit, Children’s Medical Research Institute, The University of Sydney, Westmead, Australia Received March 10, 2010

Large-scale comparative phosphoproteomics studies have frequently been done on whole cells or organs by conventional bottom-up mass spectrometry approaches, that is, at the phosphopeptide level. Using this approach, there is no way to know which protein isoforms the phosphopeptide signal originated from. Also, as a consequence of the scale of these studies, important information on the localization of phosphorylation sites in subcellular compartments is not surveyed. As a case study, we investigated whether the isoforms of dynamin I (dynI), at the whole brain and subcellular level, had differential phosphorylation. We first established that the dynI isoforms xa, xb, and xd were expressed in nerve terminals. Our investigation revealed that dynI xa was constitutively phosphorylated to a higher extent than the other isoforms despite identical sequences in the phosphorylated subdomains. DynI xa had a 10-fold higher stoichiometry of diphosphorylation at Ser-774 and Ser-778 than dynI xb and xd combined. Diphosphorylation was 2-fold enriched in nerve terminals relative to whole brain and was preferentially targeted for stimulus-dependent dephosphorylation. Phospho-Ser-851 and Ser-857 were depleted from nerve terminals. Our data reveals major differential phosphorylation of dynI phosphosites in different variants and in different neuronal compartments that would be completely imperceptible to a large-scale phosphoproteomics approach. Keywords: dynamin • synaptic vesicle endocytosis • phosphorylation • isoforms • iTRAQ • mass spectrometry • phosphoproteomics • nerve terminals

Introduction With the advent of new technology and methods, large lists of phosphorylation sites can be obtained and simultaneously monitored in comparative studies.1,2 In the largest quantitative phosphoproteomics study to date, greater than 20 000 phosphorylation sites were monitored during mitosis.3 Despite these advances, there are issues that limit the usefulness of this data, such as under-sampling of the phosphoproteome and false positive identifications. These issues have been acknowledged.2 Less well acknowledged or documented is how protein isoforms can confound the interpretation of the large-scale comparative phosphoproteomics experiments. The typical subjects of LC-MS/MS phosphoproteomics analyses are tryptic phosphopeptides, using the bottom-up proteomics approach.4,5 Unless the peptide sequence is specific to the protein isoform, then the isoform-specific identity of the sequence is lost after proteolysis. A phosphopeptide may have originated from multiple phosphoprotein isoforms of varying function. Therefore, any measured change in the level of a phosphorylation site on a particular phosphopeptide is the sum of the changes on all isoforms, resulting in a measurement that * To whom correspondence should be addressed. Dr. Mark E. Graham, Cell Signalling Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia, Tel. +61-2-9687-2800; Fax. +61-2-96872120; E-mail: [email protected].

4028 Journal of Proteome Research 2010, 9, 4028–4037 Published on Web 06/18/2010

may have reduced biological relevance. Such circumstances are not rare, since alternative splicing is more the rule than the exception. It is estimated that 80% of known genes are subject to alternative splicing,6,7 although not all splice variants result in protein expression and their expression may be specific to a cell type. Possibly further mitigating this effect is the finding that alternative splicing often results in the gain or loss of posttranslational modifications8 suggesting that phosphorylation sites often occur in isoform specific sequences. Nevertheless, an unknown but significant proportion of the information from large-scale quantitative phosphoproteomics may be of limited or no use in understanding the biology of the proteins involved because the information is not isoform-specific. Another concern relates to the scale at which phosphoproteomics experiments are conducted. Large-scale phosphoproteomics experiments have typically been done on whole cells3 or whole organs.9 This has enabled global changes to phosphoregulated signaling networks to be defined. However, it is obvious that this information is disconnected from the cellular architecture. Ideally, it would be useful to know if a particular phosphorylation site and phosphoprotein isoform is localized to a subcellular compartment to better describe its phosphoregulated function. We tested the potential for hidden complexity in the brain phosphoproteome as a result of alternative splicing and compartmentalization using the protein dynamin I (dynI) as a 10.1021/pr100223n

 2010 American Chemical Society

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Differential Phosphorylation of Dynamin I Isoforms

Figure 1. Domain structure of dynI isoforms and sequence of each proline rich domain.

case study. DynI is a key protein in the process of synaptic vesicle endocytosis (SVE). SVE follows nerve terminal depolarization and the exocytic release of neurotransmitters. Synaptic vesicles are reassembled from the membrane by the SVE protein machinery. DynI forms a helix at the neck of budding synaptic vesicles and constricts upon GTP hydrolysis, pinching them off.10 Phosphorylation of dynI by cyclin-dependent kinase 5 at Ser-778 and at Ser-774 by glycogen synthase kinase 311,12 in the phospho-box of the proline-rich domain (PRD, see Figure 1A) appears to be an inhibitory control mechanism for SVE. It is established that syndapin I binding to dynI is regulated by phosphorylation at Ser-774 and Ser-778.13,14 Thus, the function of these major dynI phosphosites could be said to be well understood. However, these studies were done using transfection of a single isoform of dynI or by the analysis of the pooled endogenous dynI isoforms. It should not be assumed that these phospho-box sites have the same function and the same phospho-regulation on all the dynI isoforms. This type of detailed study is essential to fully understand the role of dynI phospho-regulation. There is the potential for multiple dynI isoforms to exist in the brain. The rat dynI gene is alternatively spliced at three sites and mRNAs for eight alternate splice variants of rat dynI (UniProtKB accession number: P21575) have been reported (see Figure 1A).15 The first splice site is within the middle domain of dynI and has two alternative splicing sequences of equal length giving rise to dynI ax and bx (or isoforms 1-4 and 5-8). The second splice event begins at residue 766 and would truncate the protein at residue 814 (dynI xc or isoforms 3 and 7) producing 92.4-92.5 kDa proteins. The third splice site is at the C-terminus which would give rise to three alternative splicing sequences beginning at residue 845 (dynI xa or isoforms 1 and 5, dynI xb or isoforms 2 and 6, and dynI xd or isoforms 4 and 8) producing 96-97 kDa proteins. Clear evidence for the existence in the brain of only one of the eight alternative sequences has been reported (dynI xb).16 DynI is well-known to migrate as a doublet using SDS-PAGE of brain lysates and synaptosomes.17,18 The presence of dynI xb in the lower doublet was confirmed with antibodies specific to the

C-terminal sequences.16 However, the isoform present in the upper band has not been identified. Although dynII and III, which are products of different genes, are known to be expressed in the brain, the overwhelming majority of the expressed dyn protein is dynI.16,19 DynI has five regions: a GTPase domain, middle region, pleckstrin homology (PH) domain, GTPase effector domain (GED) and PRD. Alternative splicing of dynI results in isoforms that differ in the middle region (dynI ax and bx) and at the C-terminus in the PRD (dynI xa, xb, xc and xd; xc is a truncation) producing up to eight possible variants at four possible lengths. This work concerns the dynI isoforms with different sequences in the PRD (dynI xa, xb and xd) in rat brain. DynI xc was not detected. The PRD of dynI xa, xb and xd have three common in vivo phosphorylation sites (Ser-774, -778 and -822). DynI xa has two additional in vivo phosphorylation sites in the PRD (Ser-851 and -857). Much is known about the in vivo phosphorylation sites of dynI. Seven in vivo phosphorylation sites were discovered by analysis of the pooled dynI in isolated rat brain presynaptic nerve terminals (synaptosomes), and the extent to which they are dephosphorylated following depolarization was measured by tracking 32P levels after 1 h of metabolic labeling.20 Three tyrosine phosphorylation sites, at Tyr-80, -125 and -354, were observed on unresolved mouse brain dynI using large scale phosphoproteomics screens for tyrosine phosphorylation.21 Ser-774 and Ser-778 are the major phosphosites in synaptosomes (69% of all the 32P), having both a high turnover and strong response to depolarization.20 They are common to the amino acid sequence of six of the eight dynI isoforms, but they are spliced out of the truncated form dynI xc. Ser-851 and Ser857 are only found in the long tail (dynI xa). These extra phosphosites provide an avenue for unique phospho-regulation compared with the shorter isoforms. Hwang and co-workers22 reported that Ser-857 is phosphorylated by dyrk 1A in vitro and confirmed the presence of Ser-857 in vivo with a phosphospecific antibody. They also reported that phosphorylation at Ser-857 could affect amphiphysin I (amphI) binding.22 DynI has the potential to bind multiple proteins containing Src-3 Journal of Proteome Research • Vol. 9, No. 8, 2010 4029

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homology (SH3) domains (e.g., amphI, endophilin I, syndapin I, Grb2 and p85) at different stages, or during different modes of endocytosis using shared or different short binding motifs within the PRD.23-26 This raises the possibility that the dynI isoforms have different functional roles. Nothing is known about the functional role of the remaining phosphorylation sites which were previously reported to have low or no 32P incorporation (Ser-822, -347 and -512). In the process of demonstrating the hidden complexity of the phosphoproteome, we have determined the detectable dynI isoforms in the brain and used iTRAQ (isobaric tags for relative and absolute quantification), after in-gel digestion27 and prior to enrichment of phosphopeptides with titanium dioxide,28 to quantify the relative phosphorylation status of the dynI isoforms (see workflow in Figure 2). The quantification of differential phosphorylation on protein isoforms has not been attempted previously, to our knowledge.

Materials and Methods Materials. Acrylamide and N-N′-methylene-bis-acrylamide powder were from BioRad (Sydney, Australia). Milli-Q water was used in all experiments (Milli-Q UF PLUS; Millipore, Billerica, MA). Titanium dioxide beads were obtained from a disassembled Titansphere column (GL Sciences, Tokyo, Japan). 32 P orthophosphate was from Perkin-Elmer Life Sciences (Boston, MA,). Trypsin (porcine, modified) was from Promega (Sydney, Australia). The plasmid expressing GST-amphI SH3 domain (human, amino acids 545-695) was as previously reported.13 Purification of DynI from Synaptosomes and Whole Brain. Crude (P2) synaptosomes16 were prepared from two rat brains and labeled with 32Pi as described previously.11 Briefly, synaptosomes in low calcium Krebs-like buffer (118.5 mM NaCl, 4.7 mM KCl, 1.18 mM MgCl2, 0.1 mM K2HPO4, 20 mM HEPES, 10 mM glucose, with 0.1 mM calcium, pH 7.4) were incubated with 0.75 mCi/ml 32Pi for 1 h at 37 °C and washed. The synaptosomes were depolarized by addition of KCl to reach 41 mM for 3 s and were immediately lysed in 25 mM Tris, pH 7.4, containing 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 2 mM EDTA, 50 mM NaF, 20 µg/mL leupeptin, 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche, Sydney, Australia). DynI was purified from the lysates using the GST-amphI SH3 domain bound to glutathione sepharose beads as described previously.11 The beads were washed extensively, eluted in 2× concentrated SDS sample buffer and resolved on SDS gels. DynI was purified from whole brain by homogenizing a freshly isolated rat brain in 8 mL of the same lysis buffer. The homogenate was centrifuged at 23 000× g for 30 min at 4 °C and the supernatant collected. The supernatant was either frozen at -80 °C or used immediately in GST-amphI SH3 pulldowns to extract all of the available dynI.20 Modified SDS-PAGE Procedure. The extracted dyn from either the synaptosome preparation or a whole brain preparation was run in a 1 mm thick large format gel (20 cm, length) using the BioRad Protean II system. Starting with 1 rat brain, the amount loaded in each lane was 1% of the final volume of the synaptosome preparation and 2% of the whole brain preparation. A sample was run using a “standard” SDS-PAGE system with a 7.5-15% acrylamide and 0.2-0.4% bis-acrylamide linear gradient. For the remainder of this study we used a modified SDS-PAGE system which optimized resolution of dynI isoforms. The modified gels had (a) fixed acrylamide concentration (12%) instead of a gradient, (b) reduced con4030

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Figure 2. Generalized workflow used to obtain differential quantitative phosphorylation site data on dynI isoforms from rat brain. Specific workflows for each experiment, with minor variations, are provided in Supporting Information.

centration of bis-acrylamide (0.08%)29 and (c) elevated pH in the separation buffer (pH 9.2)30 with standard amounts of reagents for polymerization. The gels were run at a constant 300 V at 10 °C for approximately 6 h until the prestained ovalbumin marker (54.4 kDa, BioRad, prestained SDS-PAGE standards, broad range) ran off the bottom of the gel. The gels were stained with colloidal Coomassie Blue.31 Autoradiography and densitometry was done using quantitative imaging (Typhoon TRIO and ImageQuant TL software, GE Healthcare). Tryptic Digestion, iTRAQ Labeling and Phosphopeptide Enrichment. DynI gel bands were excised from the gels, diced to 1 mm3 and destained by vortexing with three 1 mL aliquots of 50% acetonitrile in water until the bands were translucent

Differential Phosphorylation of Dynamin I Isoforms (approximately 4 h) and digested with trypsin in 50 mM triethylammonium bicarbonate at 37 °C for 16 h. The solution was then made up to 50% acetonitrile and the tryptic peptides were extracted after 15 min of vortexing. A second extraction was obtained with 80% acetonitrile solution with a further 15 min of vortexing. Samples were dried in vacuo (ALPHA-RVC IR, CHRIST, Germany). Different combinations of bands, for example, bands from synaptosomes versus rat brain, or bands with different mobility were pooled to increase the amount of sample for later analyses. Typically, two to six bands (estimated at 1-4 µg by comparison to known amount of marker protein) were dissolved in 3 µL of 1 M triethylammonium bicarbonate. Another 3 µL of water was added, then 7 µL of ethanol and 7 µL of one of the iTRAQ reagents (iTRAQ 4-plex reagents, each reagent vial was resuspended in 70 µL of ethanol). The iTRAQ labeling proceeded for 1 h at room temperature (22 °C). The iTRAQ labeled samples were dried in vacuo, dissolved in 20 µL titanium dioxide loading buffer (5% trifluoroacetic acid, 50% acetonitrile in water) and then mixed together. The dynI phosphopeptides were enriched from the mixture using a titanium dioxide microcolumn as described previously32 except that loading and washing buffer was 5% TFA in 50% acetonitrile. The phosphopeptides were eluted with 20% ammonium hydroxide (29% ammonia in water) 20% acetonitrile solution into a tube containing 1.3 µL of 20% formic acid (to neutralize the base) and were immediately dried. The nonbinding peptides, presumably nonphosphopeptides, of the titanium dioxide microcolumn were collected, dried, redissolved and applied to a strong cation exchange (SCX) microcolumn to remove the excess iTRAQ reagent. The SCX microcolumn was made by packing the SCX material supplied with the iTRAQ Reagents Method Development Kit (removed from the supplied column) into a gel loading tip (Prot/Elec Tips, Bio-Rad, Sydney, Australia) to a volume of 6 µL. The loading, washing and eluting was done as described in the iTRAQ chemistry reference guide (Applied Biosystems) except that the volumes were reduced 10fold. Nanoliquid Chromatography Mass Spectrometry. The iTRAQ-labeled, titanium dioxide-enriched, dynI phosphopeptides were analyzed using fixed precursor ion selection for the known dynI phosphopeptides using LC-MS/MS as previously described.20 Briefly, samples were loaded onto the HPLC system (LC Packings Ultimate HPLC system, Dionex, Netherlands) with a 75 µm inside diameter precolumn of C18 reversed phase material (ReproSil-Pur 120 C18-AQ, 3 µm beads, Dr Maisch, Germany) in 0.1% formic acid 99.9% water. The samples were eluted through a 50 µm inside diameter C18 column of the same material at 100 nL/min. The main part of the gradient was from 10% phase B (90% acetonitrile, 0.1% formic acid and 9.9% water) to 65% phase B in 28 min. The eluate was sprayed through a 10 µm i.d. distal coated SilicaTip (New Objective, Woburn, MA) into a QSTAR XL quadrupole-TOF MS (MDS Sciex/Applied Biosystems). The precursor ion selection was fixed at multiple different m/z for different elution times to produce multiple 2 s scan MS/MS spectra of the same phosphopeptide: m/z 641.3, 681.3, 479.9, and 506.6 for 0-35 min; m/z 717.0 and 743.7 for 35-75 min. Typical peak widths of the product ion current chromatograms were 0.4-0.6 min at the base. The MS/MS spectra for each phosphopeptide (fixed precursor ion) were summed using the portion of the peak which was within 75% of the height of the peak apex, to produce high quality data for identification of the peptide and the iTRAQ reporter ions (Analyst QS 1.1, MDS Sciex/Applied

research articles Biosytems). This resulted in final spectra that were the sum of approximately three to four scans for the phospho-box peptides (four precursor ions monitored at once) and spectra that were the sum of six to eight scans for the dynI xa C-terminal peptides (two precursor ions monitored). The summed spectra were converted to peak lists using the script Mascot.dll version 16b21 or 16b23. The iTRAQ reporter ion peak areas, calculated by integration using Analysis QS 1.1, were manually inserted into the peak lists before searching, since the earlier version of the script used peak height for intensity and deisotoped the iTRAQ reporter ion m/z region. The quantitative data on phospho-Ser-774 and -778 was derived from dynI 774-783 (zero missed cleavages) and dynI 774-784/773-783 (one missed cleavage resulting in an extra Arg at the C- or N-terminal end of peptide) to ensure that there was no bias in reporting only one type of trypsin cleavage product. To separate the signals from the phospho-Ser-774 and -778 sites, which occur on the same tryptic peptide, we relied on partial separation of the differently phosphorylated peptides. The peptide 774-SPTS(phospho-Ser-778)PTPQR-783 eluted slightly earlier (5-10 s) than the peptide 774-(phospho-Ser774)PTSSPTPQR-783 (see Supplementary Figure S4, Supporting Information). A spectrum for the phospho-Ser-778 peptide could be extracted without contamination from the dominant, slower eluting phospho-Ser-774 peptide signal. Minor contamination of the phospho-Ser-774 signal by phospho-Ser-778 signal was ignored, since the intensity was at least 5-fold different. The nonphosphopeptides from the SCX eluate were desalted with a C18 microcolumn, as described previously.33 The LC-MS/MS analysis was done in information dependent data acquisition mode as described previously,34 except that the slope of the line used to automatically determine the collision energy was increased by 20% above the standard parameters. The standard parameters were (CE ) slope × m/z + intercept); 2+, slope )0.06, intercept ) 9; 3+ slope ) 0.05, intercept ) 4; slope ) 0.048, intercept ) 3; 4+ and higher, slope ) 0.045, intercept ) 3. The method of determining the iTRAQ ratios for the nonphosphopeptides was not conventional. Rather than carefully selecting several MS/MS spectra of nonmodified dynI peptides to use in estimating the amount of protein, all of the MS/MS spectra (approximately 500 spectra) were summed to produce a single representative spectrum. The area for each iTRAQ reporter ion from this single spectrum was used to determine the amount of protein in each sample under comparison (after correction for impure iTRAQ reagents using Mascot). This nonselective method was simpler, faster and gave a similar result (data not shown) to the conventional method. Once established, the relative amount of protein was used to normalize the relative amount of phosphopeptide by division of the areas (see Figure 2), using Microsoft Office Excel 2007, so that the result could be expressed as the relative amount of phosphopeptide for equivalent amount of protein. Protein Database Searching. The parameters/settings for creating peak lists in the mascot.dll version 16b21 or 16b23 script were: precursor mass tolerance for grouping was 1 unit of m/z, maximum number of cycles between groups was 10, minimum number of cycles per group was 1, all MS/MS data was centroided and deisotoped, peaks were removed if less than 0.1% of maximum and spectra were rejected if they had less than 10 peaks, attempt to determine charge state from survey scan was on and default precursor charge states were 2+, 3+, 4+ and 5+. If mascot.dll version 16b23 was used, Journal of Proteome Research • Vol. 9, No. 8, 2010 4031

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Table 1. Identification of DynI Isoform Specific Peptides in Coomassie Stained Bands from a Modified SDS-PAGE Method using LC-MS/MS sequencea

c

m/z (error, ppm)

charge

Mascot score

3+

31

0.25

50

2+

32

0.21

167

2+

59

0.0004

35

-

-

-

-

expect

MS/MS error (ppm)

protein name

SGQASPSRPESPRPPFDL (847-864) iTRAQ-ITISDPb (846-851) APPGVPSQPIGSGK (839-852) -

642.3 (-34) 395.2 (-69) 646.4 (72) -

TGLFTPDLAFEATVKK (400-415)

580.0 (25)

3+

47

0.0036

37

DynI xa (long) DynI xb (short) DynI xd (int) DynI xc (trunc) DynI ax

TGLFTPDMAFETIVKKc (400-415)

605.4 (135)

3+

60

0.00026

42

DynI bx

a Mass spectra of identified peptides are shown in Supplementary Figure S2. Underlined methionine in the dynI xb 400-415 peptide was oxidized.

the iTRAQ reporter region was not deisotoped and peak area was reported. Database searching was performed using a local copy of Mascot version 2.2 (Matrix Science, London, U.K.). The searched database was the rat international protein index version 3.53 (39924 sequences; 20363580 residues). The searches were done with the variable modifications; deamidation (NQ), oxidation (M), phosphorylation (ST) and phosphorylation (Y). In iTRAQ experiments the Mascot iTRAQ 4-plex quantitation method was used. The precursor ion mass tolerance was 150 ppm and fragment ion tolerance was 0.1 Da. Enzyme specificity for tryptic digests was selected to trypsin with 1 missed cleavage. A Mascot Expect (probability) of 0.05 was considered acceptable for identification. Two isoform-specific C-terminal peptides did not reach this criterion (see Table 1). However, these peptides would not be expected to produce ideal fragmentation patterns because they do not have a C-terminal Lys or Arg. All spectra were manually validated. Novel spectra are supplied in Supporting Information (Supplementary Figure S2). No new phosphopeptides were sequenced in this work. The previously published (non-iTRAQ labeled) phosphopeptide spectra were readily available for comparison.20 The data associated with this manuscript may be downloaded from the ProteomeCommons.org Tranche network using the following hash: gTItabbBZD0hVqz+s8TWbb5c+ZYJwmKFCQ 06GeOTvvvrvbpPMxkd1KsmudYpa9Zs+d+9PJtP1SqlHDmd WNUmvpHvlGUAAAAAAAAVSw)). Quantitative Analysis. For most experiments, both multiple biological and technical replicates were done to improve the reliability of the data (see workflows in Supplementary Figure S1A-C, Supporting Information). Two types of biological replicates were produced: (i) samples were obtained from synaptosomes made from different rats (but were treated in the same way) or (ii) samples were obtained from synaptosomes made from the same rat, but were from different aliquots and were stimulated separately. Technical replicates arose from loading the same biological sample into different SDS-PAGE gels and then continuing with separate analyses. Spectra where the most abundant iTRAQ reporter ion had a signal-to-noise ratio of less than 10 were discarded. The removal of outlying data points was not required. One way analysis of variance was used to determine if there was any significant difference 4032

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b

accession number and isoform

P21575-1 P21575-5 P21575-2 P21575-6 P21575-4 P21575-8 P21575-3 P21575-7 P21575-1 P21575-2 P21575-4 P21575-5 P21575-6 P21575-8

historical name

predicted MW (kDa)

aa ba ab bb ad bd ac bc aa ab ad ba bb bd

97 295 97 424 95 927 96 057 96 270 96 399 92 376 92 505 97 295 95 927 96 270 97 424 96 057 96 399

DynI xb peptide, 846-851, could only be detected after iTRAQ labeling.

between the sample normalized to 100% and the sample under comparison.

Results and Discussion SDS-PAGE Separation and Identity of DynI Isoforms. Our first aim was to achieve a separation of the dynI C-terminal differentiated isoforms (Figure 1A) using an SDS-PAGE approach that would allow further quantitative analysis of the phosphorylation of the main isoforms. The GST-amphI SH3 affinity purified dynI isoforms typically migrate as one band (Figure 3A, first lane) in the acrylamide gradient gels that are routinely used in our laboratory (7.5% acrylamide 0.2% bisacrylamide to 15% acrylamide 0.4% bis-acrylamide at pH 8.8). A partially separated doublet can be achieved with reduced protein (50 amino acid residues each side), we would have expected that Ser-774 and -778 phosphorylation would be equally accessible to phospho-regulation in the context of any dynI isoform. This study shows that this assumption is incorrect. Since the only difference between the PRD of dynI xa, xb and xd is in the sequence at the C-terminus, it follows that these varying tail sequences may regulate the phosphorylation and function of the phospho-box, presumably by differential targeting of the isoforms to different locations or by binding with different partners. It also demonstrates that protein isoform diversity offers more than simply an opportunity for splicing in or out additional phosphosites, alternative splicing can also change the function of phosphorylation sites in common sequences. This work raises new questions about how dynI isoforms may be divided into subcellular pools that are phospho- and isoform-specific and what may be their functional impact. Previous studies using pooled dynI isoforms undoubtedly resulted in progress in understanding dynI and SVE. However, the lack of understanding of the phospho-regulation and subcellular localization of individual isoforms and the use of single isoforms to probe the function of dynI has limited progress. Likewise, the information from large-scale quantitative phosphoproteomics experiments is useful, but has limited application because of experimental design limitations. Global quantitative information can be used to make global conclusions. However, our work demonstrates that quantitative information on individual phosphosites which are present in multiple isoforms, without prior separation of the isoforms, may defy interpretation. Also, important isoform and locationspecific functional information may be overlooked. These problems have not previously been documented, to the extent of our knowledge. Our approach can be used to obtain information on the differential phosphorylation of isoforms of many other proteins and demonstrates the need to study subcellular compartments. 4036

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Chan et al. Targeted studies such as ours can follow global studies. However, it is preferable that a higher throughput approach is developed. Top-down proteomics5 offers an alternative and complementary approach to bottom-up proteomics, which preserves isoform information. But it is not yet amenable to large-scale high through-put analysis. Nevertheless, given the high predicted rate of alternative splicing, a greater emphasis on defining and accounting for protein isoforms is required in future large-scale studies to provide a true description of the dynamic phosphoproteome.

Acknowledgment. This work was supported by a Project Grant from the National Health and Medical Research Council of Australia (P.J.R. and M.E.G.) and a Career Development Award from the National Health and Medical Research Council of Australia (M.E.G). Additional support was from the Cancer Institute New South Wales. We thank our colleagues who have generously provided materials for this study. Supporting Information Available: Supplementary Figure S1, specific workflows for proteomics experiments. Supplementary Figure S2, mass spectra of dynI xa, xb, xd, ax and bx isoform-specific peptides. Supplementary Figure S3, the distribution of dynI xd in SDS-PAGE bands. Supplementary Figure S4, partially separated phospho-Ser-774 and -Ser-778 peptides for quantification. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Dunn, J. D.; Reid, G. E.; Bruening, M. L. Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry. Mass Spectrom. Rev. 2009, 29, 29–54. (2) Macek, B.; Mann, M.; Olsen, J. V. Global and site-specific quantitative phosphoproteomics: principles and applications. Annu. Rev. Pharmacol. Toxicol. 2009, 49, 199–221. (3) Olsen, J. V.; Vermeulen, M.; Santamaria, A.; Kumar, C.; Miller, M. L.; Jensen, L. J.; Gnad, F.; Cox, J.; Jensen, T. S.; Nigg, E. A.; Brunak, S.; Mann, M. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci. Signal. 2010, 3 (104), ra3. (4) Aebersold, R.; Mann, M. Mass spectrometry-based proteomics. Nature 2003, 422 (6928), 198–207. (5) Chait, B. T. Chemistry. Mass spectrometry: bottom-up or topdown. Science 2006, 314 (5796), 65–66. (6) Kampa, D.; Cheng, J.; Kapranov, P.; Yamanaka, M.; Brubaker, S.; Cawley, S.; Drenkow, J.; Piccolboni, A.; Bekiranov, S.; Helt, G.; Tammana, H.; Gingeras, T. R. Novel RNAs identified from an indepth analysis of the transcriptome of human chromosomes 21 and 22. Genome Res. 2004, 14 (3), 331–342. (7) Matlin, A. J.; Clark, F.; Smith, C. W. Understanding alternative splicing: towards a cellular code. Nat. Rev. Mol. Cell. Biol. 2005, 6 (5), 386–398. (8) Furnham, N.; Ruffle, S.; Southan, C. Splice variants: a homology modeling approach. Proteins 2004, 54 (3), 596–608. (9) Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Phosphoproteomic analysis of the developing mouse brain. Mol. Cell. Proteomics 2004, 2, 1093–1101. (10) Sweitzer, S. M.; Hinshaw, J. E. Dynamin undergoes a GTPdependent conformational change causing vesiculation. Cell 1998, 93, 1021–1029. (11) 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.; Robinson, P. J. Cdk5 is essential for synaptic vesicle endocytosis. Nat. Cell Biol. 2003, 5, 701–710. (12) Clayton, E. L.; Sue, N.; Smillie, K. J.; O’Leary, T.; Bache, N.; Cheung, G.; Cole, A. R.; Wylie, D. J.; Sutherland, C.; Robinson, P. J.; Cousin, M. A. Dynamin I phosphorylation by GSK3 controls activitydependent bulk endocytosis of synaptic vesicles. Nat. Neurosci. 2010, DOI: 10.1038/nn.2571. (13) Anggono, V.; Smillie, K. J.; Graham, M. E.; Valova, V. A.; Cousin, M. A.; Robinson, P. J. Syndapin I is the phosphorylation-regulated

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