Mapping the Phosphorylation Sites of Ulk1 Frank C. Dorsey,*,† Kristie L. Rose,§,| Silvia Coenen,‡ Stephanie M. Prater,† Valerie Cavett,§ John L. Cleveland,† and Jennifer Caldwell-Busby*,§,| Departments of Cancer Biology and Molecular Therapeutics and The Translational Research Institute, The Scripps Research Institute, Jupiter, Florida 33458, and Max Planck Institute of Biochemistry, Martinsried, Germany Received July 2, 2009
Ulk1 is a serine/threonine kinase that controls macroautophagy, an essential homeostatic recycling pathway that degrades bulk cytoplasmic material and directs the turnover of organelles such as peroxisomes and mitochondria. Further, macroautophagy is potently induced by signals that trigger metabolic stress, such as hypoxia and amino acid starvation, where its recycling functions provide macromolecules necessary to maintain catabolic metabolism and cell survival. Substrates for Ulk1 have not been identified, and little is known regarding post-translational control of Ulk1 kinase activity and function. To gain insights into the regulatory mechanisms of Ulk1, we developed a robust purification protocol for Ulk1 and demonstrated that Ulk1 is highly phosphorylated and requires autophosphorylation for stability. Importantly, high-resolution, tandem mass spectrometry identified multiple sites of phosphorylation on Ulk1, including several within domains known to regulate macroautophagy. Differential phosphorylation analyses also identified sites of phosphorylation in the C-terminal domain that depend upon or require Ulk1 autophosphorylation. Keywords: Autophagy • Ulk1 • Atg13 • FIP200 • kinase • phosphorylation • Protein kinase A • PKA • mTOR
Introduction Macroautophagy (hereafter referred to as autophagy), is an ancient, essentially cannibalistic pathway where components of the cell, including bulk cytoplasmic material and organelles, are engulfed within double-membraned vesicles called autophagosomes, which then deliver their cargo to the lysosome for degradation.1 Under homeostatic conditions, autophagy directs the degradation of long-lived proteins and of unwanted or damaged organelles such as peroxisomes and mitochondria. However, during times of starvation or stress, this pathway provides an essential recycling center that disassembles existing macromolecular structures into monomers, which are used to sustain cell survival. Although controversial, autophagy has also been proposed as a cell death mechanism,2 and indeed it is easy to envision that, if left unchecked, autophagy would lead to the annihilation of the cell. The serine/threonine kinase Atg1 was originally identified as a gene required for the survival of Saccharomyces cerevisiae during nitrogen starvation.3 In yeast, Atg1 associates with Atg13 and Atg17 to form an active kinase complex, and the formation of this complex is inhibited by the master metabolic kinase TOR (Target of Rapamycin).4,5 When nutrients are abundant, activated TOR leads to the phosphorylation of both Atg1 and Atg13, which triggers their dissociation and dampens Atg1 kinase * To whom correspondence should be addressed. E-mail: (J.C.-B.)
[email protected]; (F.C.D.)
[email protected]. † Department of Cancer Biology, The Scripps Research Institute. § The Translational Research Institute, The Scripps Research Institute. | Department of Molecular Therapeutics, The Scripps Research Institute. ‡ Max Planck Institute of Biochemistry. 10.1021/pr900583m CCC: $40.75
2009 American Chemical Society
activity. Accordingly, the TOR inhibitor rapamycin potently activates the autophagic response.4-6 Although evidence suggests that Atg1 is phosphorylated in response to TOR signaling, these phosphorylation sites have not been identified, and there are currently no known substrates of Atg1. Under nutrient replete conditions, Atg1 is found throughout the cytoplasm, yet under nutrient limiting conditions, Atg1 redistributes to the preautophagosomal structure (PAS) where it regulates autophagosome formation.7-9 The necessity of Atg1 kinase activity for autophagy has been contested, yet recent data suggest both kinase-dependent and -independent roles for Atg1 in regulating autophagy.9 For example, while a kinasedead mutant of Atg1 can recruit Atg8, Atg17, and Atg29 to the PAS, Atg1 kinase activity is required to disassemble these autophagic components, and subsequently drive the assembly of fully formed autophagosomes. Thus, Atg1 appears to have both scaffold and enzymatic roles in directing the formation of autophagosomes. Interestingly, the redistribution of Atg1 to the PAS is regulated at least in part by the cAMP-dependent protein kinase (PKA), where PKA phosphorylates serine residues 508 and 515 in Atg1, thereby preventing its association with the PAS.10 Accordingly, alanine mutations of 508 and 515 trigger constitutive association of Atg1 with the PAS. Mammals have at least five closely related Atg1 homologues, Unc-51-like kinases-1, -2, -3, -4, (Ulk1, Ulk2, Ulk3, and Ulk4) and Fused.11 Ulk1 and Ulk2 are widely expressed, whereas Ulk3 is restricted to select tissues and lacks any homology outside the kinase domain. Similarly, both Ulk4 and Fused show significant homology to Atg1 only within the kinase domain, and the roles of Ulk3, Ulk4 and Fused in Journal of Proteome Research 2009, 8, 5253–5263 5253 Published on Web 10/06/2009
research articles autophagy are unclear. Mammalian Ulk1 was first identified as a homologue of Caenorhabditis elegans Unc-51, which plays important roles in the elongation and guidance of newly formed axons.12,13 More recently, Ulk1 and Ulk2 have been linked to the regulation of autophagy,14 yet Ulk2 forms distinct subcellular complexes. In addition, in an siRNA kinase screen, Ulk1 was shown to be necessary for starvationdependent autophagy, yet Ulk2 knockdown had no effect on this response.15 Further, similar to its function in yeast, Ulk1 regulates the redistribution of other autophagy proteins such as Atg9 in response to amino acid starvation.16 Finally, Ulk1 also plays required roles in the clearance of mitochondria from maturing reticulocytes, and erythrocyte development in Ulk1-/- mice is abnormal, indicating that Ulk2 cannot complement this function of Ulk1.19 However, some redundancy between Ulk1 and Ulk2 must exist, as Ulk1-/- mice are viable, whereas Beclin1,20,21 Atg522 or Atg723 deficient mice are either embryonic or perinatal lethal, and Ulk1-/mouse embryonic fibroblasts (MEFs) can activate the autophagic response following glucose withdrawal.19 Ulk1 contains an N-terminal kinase domain, a serine/ proline-rich central region, a conserved C-terminal domain (CTD), and a PDZ-binding domain.13,15 As in yeast, mTOR regulates the Ulk1/Atg13/FIP200 complex.24-26 In mammals, mTOR forms two complexes, mTORC1 and mTORC2, yet only mTORC1 is sensitive to the nutrient status of the cell and inhibition with rapamycin.25 The mTORC1 complex directly associates with a large Ulk1-containing complex that contains Ulk1, Atg13, and FIP200. Further, Ulk1 and Atg13 are substrates phosphorylated by mTOR in vitro, and Ulk1 can directly phosphorylate Atg13 and FIP200.26 In addition, inhibition of mTORC1 with either rapamycin or amino acid starvation removes mTORC1 from the Ulk1 complex, initiating the Ulk1dependent phosphorylation of FIP200 and the activation of autophagy.26 Finally, the association of FIP200 with Ulk1 is Atg13-dependent, and FIP200 somehow controls Ulk1 turnover.24-27 Together, these data support a model whereby regulatory phosphorylations play direct roles in regulating the scaffolding and kinase functions of Ulk1 to coordinate the autophagic response. Here, we report a large cast of new serine phosphorylation sites on mouse Ulk1. We show that purified Ulk1 can phosphorylate itself as well as the promiscuous substrate myelin basic protein (MBP), and that Ulk1 kinase activity plays an important role in its turnover, as a kinase-dead (Ulk1-K46R) mutant of Ulk1 is much less stable. Finally, by employing an LC-coupled mass spectrometric (MS) approach, including both high-resolution MS and collision activated dissociation (CAD) tandem mass spectrometry (MS/MS), we mapped 16 novel phosphorylation sites on Ulk1, and identified multiple sites of Ulk1 autophosphorylation. Interestingly, using relative abundance measurements, we also clearly demonstrate that the kinase-dead Ulk1-K46R mutant is differentially phosphorylated on its C-terminus. These findings lay the foundation for deconvoluting the regulatory circuits that control Ulk1 function.
Methods Cloning and Protein Expression. Mouse Ulk1 was PCR amplified using the following primers, 5′-GGAATTCGAGCCGG GCCGCGGCGGCGTC-3′ and 5′-GCCTCGAGTCAGGCATAGACACCACTCAG-3′, and was cloned into the pNTAP-B Taptagged vector (Stratagene) using EcoRI and XhoI restriction 5254
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Dorsey et al. sites. To generate a Ulk1-K46R kinase-dead mutant, we used the Quikchange II site-directed mutagenesis kit (Stratagene) using the following mutagenic oligonucleotides: 5′-CTGGAGGTGGCCGTCAGATGCATTAACAAGAAGA-3′ and 5′-TCTTCTTGTTAATGCATCTGACGGCCACCTCCAG-3′. Protein production was performed following calcium phosphate-mediated transfection into 293T cells grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. In brief, 20 10-cm dishes were plated at 3 million cells per plate and were then transfected the following day with 12 µg/plate of either NTAP vector alone, NTAP-Ulk1, or NTAP-Ulk1-K46R. Twenty-four hours later, media was exchanged for fresh, prewarmed media. The following day, cells were trypsinized, washed in cold PBS, and pelleted. TAP-tagged proteins were purified using only the streptavidin resin from the NTAP purification kit (Stratagene), and were then washed and eluted according to the manufacturer’s instructions. All lysis and wash buffers were supplemented with Halt phosphatase inhibitor cocktail (Pierce) and Complete Mini protease inhibitor cocktail (Roche), except the elution buffer, which contained phosphatase inhibitors, but lacked protease inhibitors. Phosphatase Treatment. NTAP-Ulk1 and NTAP-Ulk1-K46R were transfected into one 10-cm dish of 293T cells, were harvested after 48 h, and lysed in NTAP lysis buffer containing protease and phosphatase inhibitors. NTAP-Ulk1 and NTAPUlk1-K46R were purified using streptavidin resin using the NTAP purification kit (Stratagene). Resin was then washed 3 times with lysis buffer, and 3 times in phosphatase buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35). Resin was then split into two aliquots and incubated at 37 °C for 2 h with or without 4000 units of Lambda protein phosphatase (New England BioLabs). Reaction was stopped by adding an equal volume of 2× sample buffer. In Vitro Kinase Assay. In vitro kinase reactions were performed as follows: 500 ng of purified NTAP-Ulk1 or NTAPUlk1-K46R was incubated in kinase buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 2 mM EGTA, 6 mM β-mercaptoethanol) supplemented with 100 µM nonradioactive ATP, phosphatase inhibitors (Pierce), and 1 µCi/µL γ-32P-labeled ATP at 37 °C for 15 min. Kinase reactions were stopped by adding an equal volume of 2× sample buffer, and samples were immediately boiled and separated on SDS-PAGE gels, which were then fixed in 30% methanol 10% acetic acid, stained with Coomassie blue, dried, and exposed to Kodak MR resolution film. To assess Ulk1 autophosphorylation, no substrate was added to the reactions. To assess kinase activity toward a substrate, 1 µg of myelin basic protein fragments (Sigma), a promiscuous kinase substrate, was added to the reactions. Pulse Chase Analysis. 293T cells were plated at 3 million cells per 10-cm dish per time point, and were transfected the following day with either 12 µg of NTAP-Ulk1 or NTAP-Ulk1K46R using calcium phosphate-mediated transfection. The following day, media was exchanged for prewarmed media. After 48 h the cells were washed twice and incubated with prewarmed methionine- and cysteine-free DMEM with 2 mM glutamine, and then incubated in this medium for 15 min at 37 °C (5% CO2). Media was then removed from cells and replaced with 4 mL of prewarmed methionine- and cysteinefree DMEM supplemented with 2 mM glutamine containing 70 µCi/mL S35-Translabel (MP Biomedicals) and incubated for 15 min at 37 °C with 5% CO2. Cells were then either harvested immediately or were washed 3 times in prewarmed complete
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Ulk1 Phosphorylation Sites DMEM media lacking radioactivity for indicated times. Collected cells were washed in cold PBS and lysed, and NTAPUlk1 and NTAP-Ulk1-K46R were purified using the NTAP purification method described above. Purified proteins were resolved on an SDS-PAGE gel, fixed in 30% methanol/10% acetic acid, washed 3 times for 30 min in DMSO, and incubated with 20% diphenyloxazole in DMSO for 1 h. Gels were then washed in water for 1 h, dried, and exposed to Kodak MR resolution film. Protein Isolation and Digestion. In-solution digests of affinity-purified Ulk1 were performed with trypsin (Promega, Madison, WI), chymotrypsin (Roche, Indianapolis, IN), or Glu-C (Roche) according to the manufacturer’s recommendations. All in-solution digests were performed for approximately 16 h at 37 °C and quenched by the addition of acetic acid. In-gel trypsin digests were performed via standard protocols. Briefly, samples were reduced, alkylated, and dehydrated prior to rehydration with approximately 50 ng of trypsin in 20 mM ammonium bicarbonate. Samples were digested for approximately 16 h at room temperature, and peptides were extracted by sequential dehydration and rehydration. Where subdigestion of the in-gel trypsin digests was desired, extracted tryptic peptides were treated with approximately 1 pmol of either Glu-C or chymotrypsin and digested at 37 °C for 6 h before quenching with the addition of acetic acid. Liquid Chromatography and Mass Spectrometry. For analysis, peptides from either in-solution or in-gel digestions were loaded onto a 360 × 100 µm fused silica precolumn packed in-house with 3 cm of C18 packing material (YMC-ODS A 5-15 µm). Salts were removed by washing with mobile phase A (0.1 M acetic acid in 1% acetonitrile), and the column was placed in line with a 360 × 75 µm fused silica capillary (PolyMicro Technologies, Phoenix, AZ) column packed in-house with 20 cm of C18 material (YMC ODS-AQ, 5 µm). Peptides were eluted along a gradient of 5-55% acetonitrile with a flow rate of 300 nL/min into a nanoelectrospray ionization (nESI) source operated at approximately 2 kV. Initial experiments were performed on an LTQ mass spectrometer (ThermoFisher, San Jose, CA) using data-dependent scanning with dynamic exclusion enabled. Full scan spectra (m/z 300-2000) were collected and used to select the five most abundant ions for collision activated dissociation (CAD). High resolution runs were performed on an LTQ-Orbitrap mass spectrometer (ThermoFisher) using data-dependent scanning with dynamic exclusion enabled. Full scan spectra (m/z 300-2000) were collected in the Orbitrap at 30 000 or 60 000 resolution, and CAD spectra were acquired in the LTQ. Selected runs were performed using a top three data-dependent scanning method with MS3 fragmentation applied to the most intense ion with m/z greater than the initial precursor. Data were processed using an in-house developed pipeline that extracts tandem mass spectra from the raw data files, which filters the data for spectral quality using the SPEQUAL28 algorithm, and concatenates the high-quality spectra for database searching via a clustered version of Sequest (v1.7, ThermoFisher). Searches were performed against the human and mouse IPI databases downloaded from EBI29 (v3.32, 67 546 entries and v3.32, 49 441 entries, respectively), and against the protein sequence translated from mouse Ulk1 protein with fixed carbamidomethylation of cysteine, variable oxidation of methionine, and variable phosphorylation of serine, threonine and tyrosine. Output of the Sequest searches were loaded into Scaffold (v1.7, Proteome Software, Portland, OR) for statistical
analysis via Peptide Prophet and Protein Prophet followed by manual verification of all peptide assignments. To investigate differential phosphorylation at the C-terminus of wild-type Ulk1 compared to the Ulk1-K46R mutant, accurate mass measurements acquired in the Orbitrap were used to generate selected ion chromatograms (SICs) of the unmodified and phosphorylated forms of Ulk1 peptide 1041-1051. A window of (5 ppm around the theoretical monoisotopic m/z values of the [M + H]+1 and [M + 2H]+2 ions was used to generate the SICs, and areas under each peak were calculated using Xcalibur version 2.0. The relative abundance was determined by dividing the area of each form of peptide 1041-1051 by the sum of the areas of all forms identified within a sample.
Results Purification and Characterization of Mouse Ulk1 and Ulk1-K46R. Under nutrient rich conditions, Ulk1 kinase activity is repressed by phosphorylation events downstream of nutrient-sensing kinases such as mTOR. These repressive phosphorylation events modulate Ulk1 kinase activity and/or its ability to interact with cofactors such as Atg13 or FIP200, thereby regulating autophagosome formation.24-27 To identify phosphorylation sites on Ulk1 that may mediate these effects, we generated a tandem affinity purification (TAP)-tagged mouse Ulk1 that harbors both streptavidin and calmodulin affinity tags in tandem on the N-terminus of Ulk1 (NTAP-Ulk1), and a similarly tagged kinase-dead mutant of Ulk1 (NTAP-Ulk1K46R). As Ulk1 is known to oligomerize, we used a mouse Ulk1 cDNA clone to generate these constructs, as the majority of trypsin-generated mouse Ulk1 peptides differ from human Ulk1 by at least one amino acid, making it possible to distinguish endogenous Ulk1 from the overexpressed NTAP-Ulk1 constructs. High levels of expression of NTAP-Ulk1 and NTAPUlk1-K46R were documented in transfected 293T cells maintained in nutrient rich media. Further, using only a single purification step, we were able to purify NTAP-Ulk1 (Supplementary Figure S1) and NTAP-Ulk1-K46R (data not shown) to greater than 90% purity. The yield of the kinase-dead mutant NTAP-Ulk1-K46R was invariably lower than those of NTAP-Ulk1 in 293T cells, typically less than half of the wild-type kinase (data not shown). The kinase-dead mutant also migrated much faster in an SDS-PAGE gel, suggesting that it may lack specific phosphorylation events or other post-translational modifications that are found in the wild-type kinase (Figure 1A). Importantly, this difference in mobility was seen when the TAP-Tag was either on the N- or C-terminus and thus was not affected by the tag (data not shown). To characterize the enzymatic activity of purified NTAP-Ulk1 and NTAP-Ulk1-K46R, we tested their ability to both phosphorylate themselves as well as purified fragments of the promiscuous substrate myelin basic protein (MBP). As predicted, NTAP-Ulk1 was able to phosphorylate itself (Figure 1A) and myelin basic protein (Figure 1B), whereas the activity of the kinase-dead mutant was greatly attenuated. As purified Ulk1 potently autophosphorylated itself in vitro, we surmised that the altered mobility of Ulk1-K46R was due to the inability of this kinase to autophosphorylate in vivo. To test whether wild-type Ulk1 migrated more slowly in gels due to hyperphosphorylation, we treated equal amounts of both the Ulk1 and the Ulk1-K46R mutant with a Lambda protein phosphatase. Phosphatase treatment canceled the retarded mobility of Ulk1; thus, its slower migration on gels Journal of Proteome Research • Vol. 8, No. 11, 2009 5255
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Figure 1. Ulk1 is a highly phosphorylated protein whose kinase activity controls its turnover. (A) Upper panel, an in vitro kinase assay using either purified NTAP-Ulk1 (Ulk1) or a purified kinasedead NTAP-Ulk1-K46R mutant (Ulk1-K46R). Ulk1 can autophosphorylate itself, whereas the kinase-dead Ulk1-K46R mutant cannot. Experiment shown is representative of three independent analyses. Lower panel, Western blot demonstrating that equivalent amounts of Ulk1 and Ulk1-K46R were used in each reaction. (B) Upper panel, in vitro kinase assay demonstrating that purified Ulk1 can phosphorylate purified fragments of myelin basic protein (MBP), whereas purified Ulk1-K46R cannot. Experiment shown is representative of three independent analyses. Lower panel, a Coomassie-stained gel demonstrating the equivalent amounts of MBP were added to the in vitro kinase reaction. (C) Western blot analysis of purified NTAP-Ulk1 and NTAP-Ulk1-K46R either left untreated or treated with Lambda protein phosphatase, demonstrating that the molecular weight difference between the kinase and kinase-dead mutant is due to phosphorylation. Experiment shown is representative of three independent analyses. (D) Pulse-chase analysis of Ulk1 and Ulk1-K46R. 293T cells expressing NTAP-Ulk1 and NTAP-Ulk1-K46R were pulsed with 35 S-Translabel for 15 min in methionine-free DMEM (0 time point), and then chased in complete DMEM media for 1, 4, 8, and 24 h. Following affinity purification, equal cell numbers were loaded on the gel and the samples were subjected to autoradiography. Experiment shown is representative of two independent analyses of Ulk1 versus Ulk1-K46R.
was indeed due to phosphorylation events that were not present in the kinase-dead Ulk1-K46R mutant at steady state (Figure 1C). The yield of kinase-dead Ulk1-K46R was consistently much lower than that of wild-type Ulk1, suggesting that reduced levels of Ulk1-K46R might correlate with increased rates of turnover. To define the turnover rates of Ulk1 and the kinase-dead Ulk1K46R mutant, as well as their ability to acquire post-translational modifications, pulse-chase analyses were performed. 5256
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Dorsey et al. 293T cells expressing either NTAP-Ulk1 or NTAP-Ulk1-K46R were pulsed with 35S-Translabel for 15 min (0 h), and chased for 1, 4, 8, and 24 h in prewarmed media lacking radioactive label. Cell pellets were then lysed and TAP-tagged proteins were purified using streptavidin resin, resolved on SDS-PAGE gels, treated with diphenyloxazole, dried and exposed to film. These studies revealed that wild-type Ulk1 was indeed more stable than that of Ulk1-K46R. Further, at the end of a 15 min pulse, newly synthesized Ulk1 had already received post-translational modifications that were largely absent from Ulk1-K46R (Figure 1D). Although Ulk1-K46R was modified much more slowly, a significant portion of these molecules also appeared to receive post-translational modifications, suggesting that either endogenous Ulk1 or other kinases such as mTOR can phosphorylate this kinase-dead Ulk1 mutant. As these modifications were not evident in the steady-state pool of Ulk1-K46R (Figure 1A), modified Ulk1-K46R must also be less stable than wild-type Ulk1, suggesting that specific modifications play a role in Ulk1 turnover (Figure 1D). Collectively, these findings indicate that under nutrient-replete conditions Ulk1 is a highly phosphorylated kinase whose autophosphorylation controls its stability. Phosphomap of Ulk1. To generate a phosphomap of Ulk1 in its inhibited state, NTAP-Ulk1 was purified from 293T cells under nutrient-rich conditions. Following affinity purification, Ulk1 was proteolytically digested and analyzed using highresolution and tandem mass spectrometry. Ulk1 is a large, 112 kDa protein that contains three domains: an N-terminal kinase domain, a central serine/proline-rich region, and a C-terminal domain (CTD) that contains a PDZ-binding motif (Figure 2A). Accordingly, Ulk1 has significant diversity in its amino acid composition, which necessitated digestion of Ulk1 with multiple proteases including trypsin, chymotrypsin, and Glu-C. Ulk1 peptides were analyzed using LC-coupled tandem mass spectrometry and were fragmented via CAD. These mass spectrometric analyses afforded examination of 60% of the Ulk1 protein sequence (Supplementary Table S1) and identification of 16 novel sites of Ulk1 phosphorylation (Figure 2A-C). Importantly, all of the experimental peptide precursor masses were obtained using high-resolution MS analyses and were accurate to within 5 ppm of the theoretical masses for each phosphopeptide. Identified phosphorylated Ulk1 peptides, along with their site(s) of phosphorylation, are shown in Figure 2C. Modifications of the N-Terminal Kinase Domain of Ulk1. While conventional LC-coupled MS/MS analyses enabled the identification of many phosphorylation sites within the multiple domains of Ulk1, a more advanced methodology was required to map phosphorylation sites in the Ulk1 kinase domain. In a typical MS/MS analysis, doubly protonated (+2) peptide precursors generated with trypsin are dissociated via CAD to yield complete or nearly complete sequence coverage of the b- and y-type product ion series. When efficient fragmentation occurs for a phosphorylated peptide, the MS/MS spectrum affords unambiguous identification of the peptide and of the specific site(s) of phosphorylation. However, phosphorylation sitemapping of a significant portion of the Ulk1 kinase domain was challenging because trypsin-mediated proteolysis of this region produces large, highly charged peptides that are not often amenable to efficient sequencing via CAD MS/MS (or MS2) analysis. For example, the b- and y-type product ions observed in the MS2 spectrum of phosphorylated Ulk1 peptide 72-108 did not afford definitive localization of the phosphorylated residue (Figure 3A). This Ulk1 peptide is a large, 37-
Ulk1 Phosphorylation Sites
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Figure 2. Ulk1 phosphorylation map. (A) A schematic of Ulk1 illustrating its kinase domain (red), serine/proline-rich region (blue), and C-terminal domain (CTD) (green) and their associated phosphorylation sites. Structure-function analyses of Ulk1 have identified several regions that are critical for its interaction with its binding partners LC3, GABARAP, Gate-16, Atg13 and FIP200 (purple). (B) The primary sequence of Ulk1 with the 16 phosphorylation sites identified in the studies presented herein are boxed and highlighted in red, blue, or green to indicate the kinase, serine/proline-rich, and C-terminal domains, respectively. The Ulk1 kinase domain is underlined in black, and the extreme C-terminal PDZ binding domain is in bold. (C) A comprehensive list of all of the identified phosphorylated peptides from Ulk1, along with the corresponding theoretical masses, observed masses, and associated ppm errors. The start residue of the identified peptide, the charge state of each peptide, and the phosphorylated amino acid residue(s) are provided as well. The identification of phosphorylated peptides of Ulk1 and the defined sites of phosphorylation were derived from more than three separate experiments.
residue peptide and exists as a +4 peptide precursor. Therefore, we employed MS3 scanning in tandem following acquisition of the MS2 spectrum. The method applied for this analysis involved the data-dependent acquisition of an MS3 spectrum (Supplementary Figure S2) from the most intense ion in the
MS2 spectrum with an m/z greater than the initial precursor. For the 72-108 Ulk1 peptide, this resulted in the selection and fragmentation of the y24 product ion (m/z 1409), which is the most abundant ion in the MS2 spectrum, with m/z greater than the precursor (m/z 1102.7). Together, the MS2 and MS3 spectra Journal of Proteome Research • Vol. 8, No. 11, 2009 5257
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Figure 3. Identification of phosphorylation sites in the Ulk1 kinase domain. (A) MS/MS spectrum of phosphorylated Ulk1 peptide 72-108. The [M + 4H]+4 ion with m/z 1102.7 was selected for dissociation, and the phosphorylated residue was identified as serine-87. (B) MS/MS spectrum of phosphorylated Ulk1 peptide 187-198. The [M + 2H]+2 ion with m/z 774.8 was selected for dissociation, and the phosphorylated residue was identified as serine-195. (C) MS/MS spectrum of the phosphorylated Ulk1 peptide 219-230. The [M + 2H]+2 ion with m/z 698.8 was selected for dissociation, and the phosphorylated residue was identified as serine-224. The amino acid sequences are shown above each spectrum in panels A-C, and the masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the singly protonated, monoisotopic product ion masses. The observed singly protonated b- and y-type ions are underlined, and doubly protonated ions are denoted with filled circles. Asterisks indicate product ions that result from neutral loss of H3PO4. The phosphorylated serine residues are indicated with “phos”. 5258
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Ulk1 Phosphorylation Sites
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Figure 4. Putative autophosphorylation site in Ulk1. MS/MS spectrum of the phosphorylated Ulk1 peptide 327-342. The [M + 2H]+2 ion with m/z 836.4 was selected for dissociation, and the amino acid sequence of the peptide is shown above the spectrum. The masses above and below the sequence correspond to the theoretical b- and y-type product ions, respectively. The masses provided are the monoisotopic, singly protonated masses of the product ions. The observed singly protonated product ions are underlined, and the observed doubly protonated ions are denoted with filled circles. Asterisks indicate product ions that result from neutral loss of H3PO4. The product ions corresponding to neutral loss of H2O are indicated with ∆. As indicated in the sequence, the phosphorylated residue was identified as serine-341. (B) A Sequence Logo graphical representation of an alignment of all the identified phosphorylated residues within the serine/proline-rich region. The height of an individual amino acid indicates the relative frequency of an amino acid in that position (http://weblogo.berkeley.edu/). Note the prevalence of glycine and proline residues flanking the phosphorylated serine residues of Ulk1, suggesting that this domain is a target of a proline-directed kinase.
of peptide 72-108 allowed increased sequence coverage of the peptide and for the unambiguous identification of serine-87 as the site of phosphorylation. Using chymotrypsin and trypsin, two additional phosphorylation sites were also identified within the N-terminal kinase domain, pSer195 within Ulk1 peptide 187-198 and pSer224 within Ulk1 peptide 224-235 (Figure 3B,C). Phosphorylations in the Serine/Proline-Rich Region of Ulk1. Juxtaposed to the N-terminal kinase domain, Ulk1 contains a serine/proline-rich region that is conserved from C. elegans to man. Previous truncation studies of mouse Ulk1 demonstrated that Ulk1 can autophosphorylate a site within residues 287-351 of this region.13 The N-terminal portion of this region is serine rich, and detecting peptides from this region was challenging due to limited proteolytic cleavage sites. However, a tryptic peptide (residues 327-342) was identified within this region following tandem MS analysis, which yielded near complete sequence coverage of product ions and identification of pSer341 (Figure 4A).
C-terminal to pSer341, we identified eight additional phosphorylation sites within the serine/proline-rich region (Figure 2B,C). Both MS2 and MS3 spectra (Supplementary Figures S3A-E and S4A-C) were acquired to obtain extensive sequence analysis and definitive localization of the phosphorylated residue(s) within each peptide. Interestingly, nearly all of the phosphorylation sites in the serine/proline-rich region are flanked by glycine or proline residue, or by both (Figure 4B). While this domain is admittedly rich in proline residues, there are several unphosphorylated serine residues that are not flanked by proline residues, suggesting that this domain may be a target of a proline-directed kinases such as mTOR, those of the CMGC family (Cyclin-dependent kinases, Mitogenactivated protein kinases, Glycogen synthase kinases, and CDKlike kinases), and/or potentially Ulk1 or its family members Ulk2 or Ulk3. Phosphomap of the C-Terminal Domain (CTD) of Ulk1. Four phosphorylation sites were identified in the C-terminal domain (CTD) of Ulk1. First, pSer867 was identified within Ulk1 Journal of Proteome Research • Vol. 8, No. 11, 2009 5259
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Figure 5. Phosphorylation of the extreme C-terminus of Ulk1. MS/MS spectra of phosphorylated forms of Ulk1 peptide 1041-1051. The spectra in (A) and (B) afforded the identification of two singly phosphorylated peptides that contain either pSer1043 or pSer1047, respectively. The [M + 2H]+2 ions (m/z 615.32) were selected for dissociation, and the amino acid sequence and the mapped phosphorylated residues are shown above the spectra. The masses above and below the sequence correspond to the theoretical band y-type product ions for the two phosphorylated peptides. The singly protonated, monoisotopic masses of the product ions are provided. The observed singly protonated product ions are underlined, and the observed doubly protonated ions are denoted with filled circles. Asterisks indicate product ions that result from neutral loss of H3PO4.
peptide 866-893 and pSer913 was identified in Ulk1 peptide 907-923 (MS/MS spectra in Supplementary Figures S3 F,G). In addition, we examined the extreme C-terminus of Ulk1 for phosphorylation as it has been implicated in the regulation of Ulk1 activity.14,15 Tryptic cleavage of this region generates two forms of the Ulk1 C-terminus due to contiguous arginines at residues 1040 and 1041 that promote a missed cleavage, which commonly occurs when tryptic cleavage sites are in close proximity. The correctly cleaved phosphorylated Ulk1 peptide 1042-1051 contains no basic residues and is singly protonated. Therefore, this peptide fragmented poorly, and the resulting MS/MS spectrum contains limited b- and y-type product ions (Supplementary Figure S5A). In contrast, the mis-cleaved peptide 1041-1051 contains an arginine residue, and as expected, this doubly protonated peptide fragmented well upon CAD. An increased number of b- and y-type product ions was observed and enabled the identification of two singly phosphorylated peptides containing either pSer1043 (Figure 5A) or pSer1047 (Figure 5B). 5260
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Differential Phosphorylation of the Wild-Type and KinaseDead Ulk1 Mutant. Our data clearly demonstrate that Ulk1 can autophosphorylate (Figure 1A) and that the Ulk1-K46R kinasedead mutant is hypophosphorylated compared to wild-type Ulk1 (Figure 1C). These data suggest that the Ulk1-K46R mutant is differentially phosphorylated, and at least a subset of these phosphorylated residues result from autophosphorylation. Therefore, we quantitatively interrogated the peptides listed in Figure 2C from both wild-type Ulk1 and the Ulk1-K46R mutant for differences in relative abundance. The majority of these phosphorylated peptides were only marginally different in abundance, defined as less than a 2-fold change (data not shown). Remarkably, phosphorylation of the two serine residues at the extreme C-terminus of Ulk1 (Ser1043 and Ser1047) was suppressed in the Ulk1-K46R kinase-dead mutant when compared to wild-type Ulk1. Upon thorough examination, a lower abundant doubly phosphorylated form of the peptide 1041-1051 was also identified (MS/MS spectrum in Supple-
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Ulk1 Phosphorylation Sites
Figure 6. Differential phosphorylation of wild-type versus kinase-dead Ulk1 C-terminal peptide. Selected ion chromatograms (SICs) of the C-terminal peptides (1041-1051), RLSALLSGVYA, generated from Ulk1 (A) and Ulk1-K46R (B), demonstrate the reduction and/or loss of phosphorylation in the Ulk1-K46R kinase-dead mutant. The theoretical m/z values of the [M + H]+1 and [M + 2H]+2 ions for each of the unmodified and phosphorylated forms of the peptide were used to generate the SICs, and these m/z values are as follows: RLSALLSGVYA, 575.3350, 1149.6626; RLpSALLSGVYA and RLSALLpSGVYA, 615.3181, 1229.6290; RLpSALLpSGVYA, 655.3013, 1309.5953. The ion current intensities for each SIC are provided on the y-axis of each chromatogram. Note the SIC in the bottom panel of B is void, due to the absence of the doubly phosphorylated peptide in the Ulk1-K46R kinase-dead sample. (C) A bar graph is shown of the percent relative abundance of the phosphorylated forms of the RLSALLSGVYA peptide. The standard error of the mean was generated from two independent purifications and LC-MS/MS analyses.
mentary Figure S5B) in wild type Ulk1. Selected ion chromatograms, shown in Figure 6A, illustrate the presence and relative abundance of the unphosphorylated peptide (RLSALLSGVYA), the two singly phosphorylated peptides (RLsALLSGVYA and RLSALLsGVYA), as well as the doubly phosphorylated form (RLsALLsGVYA) in wild-type Ulk1. Interestingly, all the phosphorylated forms of the RLSALLSGVYA peptide were reduced in the Ulk1-K46R mutant. In fact, the singly phosphorylated form with pSer1047 and the doubly phosphorylated (pSer1043 and pSer1047) form were completely absent in the Ulk1-K46R kinase-dead mutant (Figure 6B). Quantifying two individual
experiments clearly established that the phosphorylation of the RLSALLSGVYA peptide from the kinase-dead Ulk1-K46R mutant is repressed compared to wild-type Ulk1 (Figure 6C).
Discussion Kinases play a central role in regulating almost every aspect of biology, from metabolism to cell signaling to cell-cell communication. Kinase domains are bipartite in their structures, containing an N-terminal antiparallel β-sheet and a C-terminal R-helical domain connected by a hinge region.30 Journal of Proteome Research • Vol. 8, No. 11, 2009 5261
research articles This structure can be further divided into 12 smaller subdomains, where subdomains I-IV form the β-sheet domain that both anchors and orients ATP into the active site. Subdomain V spans the N-terminal and C-terminal domains, lining a deep cleft that is recognized as the site of catalysis, whereas the remaining subdomains form the R-helical region that controls both kinase activation and substrate recognition. Within the Ulk1 kinase domain, we identified serine-87, -195, and -224 as sites of phosphorylation. Serine-87 lies between domains IV and V, and to our knowledge, phosphorylation in this region has not been reported in any other kinases. However, kinase activity requires the juxtaposition of the γ-phosphate of ATP and the acceptor amino acid with the exclusion of water from the active site. As a result, kinases undergo extensive conformational changes upon substrate binding.31 In addition to its role as the catalytic center, subdomain V is the bridge between the β-sheet and R-helical domains, which rotate with respect to one another in response to activation.32,33 Therefore, the phosphorylation of serine-87 may function to hold Ulk1 in an inactive state by preventing substrate-dependent conformational changes associated with catalysis. Conformational changes in kinase domains are also provoked by phosphorylation of the activation loop, which is defined as the region between DFG and APE peptide motifs of subdomains VII and VIII, respectively.31,34 This region is a wellcharacterized site of regulation in a wide array of kinases, where typically phosphorylation within this region activates kinase activity, while dephosphorylation abrogates catalysis through disruption of the active site. Subdomain VIII also plays a significant role in substrate recognition, and serine-195 is located at the extreme C-terminal end of subdomain VIII of Ulk1, suggesting it may play a role in either kinase activation and/or substrate recognition. In addition, serine-224 is located at the very end of subdomain IX, which contains a nearly invariant aspartate residue that stabilizes the catalytic loop through hydrogen bonding, suggesting that phosphorylation of serine-224 may disrupt Ulk1’s active site. Phosphorylation of serine-87, -195, and -224 may act separately or in concert to regulate Ulk1 kinase activity, and thus autophagy. Importantly, Ulk1 phosphorylation is not restricted to the kinase domain; indeed, the majority of phosphorylation events occurred within the serine/proline-rich region. Progressive C-terminal deletions of Ulk1 have demonstrated that residues 287-351 in the N-terminal portion of this domain are required for Ulk1 autophosphorylation.13 Indeed, we identified serine341 as a site of phosphorylation, suggesting that this site may result from autophosphorylation. Interestingly, serine-341 also lies within a region required for the binding of Ulk1 with microtubule associated protein A/B light chain 3 (LC3, a.k.a. Atg8), and its closely related family members γ-aminobutyric acid receptor associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (Gate-16).35,36 Notably LC3, GABARAP and Gate-16 are required for the formation of autophagic vesicles and/or for vesicular transport,36 and it will be interesting to test the effects of Ulk1 pSer341 on these responses. Ulk1 is negatively regulated by the mTORC1 complex, as inhibition of this complex with rapamycin potently activates the autophagy pathway. The regulation of Ulk1 by mTOR is mediated, at least in part, through repressive phosphorylation events on both Ulk1 and Atg13.24-26 Additionally, hyperactivation of mTOR through the overexpression of the mTOR 5262
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Dorsey et al. activator Ras homologue enriched in brain (Rheb) provokes dramatic increases in both Ulk1 and Atg13 phosphorylation and concomitant reductions in Ulk1 kinase activity.26 Interestingly, the serine/proline-rich domain is rife with phosphorylated serines, all of which are flanked by proline, glycine or both, suggesting that this region is the target of a proline-directed kinase such as mTOR or CMGC family kinases.37 Mitogenic signaling pathways activate many of the CMGC kinases, and this may be a general mechanism for control of Ulk1 activity and autophagy. Finally, phosphorylation of this domain may regulate Ulk1 oligomerization and/or intramolecular interactions that regulate Ulk1 function. Interestingly, Ulk1 is heavily phosphorylated at and around serine-479 that lies within the sequence GRASPS479P, which is very similar to a region of Atg13, suggesting that this site may be an important regulatory region for both proteins. The C-terminal domain of Ulk1 directs interactions with both Atg13 and FIP200, which are also required for autophagy, yet their interactions are independent of the C-terminal PDZ domain.14 The association of FIP200 with Ulk1 is mediated at least in part by Atg13 as FIP200 binding to Ulk1 is impaired in the absence of Atg13.26 In addition, Ulk1 associates with membranes whereas Atg13 does not, yet the interaction of Ulk1 with membranes or Atg13 requires amino acids 829-1001.14 We identified serine-867 and -913 as sites phosphorylated in Ulk1, suggesting that these sites may regulate the association of Ulk1 with Atg13, FIP200, and/or membranous compartments. Kinase-dead Ulk1, or just its C-terminal domain, function as dominant negative mutants.14,15 The generation of dominant negatives through such disparate domains suggests that the C-terminal domain and the kinase domain of Ulk1 may interact and clamp the molecule in a “closed”, inactive conformation, which may either be severed or enhanced by Ulk1 autophosphorylation. Further, the dominant negative activity of the C-terminal domain of Ulk1 is abolished when one deletes the seven-residue motif IERRLS1043A, which is unnecessary for the Ulk1-Atg13 interaction.14,25 Interestingly, this motif harbors a consensus PKA sequence (RRXS, where X is any amino acid), and PKA negatively regulates autophagy from yeast to man.10,38-41 Our differential phosphorylation analyses of wildtype Ulk1 and the kinase-dead mutant Ulk1-K46R demonstrated that Ser1043 phosphorylation is dramatically reduced in the mutant. Further, Ser1047 phosphorylation and the doubly phosphorylated peptide (RLsALLsGVYA) are undetectable in the kinase-dead mutant. Together these data suggest that efficient phosphorylation of the putative PKA site (Ser1043) relies upon Ulk1 autophosphorylation, and that Ser1047 is a site of Ulk1 autophosphorylation. Here, we hypothesize that Ulk1 Ser1047 autophosphorylation promotes Ser1043 phosphorylation, presumably by PKA. Further, since our analyses were performed under nutrient-replete conditions, where the majority of Ulk1 is inactive, these data also suggest that Ser1047 and Ser1043 phosphorylations may promote the closed clamp conformation of the Ulk1 complex. Ulk1 acts as a scaffold for Atg13, Atg17, and several other autophagy proteins, and also functions as a kinase to regulate autophagic vesicle formation,14,24-27,42 yet there are currently no known substrates of Ulk1. While it is clear that Ulk1 can phosphorylate itself, and evidence suggests that it can also phosphorylate Atg13 and FIP200,26 the Ulk1 consensus site is currently unknown. The identification of phosphorylated residues in Ulk1, Atg13 and FIP200 from cells grown in nutrientreplete versus nutrient-starved media will clarify which sites
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Ulk1 Phosphorylation Sites are phosphorylated by Ulk1. Once identified, their roles in controlling their interactions, the binding of Ulk1 to LC3 family members, and the regulation of Ulk1 kinase activity and autophagy can be mechanistically interrogated.
Acknowledgment. We would like to thank Meredith A. Steeves for her critical review of the manuscript and George Hilliard, who helped initiate this work. We would also like to thank the State of Florida for providing funds that supported the development of the mass spectrometry core at Scripps Florida. F.C.D. also received support from the National Cancer Institute (NRSA Fellowship no. 5F32CA123777-04). Supporting Information Available: Supplementary Figure S1, purification of NTAP-tagged Ulk1. Supplementary Figure S2, identification of Serine-87 as a site of phosphorylation. Supplementary Figure S3, assignments of phosphorylated residues in the serine/proline-rich and C-terminal domains of Ulk1. Supplementary Figure S4, MS/MS spectrum of phosphorylated Ulk1 peptide 476-491, MS3 spectrum of the doubly-protonated y14 product ion, MS3 spectrum of the doubly-protonated y12 product ion. Supplementary Figure S5, MS/MS spectrum of singly-phosphorylated Ulk1 peptide 10421051, MS/MS spectrum of doubly-phosphorylated Ulk1 peptide 1041-1051. Supplementary Table 1, all identified peptides that contribute to Ulk1 sequence coverage. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14)
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