Characterization of protein kinase ULK3 regulation by phosphorylation

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Characterization of protein kinase ULK3 regulation by phosphorylation and inhibition by small molecule SU6668 Lagle Kasak, Mihkel Näks, Priit Eek, Alla Piirsoo, Rohit Bhadoria, Pavel Starkov, Merilin Saarma, Sergo Kasvandik, and Marko Piirsoo Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00356 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 13, 2018

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Biochemistry

Figure 1. Identification of phosphosites in ULK3. (A) Schematic representation of ULK3 domain structure and identified phosphosites. ULK3 kinase domain spans amino acids 1-270, MIT domains amino acids 279–353 and 374–449. Phosphorylated residues are shown. Asterisks depict phosphorylated residues identified in our previous study (B) List of all identified phosphoserines. (C) Structure of ULK3 kinase domain generated by homology modeling. Phosphorylated residues and serine 134 is indicated. The N- and C-terminal lobes of the domain are colored in salmon and pink, respectively. The ATP-binding site is situated between the two lobes. 251x104mm (300 x 300 DPI)

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Figure 2. Identification of active ULK3 catalytic domain and identification of phosphoserines essential for ULK3 kinase activity. (A) Schematic representation of the deletion mutants used in the study. (B) Analysis of the activity of the deletion mutants. Activity of the protein was assessed by its ability to phosphorylate itself and a generic protein kinase substrate MBP. Only deletion of sequence residing immediately 3’ of the annotated kinase domain results in a catalytically inactive protein (ULK3 ∆271–365). (C) Identification of catalytically active ULK3 kinase domain. Neither ULK3KD1 (amino acids 1-270) nor ULK3CT (amino acids 271–472) showed any kinase activity. ULK3 and ULK3KD2 (amino acids 1–317) were able to phosphorylate themselves and ULK3CT protein. Asterisks represent nonspecific phosphorylation of IgG heavy chain. (D) Analysis of the activity of the ULK3 substitution mutants, where phosphorylated residues identified in ULK3 kinase domain are mutated to alanine (S217A, S219A; S146A, S147A) and ULK3 mutants, where serines 134 and 176 are mutated to alanine (S134A; S176A) or aspartic acid (S134D; S176D). Only two phosphomimetic mutants (S134D; S176D) have shown significantly lower catalytic activity . (E) Quantification of the kinase activity assays performed in panels B and D. Activities were normalized with


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Biochemistry

protein levels in the reaction. One-way ANOVA Dunnett’s multiple comparison test was used. Mean ± SEM of five independent experiments is shown.***p < 0. 0001 ∗∗ p < 0.01 (F) GLI dependent luciferase assay in ShhLight cells in the presence of exogenous Shh, showing the reduced biological activity of S134D; S176D mutants. 195x254mm (300 x 300 DPI)


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Figure 3. Nonphosphorylated form of ULK3 is catalytically active. Purified ULK3 protein was dephosphorylated using lambda phosphatase and subjected to kinase assay (middle panel), dephosphorylation reaction was monitored by subjecting the ULK3 and dephosphorylated ULK3 to SDS– PAGE gel and stained either with Coomassie stain for total protein (upper panel) or with phosphostain (lower panel). 44x136mm (300 x 300 DPI)


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Figure 4. SU6668 inhibits ULK3 catalytic acitvity in a partially ATP non-competitive manner. Purified ULK3 protein was subjected to kinase assay at different ATP concentrations as described in Materials and Methods. (A) ULK3 Km to ATP in an autophosphorylation assay (B) ULK3 Km to ATP in MBP phosphorylation assay. (C) ULK3D2 Km to ATP in the autophosphorylation assay. (D) ULK3 autophosphorylation rate in the presence of different concentrations of SU6668. 96x162mm (300 x 300 DPI)


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Figure 5. SU6668 binds to the ATP binding region in ULK3. (A) Chemical structures of SU6668 and SUX that can be covalently crosslinked to a substrate. (B) The inhibitor properties of SUX are comparable to those of SU6668. In vitro kinase assay was performed using 1 µM ULK3 in the presence of 10 µM SU6668 or SUX (upper panel). Protein amount was estimated using Coomassie staining (lower panel) (C) Identification of SUX covalently bound to ULK3 protein. Partially purified ULK3 was mixed with SUX, UV treated and resolved in SDS–PAGE gel. Gel was stained with Coomassie for total protein, ULK3 is indicated with asterisk (lower panel) or scanned with bioimager for fluorescence indicating covalent SUX binding to ULK3 (upper panel). (D) Alignment of N-termini of ULK3 and two related kinases PKA and TBK1. Residues important in ATP binding are indicated in yellow, peptide of ULK3 bound covalently to SUX and identified using mass spectroscopy analysis is underlined. 128x97mm (300 x 300 DPI)


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Figure 6. Alternative SU6668 ligand binding in serine/tyrosine kinases. Crystal structures of TBK1 (PDB ID: 4JLC) in complex with the inhibitor SU6668 (green), insulin receptor tyrosine kinase (PDB ID: 1IR3) with an ATP analog (yellow), and the homology model of ULK3 with docked SUX (magenta) were superposed. Only the structure of ULK3 is rendered for clarity. 140x108mm (600 x 600 DPI)


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For Table of Contents use only 44x34mm (600 x 600 DPI)


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Characterization of protein kinase ULK3 regulation by phosphorylation and inhibition by small molecule SU6668 Lagle Kasak,† Mihkel Näks,† Priit Eek,† Alla Piirsoo,‡ Rohit Bhadoria,† Pavel Starkov,† Merilin Saarma‡, Sergo Kasvandik,‡ Marko Piirsoo†,‡,* †

Department of Chemistry & Biotechnology, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia

‡

Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia#

To whom the correspondence should be addressed: [email protected]


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Abstract Serine/threonine protein kinase ULK3 is implicated in a variety of cellular processes including autophagy, cell division and execution of Sonic hedgehog pathway. However, very little is known how its biological activity could be controlled. The present study is focused on unraveling biochemical insights into mechanism of inhibition and activation of ULK3. We identify novel phosphorylation sites in ULK3 and show that autophosphorylation has no impact on the kinase activity of the protein. We further demonstrate that phosphorylation of two residues in the kinase domain of ULK3 by an as yet unidentified kinase may completely abolishes its catalytic activity. We show that a small molecular weight inhibitor SU6668, designed as an ATP competitive inhibitor for tyrosine kinases, binds in the ATP pocket of ULK3, yet it inhibits ULK3 kinase activity in a partially ATP non-competitive manner. Finally, we demonstrate that ULK3 kinase domain, annotated in silico, is not sufficient for its kinase activity, and additional amino acids in the 271–300 region are required.


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INTRODUCTION Intracellular signaling largely revolves around protein phosphorylation, which is brough about by the intricate interplay of kinases and phosphatases 1. Protein kinases can activate a signaling pathway, for instance, as is the case in the receptor tyrosine kinase signaling, where a cascade of kinases (RAF– MAPK–ERK) are sequentially activated leading to subsequent phosphorylation events resulting in the activation of a specific subset of transcription factors 2. Alternatively, protein kinases can keep a signaling pathway silent in the absence of extracellular stimulus, as is the case in the Sonic hedgehog (Shh) pathway, where the catalytic activity of PKA is necessary for the full or partial degradation of GLI transcription factors in the absence of ligand 3. In many cases, however, it remains somewhat unclear how a protein kinase involved in a signaling pathway can itself be regulated. ATG family of serine/threonine protein kinases is conserved from yeasts to mammals. There are 5 genes, named ULK1-4 and STK36, coding for ATG family of kinases in mammals 4. The only family member in yeast, Atg1, and mammalian ULK1 and ULK2 share homology along the entire protein sequence, and they function downstream of mTOR pathway in the regulation of autophagy. Other members of the mammalian ATG family of kinases, ULK3-4 and STK36, are homologous to Atg1 and ULK1 and -2 only in the catalytic domain of the protein indicating that there may be a different mechanism of their regulation, and hence, how they exort their function. ULK1 is the only member of the mammalian ATG family, the regulation of which has been extensively studied. ULK1 (and perhaps ULK2) is part of a a large protein complex, that includes ATG13–FIP200/Atg17–ATG101 proteins 5. It has been shown that the activity of ULK1 is mainly regulated by phosphorylation. Under normal growth conditions, the activity of ULK1 is acutely inhibited by mTOR-mediated phosphorylation, whereas stress conditions trigger rapid dephosphorylation of mTOR sites in ULK1, and ULK1 is activated by phosphorylation in AMPK protein kinase sites 6 7. All of these regulatory phosphosites in ULK1 reside outside its catalytic domain and are not conserved in ATG family members ULK3, -4 and STK36. A number of additional phosphorylated residues have been identified in ULK1 protein, with four of them found in the catalytic domain 8 9. Important functions attributed to ULK3 involve induction of autophagy that precedes the occurrence of senescent phenotype, and control of daughter cell separation in mitosis 10 11.We have previously shown that ULK3 is involved in the regulation of key mediators of SHH signalling GLI transcription factors playing kinase activity dependent and independent roles 12 13. In resting cells, ULK3 binds to SUFU, the main negative regulator of GLI proteins. The exact consequence of ULK3/SUFU interaction is not clear, but it has been suggested that SUFU binds N-terminal domain of ULK3 thereby inhibiting its catalytic activity. Activation of the SHH pathway leads to dissociation of ULK3/SUFU/GLI complex, generation of GLI transcriptional activators via ULK3-mediated phosphorylation and subsequent initiation of SHH dependent transcriptional program. In addition, we have shown that ULK3 is phosphorylated at four sites in its C-terminal domain and the activity of the kinase is inhibited by a protein kinase inhibitor SU666813 14. Moreover, SU6668 is able to inhibit the activation of SHH pathway in cell culture. Our previous study showed that addition of SU6668 to cells results in a phenotype that is similar to ULK3 siRNA mediated knockdown, and it antagonizes the activation of GLI dependent transcriptional programs 14. SU6668 was initially designed as an ATP competitive inhibitor of FGF receptor 1 and PDGF receptor tyrosine kinases 15 and the precise mechanism of action while inhibiting ULK3 activity at the molecular level was poorly understood. Herein, we describe the underlying biochemistry of ULK3 regulation and its inhibition. We identify a number of additional autophosphorylation sites in ULK3, and show that these sites, and phosphorylation in general, are not required for its catalytic activity. We further demonstrate that it can


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be abolished by two phosphomimetic mutations in the activation or catalytic loop of the protein. Finally, we also show that small molecule SU6668 binds into the ATP pocket while partially acting in an ATP competitive manner. MATERIALS AND METHODS Expression constructs. To obtain tagless native protein, ULK3 coding sequence of was ligated into pET-47b(+) vector (Novagen), where the target protein is expressed with an N-terminal His-tag, separated by a HRV 3C protease site, so only two extra residues (Gly-Pro) are retained in the Nterminus after cleavage. The construct was further modified by exchanging 6×His tag with 10×His-tag followed yeast ubiquitin protein and HRV 3C protease site. Generation of Flag-tagged ULK3 constructs is described in Maloverjan et al13. Protein purification. ULK3 was expressed in BL21-CodonPlus(DE3)-RP cells. Bacterial biomass was obtained from fed-batch cultivation in minimal medium containing 10% glycerol. ULK3 expression was induced with 0.5 mM IPTG for 5 h at 20 °C. Cell pellet was resuspended in binding buffer (50 mM Tris HCl, 300 mM NaCl, 20 mM imidazole, 10% glycerol pH 8.0) with lysozyme and PMSF. Cell suspension was stirred and incubated on ice for 30 min, cells were lysed using a french press and centrifuged at 39,000 g for 60 min at 4 °C. Supernatant was filtered using 0.45 µm filter. During first step purification the lysate applied onto a HisTrap Ni-Sepharose column (GE Healthcare) and washed with binding buffer on an ÄKTA FPLC system (GE Healthcare). The protein was eluted with an imidazole gradient from 20 to 300 mM. Eluates containing ULK3 were supplemented with HRV 3C protease and incubated overnight at 4 °C. Samples were dialyzed against binding buffer for 2 hours with one exchange of buffer. Cleaved protein was separated from the protease and uncleaved material in a second Ni-affinity run analogous to the first one, but was collected from the flowthrough. For the final purification step, concentrated protein fractions were run on a Superdex 200 size exclusion column (GE Healthcare) with 20 mM Tris-HCl, pH 8.0, and 150 mM NaCl. Next, 10% glycerol was added to ULK3 containing fractions, purified protein was concentrated with Amicon Ultra centrifugal filters and stored at −80 °C. Cell culture. Human embryonic kidney cell line 293T (ATCC CRL-3216) was used for overexpression studies and Shh-Light2 cells were used for luciferase assay. Cells were propagated and transfected as described previously 12. Cells were analyzed 48 h post-transfection. Immunoprecipitation and luciferase assay. Assays were performed as previously described 13. Enzyme kinetics studies. In vitro kinase assays were performed as previously described 12. For ATP Km determination purified ULK3 protein (1 µM) was subjected to kinase assay at the following ATP concentrations: 0.1; 1; 5; 25; 100 and 500 µM. For SU6668 inhibition mechanism studies the following ATP concentrations were used: 5, 10, 20 and 40 µM. Kinase inhibitor SU6668 was added at concentrations 0; 0.5; 1; 2; 4 and 8 µM. Km and Vmax calculations were performed using GraphPad Prism (GraphPad Software, La Jolla, CA). Phosphoprotein analysis. Pro-Q® Diamond phosphoprotein gel stain (Invitrogen) was used according to manufacturer’s instructions. Synthesis of SUX and UV crosslinking. Full synthetic procedures and characterization data for minimalist cross-linker and SUX construct are available in Supporting Information (Supplementary File 1). 2-(3-Methyl-3H-diazirin-3-yl)ethan-1-amine hydrochloride (21 mg, 156 µmol, 2.0 equiv) was added to a pre-mixed solution of SU6668 (24 mg, 72 µmol, 1.0 equiv), EDCI (18 mg, 94 µmol, 1.2 equiv) and DIPEA (68 µL, 391 µmol, 5.0 equiv) in EtOAc and the mixture was left to stir at RT. After 12 h, the mixture was concentrated under reduced pressure and purified by flash chromatography (EtOAc/DCM/MeOH 50:50:1) to give the desired product as an orange viscous oil (16.2 mg, 41.3


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Biochemistry

µmol, 53%). 1H NMR (DMSO-d6, 400 MHz) δ 13.39 (s, 1H, NH-pyrrole), 10.74 (s, 1H, NHoxindole), 7.84 (t, 1H, J = 5.6, CONH), 7.71 (d, 1H, J = 7.9, ArH), 7.56 (s, 1H, C=CH), 7.09 (td, 1H, J = 7.6, 1.0, ArH), 6.97 (td, 1H, J = 7.5, 0.9, ArH), 6.87 (d, 1H, J = 7.6, ArH), 2.97 (q, 2H, J = 6.7, NHCH2), 2.64 (t, 2H, J = 7.6, CH2CH2CO), 2.30 (s, 3H, CH3Ar), 2.27 (s, 3H, CH3Ar), 2.21 (t, 2H, J = 7.6, CH2CO), 1.42 (t, 2H, J = 7.1, NHCH2CH2), 0.97 (s, 3H, CH3). HRMS for C22H26N5O2 [M+H]+ found 392.2091; calc. 392.2081. UV crosslinking of SUX to ULK3 was performed in GF buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 % glycerol). 10 µM ULK3 was mixed with 20 µM SUX on ice and open eppendorfs were irradiated with hand-held UV lamp UVGL-25 (UVP, USA) at 365 nm for 10 minutes in the dark. Proteomics analyses. Detailed description of the proteomics methods can be found in the Supporting Information (Supplementary File 1). Briefly, proteins were trypsinized into peptides with in-gel (ULK3-SUX samples) and in-solution digestions (non-enriched phosphoproteome samples). Peptides were desalted and injected to a nano-LC/MS/MS system operated in a data-dependent acquisition mode. Mass spectrometric raw data were processed and analyzed with the MaxQuant software suite 16. For phosphoproteomics analyses with phosphopeptide enrichment, samples were processed and enriched with the EasyPhos protocol as described elsewhere 17. Modelling. Structural analogues of the ULK3 kinase domain (residues 1-270) were searched using the HHpred server 18. Six top hits (PDB ID: 3FE3, 5YKS, 2BDW, 5IG1, 4B9D, 3H4J) were used as input for MODELLER 19 to obtain a representative homology model. The inhibitor derivative SUX was docked into the ATP-binding pocket of the homology model using the Rosetta suite (release 2018.09). The docking protocol was based on scripts provided in 20. Conformational library for SUX was generated using Confab 21. The distance between the aziridine group of SUX and Ile43 of ULK3 was restrained to 3.0 Å. Altogether 500 models were generated and analyzed. The docking script and input models are provided in Supporting Information (Supplementary File 2). UCSF Chimera was used for structure analysis and visualization 22. RESULTS ULK3 is a multiphosphorylated protein. We have previously identified four autophosphorylated residues in the C-terminal regulatory domain of ULK3 13. In the meantime, three autophosphorylated sites were identified in the kinase domain of ULK1, that shares substantial homology with ULK3 kinase domain. Furthermore, it was suggested that kinase domain autophosphorylation regulates ULK1 catalytic activity 8 9. We were therefore interested to know whether ULK3 catalytic domain is also phosphorylated. We overexpressed and immunopurified FLAG-tagged ULK3 from 293T cells. Immunopurification was performed in the presence of phosphatase inhibitors to ensure that the protein is not de-phosphorylated during purification. Purified protein was trypsinized and followed up by LC– ESI–MS/MS analysis of phosphopeptides. We were able to visualize 49 ULK3 peptides with a coverage of 85% of ULK3 protein sequence (Supplementary File 3). Our MS/MS analysis revealed nine phosphorylated serines, and four of those were situated in the catalytic domain of the protein (Fig. 1B). Two of the phosphoserines (S350 and S464) were also identified in our previous study13. Raw data of phosphopeptide analysis is shown in Supporting Information (Supplementary Files 4 and 5). In order to investigate whether the identified serines were autophosphorylated, we used ULK3 protein purified from bacteria to near homogeneity. The purified protein was subjected to in vitro kinase assay, and the phosphorylated proteins were trypsinized and followed up by LC–ESI–MS/MS analysis of phosphopeptides. This analysis revealed that out of nine identified phosphoserines, seven were autophosphorylated (Fig 1B). Only serines 176 and 467/468 were not identified in proteins subjected to autophosphorylation assay. In addition, we identified seven phosphoserine residues that we did not pick up in ULK3 immunopurified from 293T cells (S55, S146/147, S300, S339, S349, S384, S449).


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Figure 1. Identification of phosphosites in ULK3. (A) Schematic representation of ULK3 domain structure and identified phosphosites. ULK3 kinase domain spans amino acids 1-270, MIT domains amino acids 279–353 and 374–449. Phosphorylated residues are shown. Asterisks depict phosphorylated residues identified in our previous study (B) List of all identified phosphoserines. (C) Structure of ULK3 kinase domain generated by homology modeling. Phosphorylated residues and serine 134 is indicated. The N- and C-terminal lobes of the domain are colored in salmon and pink, respectively. The ATP-binding site is situated between the two lobes.

ULK3 protein has a modular structure with a N-terminal kinase domain (amino acids 1-270) and two microtubule interacting and trafficking (MIT) domains (amino acids 279-353 and 374-449) (Figure 1A). Our phosphosite mapping showed that ULK3 is a multiphosphorylated protein. In addition to previously reported residues, our present analysis revealed 12 additional phosphosites. All four phosphosites identified in our previous study were also found to be phosphorylated in this extended analysis. Six of the phosphorylated residues are situated in ULK3 kinase domain. Interestingly, we found two phosphorylated serines (S176 and S467/468) that were not picked up in ULK3 proteins subjected to in vitro kinase assay prior to ESI-MS/MS analysis, suggesting that they may be substrates for a protein kinase other than ULK3 itself. Serine176 has been identified to be a phosphoserine previously23. Modeling of the ULK3 kinase domain showed that all six autophosphorylated serines are located on the protein surface, hence making them accessible for phosphorylation (Figure 1C). In addition, we found ten phosphorylated residues in the C-terminus of ULK3 (amino acids 271-482). Phosphorylation of ULK3 in the catalytic domain results in an inactive protein. With the phosphorylation map in hand, we next explored whether phosphorylation regulates catalytic activity of ULK3. We have previously shown that the kinase domain of ULK3 lacks autophosphorylation activity. 13. We generated a series of deletion mutants spanning the C-terminal part of ULK3, leaving the catalytic domain (amino acids 1–270) intact (Figure 2A). The deletions also removed all identified C-terminal phosphosites in ULK3. The constructs encoding FLAG-tagged truncated proteins were expressed in 293T cells, immunoprecipitated and subjected to in vitro kinase assay and Western blotting. As shown in Figure 2B and 2E, only the mutant protein deficient in amino acids 271-365 had severely reduced autophosphorylation activity. We also used a generic kinase substrate myelin basic protein (MBP) in our assays. MBP was used to rule out the possibility that decreased signal in kinase assays was due to the lack of some phosphosites in deletion mutants rather than the reduced kinase activity. However, it has to be mentioned that MBP is five times worse a substrate for ULK3 kinase than ULK3


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Biochemistry

itself (Fig 4). Mutant, lacking amino acids 271-365, had also lower activity towards MBP (Figure 2B). Since the deletion mutant, where amino acids 300-365 were deleted, rendered its kinase activity, we concluded that the ULK3 kinase domain, annotated in silico, is not sufficient for its catalytic activity, and additional amino acids in the region between 271-300 are required. To verify that this could be the case, we used ULK3 kinase domain (amino acids 1-270, ULK3KD1) and C-terminal domain (amino acids 271-472, ULK3CT) 13, and generated an additional ULK3 deletion mutant lacking amino acids 317-472 (ULK3KD2). The schematic structures of these mutants are shown in Figure 2A. All mutants were expressed at comparable levels as assessed by Western blotting (Figure 2C). In contrast to ULK3KD1, ULK3KD2 phosphorylates readily both itself and ULK3CT, however, the intensity of phosphorylation is lower when compared to the full length ULK3 (Figure. 2C). The reason for this is either due to the fact that ULK3KD2 does not have full catalytic activity or/and because the protein lacks C-terminal phosphosites. These data also show that the C-terminal, heavily phosphorylated cluster of amino acids (amino acids 448-472) is not required for ULK3 kinase activity. Next, we wished to know whether phosphorylation in the kinase domain of ULK3 regulates its catalytic activity. We generated two constructs, where phosphorylated serines 146 and 147 or 217 and 219, in the ULK3 kinase domain were changed to alanine (constructs S146,147A and S217,219A, respectively). In addition, we mutated serines 134 or 176 to alanine or phosphomimetic aspartic acid (constructs S134A, S134D, S176A, S176D). Ser134 is found directly in the active site of the kinase and it could show whether phosphorylation status renders the catalytic activity of the protein. Ser176 was found to be phosphorylated in our MS/MS analysis by a yet unidentified protein kinase (Fig. 1), and has been previously found to be phosphorylated in HeLa cells 23. Also, residue corresponding to ULK3 serine 176 has been shown to be phosphorylated by GSK3beta kinase in related MARK kinase 23 giving catalytically inactive proteins 24. According to our modelling, both of these serines are situated on the protein surface (Figure 1C). All constructs were expressed in 293T cells, immunoprecipitated and subjected to in vitro kinase assay and Western blotting. As seen in Figure. 2D, proteins ULK3 S146,147A and S217,219A bearing mutations in autophosphorylation sites, demonstrated autophosphorylation activity similar to wt ULK3. Also, mutants S134A and S176A were active protein kinases. In contrast, both phosphomimetic mutants S134D and S176D had severely reduced kinase activity. Analysis of MBP phosphorylation revealed that mutants S134D and S176D also had reduced activity towards this substrate (Fig 2D). Interestingly, mutant S134A had somewhat increased activity towards MBP, while retaining activity comparable to wt ULK3 in autophosphorylation assay (Fig 2D, 2E). These data show that phosphorylation at serines 146, 147, 217 and 219 is not required for ULK3 kinase activity. In contrast, phosphomimicking serines 134 or 176 of the kinase domain at severely reduces ULK3 catalytic activity. Quantification of the kinase assays showed that mutants S134D, S176D and ∆271-365 had severely reduced autophosphorylation activity with more than threefold reduction (Fig 2E). Mutants S146,147A; S217,219A, S134A and S176A had slightly reduced activities, however, they were not statistically significant. Mutants ∆300-365, ∆373-446 and ∆448-472 had somewhat higher activity that wt ULK3, but quantification revealed statistical significance only in the case of ∆300-365. Similar trends were observed if MBP phosphorylation was quantified (Supplementary File 1, Fig 1).


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Figure 2. Identification of active ULK3 catalytic domain and identification of phosphoserines essential for ULK3 kinase activity. (A) Schematic representation of the deletion mutants used in the study. (B) Analysis of the activity of the deletion mutants. Activity of the protein was assessed by its ability to phosphorylate itself and a generic protein kinase substrate MBP. Only deletion of sequence residing immediately 3’ of the annotated kinase domain results in a catalytically inactive protein (ULK3 ∆271–365). (C) Identification of catalytically active ULK3 kinase domain. Neither ULK3KD1 (amino acids 1-270) nor ULK3CT (amino acids 271–472) showed any kinase activity. ULK3 and ULK3KD2 (amino acids 1–317) were able to phosphorylate themselves and ULK3CT protein. Asterisks represent nonspecific phosphorylation of IgG heavy chain. (D) Analysis of the activity of the ULK3 substitution mutants, where phosphorylated residues identified in ULK3 kinase domain are mutated to alanine (S217A, S219A; S146A, S147A) and ULK3 mutants, where serines 134 and 176 are mutated to alanine (S134A; S176A) or aspartic acid (S134D; S176D). Only two phosphomimetic mutants (S134D; S176D) have shown significantly lower catalytic activity . (E) Quantification of the kinase activity assays performed in panels B and D. Activities were normalized with protein levels in the reaction. One-way ANOVA Dunnett’s multiple comparison test was used. Mean ± SEM of five independent experiments is shown.***p < 0. 0001 ∗∗ p < 0.01 (F) GLI dependent luciferase assay in ShhLight cells in the presence of exogenous Shh, showing the reduced biological activity of S134D; S176D mutants.


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The negative effect of phosphomimetic substitution mutations in serines 134 and 176 on ULK3 activity was confirmed in vivo in luciferase assay using Shh-Light2 cell line containing Gli-dependent reporter. Both point mutants deficient in kinase activity (S134D and S176D) were unable to induce luciferase activity in the presence of exogenous Shh, while in the presence of wtULK3 luciferase activity was induced three times (Figure 2F). Autophosphorylation is not required for ULK3 kinase activity. The two N-terminal autophosphorylation residues in ULK3 (Ser22 and Ser55), identified in the present study, remained intact in the mutation analysis. Furthermore, it is possible that our LC-ESI-MS/MS analysis did not identify all possible phosphorylations of the protein. Therefore, we were interested, if phosphorylation per se is important for ULK3 kinase activity. To address this issue, we used bacterially purified ULK3 protein (which was autophosphorylated in bacteria) and after lambda phosphatase treatment, the dephosphorylated form of ULK3 was subjected to in vitro kinase assay. The efficiency of dephosphorylation was assessed by phosphostaining. As seen in Figure 3, dephosphorylation did not abolish ULK3 kinase activity, indicating that autophosphorylation is not required for ULK3 catalytic activity.

Figure 3. Nonphosphorylated form of ULK3 is catalytically active. Purified ULK3 protein was dephosphorylated using lambda phosphatase and subjected to kinase assay (middle panel), dephosphorylation reaction was monitored by subjecting the ULK3 and dephosphorylated ULK3 to SDS–PAGE gel and stained


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either with Coomassie stain for total protein (upper panel) or with phosphostain (lower panel). Gel filtration (GF) was used to separate ULK3 and phosphatase prior kinase assay.

ULK3 catalytic activity is inhibited by SU6668. We have previously shown that a small molecule kinase inhibitor SU6668 destroys ULK3 catalytic activity 14. Although our unpublished data indicated that SU6668 acted as a mixed type of inhibitor, we used relatively crude preparation of His-tagged ULK3, which may have hampered enzyme kinetics. Below, we extended our in vitro kinase activity analysis by using ULK3 protein purified from bacteria to near homogeneity, and rationalized the data with a help of chemical cross-linker probe and computational modeling. First, we subjected the purified ULK3 protein to in vitro kinase assay in the presence of increasing amounts of ATP, in order to verify if Km to ATP is in the similar range as we have observed previously with the crude protein preparation. We analyzed both ULK3 autophosphorylation and ULK3 mediated phosphorylation of myelin basic protein (MBP). Phosphorylated proteins were resolved in SDS-PAGE, quantified and analyzed using GraphPad software. As shown in Figure 4, Km to ATP in case of autophosphorylation was 1.9 µM (Figure 4A) and MBP phosphorylation 9.8 µM (Figure 4B). We also measured ULK3KD2 Km to ATP in an autophosphorylation assay, given that ULK3KD2 was a weaker enzyme than full length ULK3 (Figure 2C) and found it to be 6 µM (Figure 4C).


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Biochemistry

Figure 4. SU6668 inhibits ULK3 catalytic acitvity in a partially ATP non-competitive manner. Purified ULK3 protein was subjected to kinase assay at different ATP concentrations as described in Materials and Methods. (A) ULK3 Km to ATP in an autophosphorylation assay (B) ULK3 Km to ATP in MBP phosphorylation assay. (C) ULK3D2 Km to ATP in the autophosphorylation assay. (D) ULK3 autophosphorylation rate in the presence of different concentrations of SU6668.

In case of competitive inhibition Km increases and Vmax remains the same during increasing concentrations of the inhibitor. In noncompetitive inhibition Vmax decreases, while Km is unaffected. To distinguish between these two modes of inhibition, we performed kinase assays using increasing concentrations (5; 10; 20 ja 40 µM) of ATP at different fixed concentrations (0.5; 1; 2; 4; 8 µM) of SU6668. The averaged data from three independent experiments are shown in Figure 4D. In our case, Km to ATP increased with increasing concentrations of SU6668 (2.4 µM in the presence of 0.5 µM SU6668; 3 µM in the presence of 1 µM SU6668; 5.3 µM in the presence of 2 µM SU6668; 9.5 µM in the presence of 4 µM SU6668; 11.5 µM in the presence of 8 µM SU6668) and Vmax decreased by 60%


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when SU6668 concentration was increased from 0.5 µM to 8 µM SU6668. Hence, increasing concentrations of SU6668 increase ATP Km and decrease Vmax, indicating a mixed type of inhibition.

Figure 5. SU6668 binds to the ATP binding region in ULK3. (A) Chemical structures of SU6668 and SUX that can be covalently crosslinked to a substrate. (B) The inhibitor properties of SUX are comparable to those of SU6668. In vitro kinase assay was performed using 1 µM ULK3 in the presence of 10 µM SU6668 or SUX (upper panel). Protein amount was estimated using Coomassie staining (lower panel) (C) Identification of SUX covalently bound to ULK3 protein. Partially purified ULK3 was mixed with SUX, UV treated and resolved in SDS–PAGE gel. Gel was stained with Coomassie for total protein, ULK3 is indicated with asterisk (lower panel) or scanned with bioimager for fluorescence indicating covalent SUX binding to ULK3 (upper panel). (D) Alignment of N-termini of ULK3 and two related kinases PKA and TBK1. Residues important in ATP binding are indicated in yellow, peptide of ULK3 bound covalently to SUX and identified using mass spectroscopy analysis is underlined.

SU6668 binds to the ATP pocket of ULK3. Since enzyme kinetics indicated that SU6668 may act as a mixed type of inhibitor of ULK3 kinase activity, we wished to determine how SU6668 interacts with and inhibits ULK3. We prepared a modified version of the inhibitor so that it would chemically react with the protein (i.e. covalently cross-linked) on exposure to UV light. For this reason, we equipped our inhibitor with a ‘minimalist’ diazirine moiety attached to via a free acid of SU6668 (the new inhibitor was termed SUX, Figure 5A)25 26. Gratifyingly, SUX inhibited ULK3 kinase activity (Figure 5B), and was successfully photo-cross-linked to the purified ULK3 protein. The specificity of binding was verified by using a relatively crude preparation of ULK3 (Figure 5C; ULK3 is depicted with *). The covalently linked ULK3–SUX construct was treated with trypsin and the resulting mixture of oligopeptides was analyzed by LC–ESI–MS/MS. Only one ULK3-derived peptide bearing the reacted SUX unit was identified. Further analysis indicated that SUX had reacted selectively with Ile43, indicating that SU6668 binds rightfully into the catalytic pocket and does not bind elsewhere to the protein. Figure 5D shows an alignment of ULK3, TBK1 and PKA ATP binding regions with amino acid residues important in ATP binding shown in yellow. The peptide bound by SUX is depicted in bold.


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Biochemistry

Figure 6. Alternative SU6668 ligand binding in serine/tyrosine kinases. Crystal structures of TBK1 (PDB ID: 4JLC) in complex with the inhibitor SU6668 (green), insulin receptor tyrosine kinase (PDB ID: 1IR3) with an ATP analog (yellow), and the homology model of ULK3 with docked SUX (magenta) were superposed. Only the structure of ULK3 is rendered for clarity.

To visualize the possible binding mode of SU6668, we generated a homology model of the ULK3 kinase domain and docked the modified inhibitor SUX into the ATP-binding pocket. Original data files created for modelling are available in the Supporting Information. The diazirine group of SUX was restrained to a 3-Å distance from Ile43 to account for the photolabeling result. It should be noted that the side chain of Ile43 faces away from the binding cleft towards the distal side of the β-strand, so only the backbone part of the residue is sterically accessible to be tagged by the reactive carbene that forms on UV exposure.. Our aim was to look for possible alternative binding sites in a try to understand the differing inhibition mechanism. Among the best scoring results, the oxindole moiety of the inhibitor was found in several locations in the cleft. This included a position similar to SU6668 in TBK1 cystal structure, although in a reverse orientation, and the triphosphate site (data not shown). Interestingly, there were also models that had the SU6668 part buried deep inside a hydrophobic pocket between the two lobes, much deeper than the ATP site, which is located just by the surface next to the hinge that connects the lobes (Figure 6). It is feasible to suggest that in this alternative binding mode, there is enough space to simultaneously accommodate the substrate (an ATP molecule) as well, which explains the observed mixed type inhibition.

DISCUSSION


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Regulation of ULK3 activity by phosphorylation. ULK1 protein is known to be autophosphorylated at three residues in its catalytic domain, perhaps hinting at a possible way how ULK1 kinase activity is regulated 8. However, none of the ULK1 phosphoserines have a corresponding conserved counterpart in ULK3 protein. In the present study we identify sixteen phosphorylated serines in ULK3 protein, with at least ten sites phosphorylated in vivo in 293T cells. Previous reports show that some of these sites are phosphosylated in other cell types as well. ULK3 serines 176, 305 and 464 were phosphorylated in HeLa cells27 28 and serine 449 was phosphorylated in breast cancer samples29. Therefore, we believe that majority of the identified autophosphosphorylation sites are also functional in vivo. Our experiments revealed that ULK3 can be be phosphorylated at six serine residues in its catalytic domain, with five of those being autophosphorylated. Modeling of the ULK3 kinase domain showed that all of these serines are located on the surface of the protein; hence, they are readily accessible for phosphorylation. Additionally, we identified a cluster of four to five phosphorylated amino acids in the extreme C-terminus of the protein. However, based on deletion mutants and point mutations of potentially phosphorylatable serines in the ULK3 kinase domain, none of the autophosphorylated residues are crucial for the kinase activity. We further corroborated this finding by showing that dephosphorylated ULK3 is still an active kinase. Eukaryotic protein kinases are often divided into two subclasses depending on amino acid sequences of their activation loops. RD-type kinases have arginine-aspartate (RD) motif in their catalytic loop, and are generally believed to be activated through phosphorylation of serine or threonine in activationloop. In contrast, non-RD kinases lack arginine placed directly before catalytic aspartate, and the mode of their activation is unclear (although many of them do not autophosphorylate the activation loop)30. Unlike ULK1 and ULK2, ULK3 is, however, a non-RD type of kinase. Vertebrate ULK3 proteins feature a conserved leucine in place of arginine in the activation loop, while invertebrate (e.g. amphioxus and sea urchin) ULK3 proteins have a conserved methionine. This indicates that although ULK1–3 have similar kinase domains, their activation mechanisms could be very different. In order to get a glimpse into the ULK3 activation mechanisms, we generated two point mutations in the ULK3 kinase domain, which we assumed would alter its activity. We mutated Serine 134 in the active site of the protein to non-phosphorylatable amino acid alanine or phosphomimetic amino acid aspartate. Second, we made similar mutations in serine 176, which was found to be phosphorylated in our assay (Fig. 1) and in HeLa cells23. Residue corresponding to ULK3 serine 176 was also shown to be phosphorylated in a related MARK kinase and inactivates it23 24. Strikingly we found that phosphomimetic mutations in either residue inactivated the kinase activity of the protein. Therefore, we can conclude that in addition to SUFU-mediated binding, ULK3 may be inactivated via phosphorylation of the kinase domain at specific residues, and autophosphorylation does not play a role in the activation of the kinase. Interestingly, we have never found these residues to be autophosphorylated in our mass spectrometry analyses. It could be speculated that ULK3 is constitutively active kinase and its activity may be regulated by protein complexes it interacts with as well as specific phosphorylation at certain residues in the catalytic and activation loops. Mechanism of SU6668 inhibition towards ULK3. SU6668, a small molecule, was previously shown by us to inhibit ULK3 kinase in low micromolar range 14. SU6668 is known to act as an ATP competitive inhibitor, specifically inhibiting several tyrosine kinases 15. It was later also proved to bind to serine/threonine kinases,31 including mouse TBK1, for which a crystal structure has been also reported (PDB: 4JLC).32 Our initial experiments indicated that surprisingly SU6668 might act as an ATP non-competitive inhibitor towards ULK3. Therefore, we rigorously tested the mechanism of SU6668 inhibition and found that it is only partly ATP competitive towards ULK3. This phenomenon can be explained in several ways. First, it can be envisaged that SU6668 acts as a bimodal inhibitor, and it binds to ATP pocket and somewhere else in ULK3. Second possibility is that the inhibitor binds to ATP pocket, but induces inactive conformation of the kinase. The latter type of inhibitors are called


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Biochemistry

type II kinase inhibitors (reviewed in 33). To distinguish between these possibilities, we modified SU6668 and added a minimal diazirine linker to it, that allows crosslinking of the inhibitor to the protein with ultraviolet light. The cross-linked inhibitor-protein complex was trypsinized and analyzed by mass spectrometry. Our LC-ESI-MS/MS analysis identified only a single bound peptide that corresponded to the ATP-binding pocket of ULK3. In the mouse TBK1 crystal structure model, SU6668 is found in the ATP-binding site next to the hinge that connects the two lobes of the kinase domain. Its oxindole moiety is directed into the pocket, while the carboxylate group is facing outwards. This is the site that also binds the adenosine moiety of ATP, making SU6668 unambiguously a competitive inhibitor (type I, according to 33) to TBK1. However, in ULK3 the photoreactive diazirine group of SUX must be situated near Ile43, which makes it difficult for the oxindole part to acquire an analogous position. According to docking results, an alternative binding site might be located deep enough in the cleft so that simultaneous association of the inhibitor and the substrate could be considered. This can explain the observation of mixed type inhibition. Granted, the differing binding of SUX to ULK3 might be an artifact caused by the additional chemical group, but we consider this unlikely due to the similar inhibitory properties of both SU6668 and SUX. The deep cavity is also employed by type II inhibitors, which induce a conformational shift in the catalytically essential DFG motif 33 34 35. Although type II inhibitors tend to be larger and are supposed to associate with both the adenosine site and the deeper end of the cavity, the structural differences specific to ULK3 might allow SU6668 to induce such conformational changes, too. The crystal structure of a ULK3:inhibitor complex would be of high value to eradicate these speculations, however our trials to obtain the crystal structure of ULK3 have met only limited success so far. CONCLUSION We show that phosphorylation of a non RD protein kinase ULK3 does not play a role in its activation. It could be speculated that as such, ULK3 is constitutively active kinase and its activity is regulated by protein complexes it is situated in (for instance the ESCRT-III machinery and/or SHH recognition complex). We further show that ULK3 is inactivated by phosphorylation in the catalytic and activation loops. Finally, the facts that a conserved kinase domain of ULK3 (amino acids 1-270) is inactive, and that the derivative of the SU6668 inhibitor binds to ULK3 differently than to related kinase domains and acts as a mixed type inhibitor indicate that the structure of the kinase domain may differ from related kinase domains. Further structural studies are needed to solve this issue.

ASSOCIATED CONTENT Supporting Information files are: 1) Synthetic procedures and full characterization data (Supplementary File1). 2) The input files for docking with Rosetta, including the homology model of ULK3 kinase domain, Confab-generated library of SUX conformers, and the used docking protocol (ULK3_SUX_docking.zip). 3) ESI-MS-MS data for identified ULK3 peptides (Supplementary Files 3, 4, 5).

FUNDING


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This work was supported by Estonian Science Foundation (Grant 9478) and Tallinn University of Technology (Grant B40) (both awarded to M. P.); and Estonian Reseach Council Starting Grant PUT1290 and TUT Young Investigator Grant B62 (both awarded to P.S.). Priit Eek was supported by Institutional Research Funding of the Estonian Ministry of Education and Research IUT 19-9. NOTES The authors declare no competing financial interest. ABBREVIATIONS ULK3, unc-51-like kinase 3; MIT, microtubule interacting and trafficking domain; RT, room temperature; AMP-PNP, adenylyl imidodiphosphate. ACKNOLEDGEMENTS We are very grateful to Dr. Vello Tõugu for fruitful discussions.

REFERENCES

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Characterization of protein kinase ULK3 regulation by phosphorylation

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