Chapter 4 Screening of Kinase Substrates Using Kinase Knockout Mutants Taishi Umezawa Abstract Protein kinases are widely known to be major regulators of various signaling processes, particularly in eukaryotes, including plants. To understand their role in signal transduction pathways, it is necessary to determine which proteins are phosphorylated by these enzymes. Recent studies have applied a comparative phosphoproteomic approach to identify protein kinase substrates in plants. The results demonstrated that kinase knockout mutants are useful for screening protein kinase substrates via such a comparative analysis. Here some technical points are described for the experimental design and comparative analysis using kinase knockout mutants. Key words Knockout mutant, Plant, Phosphoproteomics, Protein kinase, Signal transduction


Introduction Protein kinases are enzymes that phosphorylate other proteins and are widely accepted as versatile mediators of cellular signaling. In plants, the protein kinase superfamily comprises over 1,000 members, corresponding to 1–2 % of all functional genes in the plant genome and suggesting their importance to plant life [1]. To date, multiple studies have demonstrated that protein kinases are involved in many regulatory cellular processes, including developmental processes and hormonal, stress, and defense responses to name a few. Therefore, the study of protein kinases may facilitate a more thorough understanding of many biological processes in plants. To understand how a protein kinase transduces intracellular signals, it is necessary to understand its signal transduction pathway consisting of upstream and downstream regulatory systems. Although each protein kinase is involved in a wide variety of upstream pathways, their downstream pathway seems to be relatively simple because it is usually initiated by phosphorylation of their respective “substrates.” However, it is still difficult to determine specific substrates of protein kinases because a typical kinase can recognize

Waltraud X. Schulze (ed.), Plant Phosphoproteomics: Methods and Protocols, Methods in Molecular Biology, vol. 1306, DOI 10.1007/978-1-4939-2648-0_4, © Springer Science+Business Media New York 2015



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multiple substrates and also because there are technical limitations that prevent their comprehensive analysis. Previous studies have utilized multiple methods to identify protein kinase substrates one by one. If a biological linkage between protein kinases and other proteins were to be identified, it would be a good indication of a protein kinase-substrate pair. However, performing this type of study, even for a single kinase, is usually time consuming; therefore, it is impossible to use this as a comprehensive approach for all protein kinases and substrates. According to one estimate, more than 70 % of human proteins are modified by phosphorylation, suggesting that one protein kinase can phosphorylate over 200 substrates on an average [2]. Therefore, the cellular protein phosphorylation network may be much larger than what is expected, and a systems approach will be required for its understanding. To date, several methods have been proposed to survey protein kinase substrates on a large scale. For example, protein microarrays and synthetic peptide libraries are used for predicting protein kinase substrates [3, 4]. Although these methods are based on in vitro kinase reactions, in vivo protein phosphorylation can be measured by phosphoproteomics, a recently developed large-scale analysis of phosphoproteins/peptides. An example of one such technique is the shot-gun analysis in combination with an efficient method for phosphopeptide enrichment, e.g., metal oxide chromatography (MOC) or immobilized metal affinity chromatography (IMAC), and a high-performance and high-accuracy LC-MS/MS system [5, 6]. Such technical development enabled the analysis of thousands of phosphoproteins and their in vivo dynamics, thus providing useful information to predict protein phosphorylation networks involved in a variety of signaling pathways [7, 8]. Furthermore, such a shot-gun phosphoproteomics technology has recently begun to be applied to screening for protein kinase substrates. For example, several recent studies determined specific protein kinase substrates by comparative phosphoproteomic analysis between different biological samples [9–13]. In comparative experiments, experimental design is critical for obtaining optimal results. Therefore, it is imperative to find a suitable condition under which protein phosphorylation is significantly changed between samples. For example, protein kinase inhibitors are often used to inhibit protein phosphorylation catalyzed by a protein kinase in mammals or yeast. However, such inhibitors are largely unavailable in plants. Instead, we can use knockout mutants, transgenic lines, various natural variations, or cultivars in which protein kinase-mediated phosphorylation is significantly blocked or enhanced (Fig. 1). Furthermore, it is necessary to have advanced information regarding the protein kinase under study, such as its biological function, the condition(s) that activate it, and the time course of when it is activated in plant cells. Therefore, the first step in developing these studies should be to collect such information (Fig. 2).


Screening of Kinase Substrates Using Kinase Knockout Mutants









? C





substrates (?) Fig. 1 A basic strategy of phosphoproteomics using a kinase knockout mutant. In this case, a stimulus induces phosphorylation of proteins A, B, and C, and a protein kinase (PKase) phosphorylates A and B. Comparative phosphoproteomic analysis is able to detect that phosphorylation of A and B are impaired or depressed in a kinase knockout mutant. Red circle shows a phosphate group

1) Biological functions of PKase

2) Biochemical characterization Time course

PKase activity

Wild type

PKase KO

3) Experimental design Control

Treatment (Time course)

Wild type

PKase KO

Fig. 2 Experimental design of a phosphoproteomic study using a kinase knockout mutant. It is important to collect multiple information of a protein kinase in advance, for example (1) biological function or (2) biochemical characterization. (1) Biological function may be determined by loss-of-function or gain-of-function analyses (e.g., what kind of stimuli can activate a protein kinase?). (2) Biochemical characterization includes a method by which to detect protein kinase activity, when it is activated, and so on. (3) Experiments should be designed based on such information of a protein kinase


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For example, the dynamics of kinase activity should be important for identifying protein kinase substrates. Prior to phosphoproteomic analysis, we must determine when we can observe protein phosphorylation mediated by the target kinase. Since protein kinase activity is often informative to determine a time course, it should be determined when the target protein kinase is activated in vivo. This can be performed with one of several available methods to measure protein kinase activity; for example, in-gel phosphorylation or immunoprecipitated kinase assays. Once some candidate phosphopeptides are identified by comparative phosphoproteomic analysis, the next step should be a validation of their biological significance (Fig. 3). First, it is essential to confirm whether the phosphoprotein is an actual substrate of the protein kinase. Then, the following characteristics should be determined: (1) whether the phosphoprotein is involved in signal transduction pathways, (2) whether it is a positive or negative regulator of biological responses, and (3) how the phosphorylation affects protein functions. Such experiments may take a long time to complete and require multiple different techniques. However, they are necessary to fully utilize phosphoproteome data. In other words, phosphoproteome data is just a catalog of phosphoproteins unless their importance is biologically confirmed. In this chapter, we describe a protocol for phosphoproteomic analysis using kinase knockout mutants of Arabidopsis and our recent Wild type

PKase KO

Crude extracts Trypsin digestion

Phosphopeptide enrichment Phosphosites

LC-MS/MS analysis

Peptide ID

Quantitative data

(Motif analysis)

Differential analysis

Functional analysis

Fig. 3 A scheme of comparative phosphoproteomic analysis using a kinase knockout mutant. After screening of phosphopeptides by differential analysis, functional analysis of phosphoproteins should be important to verify phosphoproteomic data

Screening of Kinase Substrates Using Kinase Knockout Mutants


study as a model case [12]. In our study, we used a triple-knockout mutant of closely related SnRK2 protein kinases to avoid their functional redundancy in plants [12, 14]. We employed a label-free quantitation of phosphopeptides enriched by hydroxyl acid-modified metal oxide chromatography (HAMMOC) [15]. In the triple mutant, SnRK2-mediated protein phosphorylation in response to ABA was clearly impaired, allowing us to identify substrate candidates that were differentially regulated between wild-type and mutant strains. Thus, we believe that a phosphoproteomic study using kinase knockout mutants represents a powerful tool with which to identify protein kinase substrates. Such a study will allow us to bring new insights to aid in the exploration of signaling pathways in plants.


Materials With the exception of plant medium, prepare all solutions using ultrapure water and use reagents with the highest purity or grade. Reagents are purchased from Sigma unless otherwise noted.

2.1 Sample Preparation

1. Petri dish: 90 mm Ø × 20 mm. 2. Germination medium (GM): For 1 L of the medium, add 1 pack of MS salt mix for 1 L (392-00591, Wako), 10 g sucrose, 0.5 g MES, and 1 mL Gamborg’s Vitamin B5 solution to 1 L of deionized water. Adjust the pH to 5.8 by 1 N KOH. For agar plates, 0.8 g bactoagar was added prior to autoclaving, and then pour it into petri dishes. 3. 0.5× GM solution. 4. 100 mM ABA solution: Dissolve 26.4 mg (±)-ABA in 1 mL ethanol and store at −20 °C. 5. Extraction buffer: 50 mM HEPES pH 7.5, 5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4, 25 mM NaF, 50 mM ß-glycerophosphate, 10 % glycerol, 2 mM dithiothreitol (DTT), and proteinase inhibitor cocktail (see Note 1). 6. Protein quantification reagents (e.g., Bio-Rad Protein Assay). 7. Liquid nitrogen. 8. Mortar and pestle. 9. Centrifuge with an angle rotor for 50 mL tubes. 10. 50 mL centrifuge tubes. 11. Miracloth. 12. 1.5 mL tubes.

2.2 Phosphoproteomic Analysis

1. Reduction buffer (prepare just before use): 10 mM DTT in 50 mM NH4HCO3. 2. Alkylation buffer (prepare just before use): 50 mM iodoacetamide in 50 mM NH4HCO3.


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3. Lysyl endopeptidase (LysC), MS grade. 4. Trypsin, MS grade. 5. C18 Stage-Tip (see Note 2). 6. HAMMOC Stage-Tip (see Note 2). 7. Solution A: 5 % acetonitrile (ACN) and 0.1 % trifluoroacetic acid (TFA). 8. Solution B: 80 % ACN and 0.1 % TFA. 9. Solution C: 300 mg/mL lactic acid in solution B. 10. 2 % TFA. 11. 10 % TFA. 12. 0.5 % Piperidine. 13. SpeedVac. 14. LC-MS/MS system. 15. Database engine (e.g., Mascot). 16. Quantitative proteomics software package (e.g., MaxQuant, MassNavigator). 17. Spreadsheet software (e.g., Excel). 2.3 In Vitro Phosphorylation Assay

1. Expression vector: For example pGEX-4T-3 (see Note 3). 2. Host: For example E. coli BL21(DE3) (see Note 4). 3. 2× YT medium: Dissolve 16 g Bacto Tryptone, 10 g Bacto Yeast Extract, and 5 g NaCl in 800 mL H2O. Adjust pH to 7.2 with 1 N NaOH, and then make up the volume up to 1 L with H2O. Sterilize by autoclaving. Add antibiotics after cooling. 4. 1 M IPTG: Dissolve 238 mg IPTG in 10 mL H2O. Sterilize by filtration and store at −20 °C. 5. Glutathione Sepharose 4B (GE Healthcare). 6. Phosphate-buffered saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 800 mL H2O. Adjust pH to 7.5 with 1 N HCl, and then make up the volume to 1 L with H2O. 7. Tris-buffered saline (TBS): Dissolve 6.05 g Tris–HCl and 8.76 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 N HCl, and then make up the volume to 1 L with H2O. 8. Protease inhibitor cocktail (Roche). 9. Sonicator. 10. 2× sample loading buffer. 11. SDS-PAGE system. 12. 10× reaction buffer: For 100 μL, mix 50 μL of 1 M Tris–HCl (pH 7.5), 10 μL of 1 M MgCl2, 10 μL of 1 M MnCl2, 0.5 μL of 100 mM ATP, and 29.5 μL of H2O.

Screening of Kinase Substrates Using Kinase Knockout Mutants


13. [γ-32P]-ATP (185 MBq/mL, Perkin Elmer). 14. Phosphorimager.


Methods Carry out all procedures at room temperatures unless otherwise specified.

3.1 Sample Preparation

Before starting the experiments, choose a strategy for MS data quantification, e.g., label free or others. In this chapter, label-free quantification is described as an example. If a different quantification method such as stable isotope labeling is chosen, additional procedures for sample preparation may be needed (see Chapter 6). 1. Sow 25–30 sterilized seeds on each GM agar plate. After 3 days at 4 °C in the dark, put the plates in a growth chamber at 21 °C on a 16-h light/8-h dark photoperiod. 2. Use 2–3-week-old seedlings for the treatment (see Note 5). 3. Store plant samples at −80 °C until use. 4. Grind 1–2 g samples to a fine powder with a mortar and pestle in liquid nitrogen. 5. Homogenize samples in 5–10 mL ice-cold extraction buffer. 6. After filtration through three layers of Miracloth, centrifuge at 8,000 × g at 4 °C for 20 min. 7. Transfer supernatant to a new tube. 8. Measure protein concentration of the extract. 9. Store at −80 °C until use.

3.2 Phosphoproteomic Analysis

This section is adapted from [17]. 1. Prepare aliquots of the extract containing 100–500 μg protein. The amount of protein, which depends on the tissues, cell type, or species, should be determined in advance. 2. Add 1 μL reduction buffer to every 50 μg protein and incubate for 30 min at room temperature. 3. Add 1 μL alkylation buffer to every 50 μg protein and incubate for 20 min at room temperature in the dark. 4. Digest proteins by LysC (1 μg for every 50 μg protein) for over 3 h at room temperature. 5. Dilute the digest with 4 volumes of 50 mM NH4HCO3. 6. Digest proteins by trypsin (1 μg for every 50 μg protein) overnight at room temperature. 7. Acidify the tryptic digests with an equal volume of 2 % TFA.


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8. Prepare a C18 Stage-Tip (1 mL) (see Note 2). 9. (Conditioning) Load 200 μL Solution B onto the Stage-Tip, and spin down at 1,000 × g for 2 min. 10. (Conditioning) Load 200 μL Solution A onto the Stage-Tip, and spin down at 1,000 × g for 2 min. 11. (Sample loading) Load the acidified sample onto the StageTip, and spin down at 1,000 × g for 2 min. 12. (Washing) Load 200 μL solution A onto the Stage-Tip, and spin down at 1,000 × g for 2 min. 13. Place the Stage-Tip on a new tube. 14. (Elution) Load 200 μL Solution B onto the Stage-Tip, and spin down at 1,000 × g for 2 min. 15. Dry the sample with a SpeedVac. 16. Resuspend the peptides in 200 μL Solution C. 17. Prepare a HAMMOC Stage-Tip (10 μL) (see Note 2). 18. (Conditioning) Load 20 μL Solution B onto a HAMMOC Stage-Tip, and spin down at 1,500 × g for 2 min. 19. (Conditioning) Load 20 μL Solution C onto the Stage-Tip, and spin down at 1,500 × g for 2 min. 20. Place the Stage-Tip on a new tube. 21. (Sample loading) Load the sample onto the Stage-Tip, and spin down at 1,500 × g for 4 min. 22. (Washing) Load 20 μL Solution C onto the Stage-Tip, and spin down at 1,500 × g for 2 min. 23. (Washing) Load 20 μL Solution B onto the Stage-Tip, and spin down at 1,500 × g for 2 min. 24. (Elution) Load 20 μL 0.5 % piperidine, and spin down at 1,500 × g for 2 min. Repeat this step twice. 25. Add 5 μL 10 % TFA to the eluate. 26. Prepare a C18 Stage-Tip (10 μL) (see Note 2). 27. (Conditioning) Load 20 μL Solution B onto the Stage-Tip, and spin down at 1,500 × g for 2 min. 28. (Sample loading) Load the sample onto the Stage-Tip, and spin down at 1,500 × g for 2 min. 29. (Washing) Load 20 μL Solution A onto the Stage-Tip, and spin down at 1,500 × g for 2 min. 30. (Elution) Load 20 μL Solution B onto the Stage-Tip, and spin down at 1,500 × g for 2 min. 31. Dry the sample with a SpeedVac. 32. Resuspend the desalted peptides in 10 μL solution A. 33. Analyze the samples by LC-MS/MS (see Note 2).

Screening of Kinase Substrates Using Kinase Knockout Mutants


34. Perform a database search to predict peptide IDs and phosphorylation sites. 35. Quantify each phosphopeptide using extracted ion current chromatogram. Some data analysis software for label-free quantitation is required, for example, MaxQuant and MassNavigator. 36. Calculate fold changes of phosphopeptide levels between samples. 37. Set the first criteria for screening of phosphopeptides that are responsive to the treatment. All phosphopeptides should be tested in this process (see Note 6). 38. Set the second criteria for screening of protein kinase substrate candidates. Phosphopeptides that met the first criteria should be tested (see Note 7). 39. After the second screening, list the selected phosphopeptides as candidates of protein kinase substrates. 40. (Optional) Analyze some specific phosphorylation motifs for further classification. Motif groups can be generated using some algorithms, such as Motif-X ( [16]. 41. (Optional) Compare quantitative data of each motif group and predict the target motif(s) of your protein kinase. 3.3 In Vitro Phosphorylation Assay

1. Prepare plasmid constructs in which a cDNA of your protein kinase gene is cloned into an appropriate expression vector. 2. Prepare plasmid constructs in which a cDNA of a fragment of a substrate gene is cloned into an expression vector. You can use a short fragment or a synthetic peptide containing phosphorylation site(s) when such information is known. 3. Introduce a mutation to the phosphorylation site in the substrate gene by site-directed mutagenesis. For example, change Ser/Thr to Ala. That mutation enabled us to confirm the phosphorylation site (see Note 8). 4. After transformation of E. coli, pick three colonies for subsequent experiments. 5. Culture E. coli in 2× YT and induce protein expression by adding 0.1–1 mM IPTG according to a standard protocol (see Note 9). 6. Prepare purified recombinant proteins of protein kinase or substrate in E. coli according to a standard protocol (see Note 9). 7. Measure protein concentration. 8. Prepare reaction mix on ice by mixing 0.1–1 μg protein kinase, 1 μg substrate proteins, 1 μL of 10× reaction buffer, and 0.5 μL of [γ-32P]-ATP. Then make up the volume to 10 μL with


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H2O. For multiple reactions, you can make a premix solution without proteins. 9. Incubate reaction mix at 30 °C for 30 min (see Note 10). 10. Take 10 μL aliquots and mix with 10 μL of 2× sample loading buffer. 11. Load 20 μL of the reaction mix onto an SDS-PAGE. 12. Wash the gel twice in 100 mL H2O at room temperature for 10 min. 13. (Optional) Incubate the gel in 100 mL 5 % glycerol at room temperature for 15 min. 14. Dry the gel. 15. Detect radioisotope activity by autoradiography.


Notes 1. It is necessary to optimize the composition of buffer depending on the samples. You must take into consideration tissue type(s), cellular localization, plant species, etc. 2. See Nakagami et al. [17]. 3. You can use any vector for expression in E. coli. 4. You can use any host strain optimized to your expression vector. 5. In the case of ABA treatment, pull out seedlings from agar plates. Do not damage roots or leaves. Place them on a new petri dish with 10 mL 1/2 GM solution overnight. Change the solution to 10 mL 1/2 GM solution containing 50 μM ABA. Collect seedlings after 0, 15, 30, and 90 min with three biological replicates for each time point. Remove water from plants, and put them rapidly into liquid nitrogen. Store at −80 °C. 6. In our case, we selected phosphopeptides which showed >3-fold upregulation in response to ABA, in at least two of the three replicates. 7. In our case, the second criteria was

Screening of kinase substrates using kinase knockout mutants.

Protein kinases are widely known to be major regulators of various signaling processes, particularly in eukaryotes, including plants. To understand th...
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