Chapter 12 Plant Protein Kinase Substrates Identification Using Protein Microarrays Shisong Ma and Savithramma P. Dinesh-Kumar Abstract Protein kinases regulate signaling pathways by phosphorylating their targets. They play critical roles in plant signaling networks. Although many important protein kinases have been identified in plants, their substrates are largely unknown. We have developed and produced plant protein microarrays with more than 15,000 purified plant proteins. Here, we describe a detailed protocol to use these microarrays to identify plant protein kinase substrates via in vitro phosphorylation assays on these arrays. Key words Protein kinase, Protein microarray, Kinase substrates, In vitro phosphorylation, Protein expression
1
Introduction Protein kinases modify their substrate targets by adding a phosphate group to them, resulting in functional change of the target proteins. These kinases regulate many cellular signal transduction pathways by such phosphorylation events. In plants, the mitogenactivated protein kinase (MAPK) cascades modulate many stress responses and developmental pathways [1–3]. The receptor-like kinases (RLKs) function in organ formation, hormone signaling, and pathogen responses [4–6]. These and other protein kinases constitute a complex signaling system in plants. In the model plant Arabidopsis thaliana, the MAPK signaling system consists of 20 MAPKs, 20 MAPK-activating kinases (MKKs), and 60–80 predicted MKK-activating kinases (MKKKs) [7]. The RLKs belong to a gene family with more than 600 members [8]. The vast number of plant kinases, together with their functional redundancy and pleiotropy associated with many of their mutants, renders it very difficult to identify their substrate targets. Traditionally, kinase assays have been carried out with individually purified proteins in a low-throughput manner. The protein microarray platform facilitates identification of plant kinases
Waltraud X. Schulze (ed.), Plant Phosphoproteomics: Methods and Protocols, Methods in Molecular Biology, vol. 1306, DOI 10.1007/978-1-4939-2648-0_12, © Springer Science+Business Media New York 2015
159
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Shisong Ma and Savithramma P. Dinesh-Kumar
substrates in a high-throughput manner [7, 9]. These protein microarrays contain more than 10,000 purified proteins printed onto glass microarray slides as individual spots. On-slide in vitro kinase assays are conducted with purified active kinase and radiolabel gamma-33P (γ-33P) ATP. After phosphorylation reaction, γ-33Phosphate group will be transferred to the kinase substrate targets, which can be identified via exposure to X-ray films. We have developed a plant protein microarray platform that has been used to identify targets of Arabidopsis MAPKs [7]. Here, we provide a detailed protocol to use these protein microarrays to identify kinase substrates.
2
Materials
2.1 Reagents for Protein Microarray Probing and Analyses
1. Protein microarrays (stored at −80 °C). We have developed Arabidopsis protein microarrays with more than 10,000 proteins [7, 9, 10], in which Arabidopsis proteins were transiently expressed and purified from Nicotiana benthamiana leaves. Purified proteins were printed onto glass slides as duplicate spots. The slides are available from Arabidopsis Biological Resource Center (ABRC, Ohio, USA) (see Note 1). 2. Hybridization chamber—The chamber should maintain 100 % humidity and provide a level surface to place the protein microarray slides. A Pyrex rectangular storage dish (8 × 6 × 2 in.) is used as a hybridization chamber. Place a petri dish inside the storage dish at the center position. Place a glass plate (4 × 7 in.) on top of the petri dish and adjust its surface to level position. Up to four protein microarray slides can be placed on top of the glass plate for in vitro phosphorylation reaction. Add a small amount of water to the bottom of the storage dish and close the cap to maintain 100 % humidity inside the chamber. 3. Kinase buffer (KB): 25 mM Tris–HCl at pH 7.5, 1 mM EGTA, 10 mM MgCl2, 1 mM DTT. 4. Washing buffer (WB): 50 mM Tris–HCl pH 7.5, 0.5 % SDS. 5. [γ-33P] ATP (25 mCi/mL; Amersham Biosciences, Amersham, UK). 6. [γ-32P] ATP (25 Amersham, UK).
mCi/mL;
Amersham
Biosciences,
7. Bovine brain dephosphorylated MBP (Upstate Biotechnologies, Cell Signaling Solutions, Lake Placid, NY). 8. Dot-blot apparatus. 9. Microarray coverslip Waltham, MA).
(LifterSlips™,
Thermo
Scientific,
Plant Protein Kinase Substrates Identification Using Protein Microarrays
161
10. Kodak X-ray film. 11. High-resolution scanner. 12. GenePix 6.0 (Molecular Devices) or similar software. 2.2 Reagents for Transient Protein Expression
1. Agrobacterium tumefaciens strain GV2260. 2. Healthy 4- to 5-week-old Nicotiana benthamiana plants. 3. LIC6 plant expression vector (available at TAIR, http://www. arabidopsis.org/servlets/TairObject?type=vector&id=501 100131). 4. Vector to express the p19 protein, SPDK785. 5. Luria Broth (LB) media. 6. Carbenicillin (250 mg/mL); kanamycin (100 mg/mL); rifampicin (25 mg/mL); streptomycin (200 mg/mL); spectinomycin (50 mg/mL) stock solutions (see Note 2). 7. Infiltration medium (IM): 10 mM magnesium chloride (MgCl2), 10 mM 2-(N-morpholino) ethane (MES), 200 μM 3′,5′-dimethoxy 4′-hydroxy acetophenone (acetosyringone) (see Note 3). 8. 1-mL syringes. 9. Razor blades.
3
Methods
3.1 Generate, Express, and Purify Active Protein Kinase In Vivo
The following section describes expressing and purifying protein kinase using a TAP tag system [9, 11]. Alternatively, the readers can choose their favorite method to produce the active protein kinase. 1. Open reading frame (ORF) of plant protein kinase of interest is cloned into LIC6 expression vector using ligation-independent cloning method [9, 12]. The LIC6 vector is a binary vector for over-expressing plant proteins, driven by 35S promoter and containing a C-terminal TAP tag. 2. Transform the LIC6 with kinase of interest into A. tumefaciens GV2260 strain using chemical or electro competent cells. Select the transformants on SSRC (100 μg/mL spectinomycin, 100 μg/mL streptomycin, 25 μg/mL rifampicin, and 50 μg/mL carbenicillin) containing LB plates. Similarly, transform tomato bushy stunt virus p19 expressing construct into A. tumefaciens GV2260 strain, and select the transformants on KSRC (100 μg/mL kanamycin, 50 μg/mL streptomycin, 25 μg/mL rifampicin, and 50 μg/mL carbenicillin) containing LB plates.
162
Shisong Ma and Savithramma P. Dinesh-Kumar
3. Grow Agrobacterium GV2260 harboring the kinase or p19 construct overnight in LB media with SSRC or KSRC. Spin down the Agrobacterium culture, discard the supernatant, and suspend in infiltration media. Adjust the concentration of the kinase culture to OD600 0.6, and the culture of p19 to 1.0 with infiltration media. Mix equal volume of kinase and p19 cultures. Incubate the mixed culture at room temperature for at least 3 h (see Note 3). 4. Infiltrate 4-week-old N. benthamiana leaves with the mixed culture. Make a small cut on the abaxial side of the leaves using a razor blade. Using a needleless 1 mL syringe infiltrate kinase plus p19 containing Agrobacterium culture into the leaves. The culture should cover the entire leaf. Use ~6 leaves for each kinase. Keep infiltrated plants for 3–5 days in the growth chamber, harvest the leaves, and store samples at −80 °C. 5. Grind the leaf samples and purify the proteins using IgG beads. Refer to http://dinesh-kumarlab.genomecenter.ucdavis.edu/ for uploads/1/8/5/3/1853874/protein_purification.doc details on purifying proteins with TAP system. 3.2 In Vitro Kinase Assay to Evaluate Kinase Activity
1. Mix purified recombinant protein kinase with bovine brain dephosphorylated MBP (0.25 μg/μL per reaction), [γ-32P] ATP (0.2 mM per mL per one reaction), and incubate for 15 min at 30 °C. Similarly set up a negative control reaction with BSA instead of protein kinase (see Note 4). 2. Add 50 μL of PBS to each reaction and transfer the mixtures to nitrocellulose using a dot-blot apparatus. Wash the membrane for 30 min in PBS–Tween (0.1 %). Repeat washing three times. 3. Expose the membrane to Kodak, BioMax XAR film for various exposure times. Compare the signals from the negative control to make sure that the purified protein has kinase activity (see Notes 5 and 6).
3.3 In Vitro Phosphorylation Assay on Protein Microarrays
1. Take out the protein microarray from −80 °C and immediately place it into a 50 mL Falcon tube, close the cap tightly, and equilibrate the slide to 4 °C in a cold room for 15 min. This step prevents the formation of condensation on top of the microarray slide surface. 2. Mix ~500 ng of purified protein kinase in 200 μL of kinase buffer (KB) with [γ-33P] ATP (25 mCi/mL). A control experiment should also be performed without any kinase added, which will detect the auto-phosphorylation event of protein kinases that are on the protein microarray (see Note 7). 3. Pipette the kinase solution with [γ-33P] ATP onto the protein microarray, cover it with a microarray coverslip, and incubate for 30 min at 30 °C in a humidified hybridization chamber.
Plant Protein Kinase Substrates Identification Using Protein Microarrays
163
4. Wash the slides three times in washing buffer (WB), 30 min each, and one time in double-distilled water. Place the slides inside a 50 mL Falcon tube, and spin-dry at 700 rpm. 5. Cover the slides in Saran Wrap and expose it to X-ray Kodak film. The exposure time can vary between 12 and 96 h. Store the cassette in −80 °C during the exposure process (see Notes 6 and 7). 6. Develop and scan the film at 1,000 pixel per cm resolution. Save the acquired image (16 bit depth, gray scale) for data analysis and substrate identification. The black spots on the film correspond to the phosphorylated target protein spots on the slides (see Note 8). 3.4
Data Processing
1. The scanned image is imported in the GenePix 6.0 Pro software and aligned to a protein list file (.gal file provided with the ordered protein array) that contains the protein ID and their positions. The grid contains control auto-phosphorylation spots at the top left corner in each block of the protein microarray to facilitate alignment. The intensities of each protein (printed in duplicate) and its background signals are measured and recorded (see Note 9). 2. The intensities of each protein spots are normalized using an intensity-dependent normalization method after removing the positional control spots. After normalization, the median intensity value of each block and the median absolute deviation (MAD) value of all blocks are adjusted to the same levels. 3. A Bayesian decision method is employed to identify phosphorylated target substrates. A normal distribution ~N[μ,δ] was used to model the background noise level, where μ and δ are the mean noise level and its standard deviation, respectively. 4. The protein spots with intensity greater than two standard deviations above the background mean noise level were selected as putative targets. A protein will be identified as target substrate if both of its duplicated protein spots meet this requirement (see Note 8).
4
Notes 1. The microarray slide surface used to spot the proteins is important for the in vitro phosphorylation assays. The GAP (Corning Inc., Corning, NY) slides works better than other slides tested for in vitro kinase assays. 2. The stock solution for carbenicillin, kanamycin, streptomycin, spectinomycin should be prepared with water, while rifampicin is dissolved in dimethyl sulfoxide (DMSO). The stock solution
164
Shisong Ma and Savithramma P. Dinesh-Kumar
should be sterilized by passing through 0.2 μM filters and stored as 1 mL aliquot in −20 °C. 3. Acetosyringone stock solution (200 mM) should be made by dissolving Acetosyringone powder with 100 % ethanol and stored at −20 °C in aliquots. Acetosyringone is a natural woundinginduced plant chemical that enhances the transformation efficiency of Agrobacterium-mediated plant transformation [13]. To achieve such enhancement, after suspending Agrobacterium culture with the infiltration media, the Agrobacterium mixture should be incubated at room temperature for at least 3 h. During the incubation, Acetosyringone induces the expression of transformation related genes’ in Agrobacteria and render it hyperactive for transformation. The bacteria solution mixture can be stored overnight at room temperature before infiltration. 4. It is critical to use an active purified protein kinase for probing experiments. The amount of purified protein should be checked with western blot analyses. The purified proteins containing a C-terminal TAP tag with a 9×Myc can be detected with an anti-cMyc antibody. Once protein expression is confirmed, kinase activity should be confirmed as described in step 2. 5. The method described here uses two different type of radio labeled ATP. [γ-32P] ATP is used to check the kinase activity of the purified protein kinase. The more expensive [γ-33P] ATP is used to perform the in vitro kinase probing on protein microarrays. Compared to [γ-32P], the radiation from [γ-33P] has less energy and smaller effective angle of radiation scatter, resulting in smaller and sharper exposed spots on the x-ray films than [γ-32P]. The protein spots are printed on the slides at very high density, and smaller and sharper spot with [γ-33P] reduces the interference from neighboring spots. 6. Often the kinase activity of the in vitro assay is quite weak due to little amount of protein used in the assay. We recommend exposing the film up to 96 h to enhance the signal intensity. 7. The appearance of the auto-phosphorylated spot is a sign for the quality of the protein microarray slides and the kinase phosphorylation experimental setup. Appearance of autophosphorylated spot on the scanned film indicates successful probing experiment. 8. Since proteins are printed as duplicated spots on the slides, true positive should include signal from both spots. Signals resulting from only one spot are possible false positives. 9. The scanned image can be cropped with PHOTOSHOP software to reduce the size of the image.
Plant Protein Kinase Substrates Identification Using Protein Microarrays
165
Acknowledgement The protein microarray work in SPD-K lab was supported by National Science Foundation grants DBI-0723722 and DBI1042344 and currently by UC Davis funds. References 1. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983 2. Jonak C, Okresz L, Bogre L, Hirt H (2002) Complexity, cross talk and integration of plant MAP kinase signalling. Curr Opin Plant Biol 5:415–424 3. Wang HC, Ngwenyama N, Liu YD, Walker JC, Zhang SQ (2007) Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19:63–73 4. Nam KH, Li JM (2002) BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell 110:203–212 5. Jeong S, Trotochaud AE, Clark SE (1999) The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase. Plant Cell 11:1925–1933 6. Gomez-Gomez L, Boller T (2000) FLS2: An LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5:1003–1011 7. Popescu SC, Popescu GV, Bachan S, Zhang Z, Gerstein M, Snyder M, Dinesh-Kumar SP (2009) MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes Develop 23:80–92 8. Shiu SH, Bleecker AB (2003) Expansion of the receptor-like kinase/Pelle gene family and
9.
10.
11.
12.
13.
receptor-like proteins in Arabidopsis. Plant Physiol 132:530–543 Popescu SC, Popescu GV, Bachan S, Zhang Z, Seay M, Gerstein M, Snyder M, Dinesh-Kumar SP (2007) Differential binding of calmodulinrelated proteins to their targets revealed through high-density Arabidopsis protein microarrays. Proc Natl Acad Sci U S A 104:4730–4735 Lee HY, Bowen CH, Popescu GV, Kang HG, Kato N, Ma S, Dinesh-Kumar S, Snyder M, Popescu SC (2011) Arabidopsis RTNLB1 and RTNLB2 reticulon-like proteins regulate intracellular trafficking and activity of the FLS2 immune receptor. Plant Cell 23: 3374–3391 Rubio V, Shen Y, Saijo Y, Liu Y, Gusmaroli G, Dinesh-Kumar SP, Deng XW (2005) An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation. Plant J 41:767–778 Dong Y, Burch-Smith TM, Liu Y, Mamillapalli P, Dinesh-Kumar SP (2007) A ligationindependent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development. Plant Physiol 145: 1161–1170 Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8:291–298
1
Introduction Protein kinases modify their substrate targets by adding a phosphate group to them, resulting in functional change of the target proteins. These kinases regulate many cellular signal transduction pathways by such phosphorylation events. In plants, the mitogenactivated protein kinase (MAPK) cascades modulate many stress responses and developmental pathways [1–3]. The receptor-like kinases (RLKs) function in organ formation, hormone signaling, and pathogen responses [4–6]. These and other protein kinases constitute a complex signaling system in plants. In the model plant Arabidopsis thaliana, the MAPK signaling system consists of 20 MAPKs, 20 MAPK-activating kinases (MKKs), and 60–80 predicted MKK-activating kinases (MKKKs) [7]. The RLKs belong to a gene family with more than 600 members [8]. The vast number of plant kinases, together with their functional redundancy and pleiotropy associated with many of their mutants, renders it very difficult to identify their substrate targets. Traditionally, kinase assays have been carried out with individually purified proteins in a low-throughput manner. The protein microarray platform facilitates identification of plant kinases
Waltraud X. Schulze (ed.), Plant Phosphoproteomics: Methods and Protocols, Methods in Molecular Biology, vol. 1306, DOI 10.1007/978-1-4939-2648-0_12, © Springer Science+Business Media New York 2015
159
160
Shisong Ma and Savithramma P. Dinesh-Kumar
substrates in a high-throughput manner [7, 9]. These protein microarrays contain more than 10,000 purified proteins printed onto glass microarray slides as individual spots. On-slide in vitro kinase assays are conducted with purified active kinase and radiolabel gamma-33P (γ-33P) ATP. After phosphorylation reaction, γ-33Phosphate group will be transferred to the kinase substrate targets, which can be identified via exposure to X-ray films. We have developed a plant protein microarray platform that has been used to identify targets of Arabidopsis MAPKs [7]. Here, we provide a detailed protocol to use these protein microarrays to identify kinase substrates.
2
Materials
2.1 Reagents for Protein Microarray Probing and Analyses
1. Protein microarrays (stored at −80 °C). We have developed Arabidopsis protein microarrays with more than 10,000 proteins [7, 9, 10], in which Arabidopsis proteins were transiently expressed and purified from Nicotiana benthamiana leaves. Purified proteins were printed onto glass slides as duplicate spots. The slides are available from Arabidopsis Biological Resource Center (ABRC, Ohio, USA) (see Note 1). 2. Hybridization chamber—The chamber should maintain 100 % humidity and provide a level surface to place the protein microarray slides. A Pyrex rectangular storage dish (8 × 6 × 2 in.) is used as a hybridization chamber. Place a petri dish inside the storage dish at the center position. Place a glass plate (4 × 7 in.) on top of the petri dish and adjust its surface to level position. Up to four protein microarray slides can be placed on top of the glass plate for in vitro phosphorylation reaction. Add a small amount of water to the bottom of the storage dish and close the cap to maintain 100 % humidity inside the chamber. 3. Kinase buffer (KB): 25 mM Tris–HCl at pH 7.5, 1 mM EGTA, 10 mM MgCl2, 1 mM DTT. 4. Washing buffer (WB): 50 mM Tris–HCl pH 7.5, 0.5 % SDS. 5. [γ-33P] ATP (25 mCi/mL; Amersham Biosciences, Amersham, UK). 6. [γ-32P] ATP (25 Amersham, UK).
mCi/mL;
Amersham
Biosciences,
7. Bovine brain dephosphorylated MBP (Upstate Biotechnologies, Cell Signaling Solutions, Lake Placid, NY). 8. Dot-blot apparatus. 9. Microarray coverslip Waltham, MA).
(LifterSlips™,
Thermo
Scientific,
Plant Protein Kinase Substrates Identification Using Protein Microarrays
161
10. Kodak X-ray film. 11. High-resolution scanner. 12. GenePix 6.0 (Molecular Devices) or similar software. 2.2 Reagents for Transient Protein Expression
1. Agrobacterium tumefaciens strain GV2260. 2. Healthy 4- to 5-week-old Nicotiana benthamiana plants. 3. LIC6 plant expression vector (available at TAIR, http://www. arabidopsis.org/servlets/TairObject?type=vector&id=501 100131). 4. Vector to express the p19 protein, SPDK785. 5. Luria Broth (LB) media. 6. Carbenicillin (250 mg/mL); kanamycin (100 mg/mL); rifampicin (25 mg/mL); streptomycin (200 mg/mL); spectinomycin (50 mg/mL) stock solutions (see Note 2). 7. Infiltration medium (IM): 10 mM magnesium chloride (MgCl2), 10 mM 2-(N-morpholino) ethane (MES), 200 μM 3′,5′-dimethoxy 4′-hydroxy acetophenone (acetosyringone) (see Note 3). 8. 1-mL syringes. 9. Razor blades.
3
Methods
3.1 Generate, Express, and Purify Active Protein Kinase In Vivo
The following section describes expressing and purifying protein kinase using a TAP tag system [9, 11]. Alternatively, the readers can choose their favorite method to produce the active protein kinase. 1. Open reading frame (ORF) of plant protein kinase of interest is cloned into LIC6 expression vector using ligation-independent cloning method [9, 12]. The LIC6 vector is a binary vector for over-expressing plant proteins, driven by 35S promoter and containing a C-terminal TAP tag. 2. Transform the LIC6 with kinase of interest into A. tumefaciens GV2260 strain using chemical or electro competent cells. Select the transformants on SSRC (100 μg/mL spectinomycin, 100 μg/mL streptomycin, 25 μg/mL rifampicin, and 50 μg/mL carbenicillin) containing LB plates. Similarly, transform tomato bushy stunt virus p19 expressing construct into A. tumefaciens GV2260 strain, and select the transformants on KSRC (100 μg/mL kanamycin, 50 μg/mL streptomycin, 25 μg/mL rifampicin, and 50 μg/mL carbenicillin) containing LB plates.
162
Shisong Ma and Savithramma P. Dinesh-Kumar
3. Grow Agrobacterium GV2260 harboring the kinase or p19 construct overnight in LB media with SSRC or KSRC. Spin down the Agrobacterium culture, discard the supernatant, and suspend in infiltration media. Adjust the concentration of the kinase culture to OD600 0.6, and the culture of p19 to 1.0 with infiltration media. Mix equal volume of kinase and p19 cultures. Incubate the mixed culture at room temperature for at least 3 h (see Note 3). 4. Infiltrate 4-week-old N. benthamiana leaves with the mixed culture. Make a small cut on the abaxial side of the leaves using a razor blade. Using a needleless 1 mL syringe infiltrate kinase plus p19 containing Agrobacterium culture into the leaves. The culture should cover the entire leaf. Use ~6 leaves for each kinase. Keep infiltrated plants for 3–5 days in the growth chamber, harvest the leaves, and store samples at −80 °C. 5. Grind the leaf samples and purify the proteins using IgG beads. Refer to http://dinesh-kumarlab.genomecenter.ucdavis.edu/ for uploads/1/8/5/3/1853874/protein_purification.doc details on purifying proteins with TAP system. 3.2 In Vitro Kinase Assay to Evaluate Kinase Activity
1. Mix purified recombinant protein kinase with bovine brain dephosphorylated MBP (0.25 μg/μL per reaction), [γ-32P] ATP (0.2 mM per mL per one reaction), and incubate for 15 min at 30 °C. Similarly set up a negative control reaction with BSA instead of protein kinase (see Note 4). 2. Add 50 μL of PBS to each reaction and transfer the mixtures to nitrocellulose using a dot-blot apparatus. Wash the membrane for 30 min in PBS–Tween (0.1 %). Repeat washing three times. 3. Expose the membrane to Kodak, BioMax XAR film for various exposure times. Compare the signals from the negative control to make sure that the purified protein has kinase activity (see Notes 5 and 6).
3.3 In Vitro Phosphorylation Assay on Protein Microarrays
1. Take out the protein microarray from −80 °C and immediately place it into a 50 mL Falcon tube, close the cap tightly, and equilibrate the slide to 4 °C in a cold room for 15 min. This step prevents the formation of condensation on top of the microarray slide surface. 2. Mix ~500 ng of purified protein kinase in 200 μL of kinase buffer (KB) with [γ-33P] ATP (25 mCi/mL). A control experiment should also be performed without any kinase added, which will detect the auto-phosphorylation event of protein kinases that are on the protein microarray (see Note 7). 3. Pipette the kinase solution with [γ-33P] ATP onto the protein microarray, cover it with a microarray coverslip, and incubate for 30 min at 30 °C in a humidified hybridization chamber.
Plant Protein Kinase Substrates Identification Using Protein Microarrays
163
4. Wash the slides three times in washing buffer (WB), 30 min each, and one time in double-distilled water. Place the slides inside a 50 mL Falcon tube, and spin-dry at 700 rpm. 5. Cover the slides in Saran Wrap and expose it to X-ray Kodak film. The exposure time can vary between 12 and 96 h. Store the cassette in −80 °C during the exposure process (see Notes 6 and 7). 6. Develop and scan the film at 1,000 pixel per cm resolution. Save the acquired image (16 bit depth, gray scale) for data analysis and substrate identification. The black spots on the film correspond to the phosphorylated target protein spots on the slides (see Note 8). 3.4
Data Processing
1. The scanned image is imported in the GenePix 6.0 Pro software and aligned to a protein list file (.gal file provided with the ordered protein array) that contains the protein ID and their positions. The grid contains control auto-phosphorylation spots at the top left corner in each block of the protein microarray to facilitate alignment. The intensities of each protein (printed in duplicate) and its background signals are measured and recorded (see Note 9). 2. The intensities of each protein spots are normalized using an intensity-dependent normalization method after removing the positional control spots. After normalization, the median intensity value of each block and the median absolute deviation (MAD) value of all blocks are adjusted to the same levels. 3. A Bayesian decision method is employed to identify phosphorylated target substrates. A normal distribution ~N[μ,δ] was used to model the background noise level, where μ and δ are the mean noise level and its standard deviation, respectively. 4. The protein spots with intensity greater than two standard deviations above the background mean noise level were selected as putative targets. A protein will be identified as target substrate if both of its duplicated protein spots meet this requirement (see Note 8).
4
Notes 1. The microarray slide surface used to spot the proteins is important for the in vitro phosphorylation assays. The GAP (Corning Inc., Corning, NY) slides works better than other slides tested for in vitro kinase assays. 2. The stock solution for carbenicillin, kanamycin, streptomycin, spectinomycin should be prepared with water, while rifampicin is dissolved in dimethyl sulfoxide (DMSO). The stock solution
164
Shisong Ma and Savithramma P. Dinesh-Kumar
should be sterilized by passing through 0.2 μM filters and stored as 1 mL aliquot in −20 °C. 3. Acetosyringone stock solution (200 mM) should be made by dissolving Acetosyringone powder with 100 % ethanol and stored at −20 °C in aliquots. Acetosyringone is a natural woundinginduced plant chemical that enhances the transformation efficiency of Agrobacterium-mediated plant transformation [13]. To achieve such enhancement, after suspending Agrobacterium culture with the infiltration media, the Agrobacterium mixture should be incubated at room temperature for at least 3 h. During the incubation, Acetosyringone induces the expression of transformation related genes’ in Agrobacteria and render it hyperactive for transformation. The bacteria solution mixture can be stored overnight at room temperature before infiltration. 4. It is critical to use an active purified protein kinase for probing experiments. The amount of purified protein should be checked with western blot analyses. The purified proteins containing a C-terminal TAP tag with a 9×Myc can be detected with an anti-cMyc antibody. Once protein expression is confirmed, kinase activity should be confirmed as described in step 2. 5. The method described here uses two different type of radio labeled ATP. [γ-32P] ATP is used to check the kinase activity of the purified protein kinase. The more expensive [γ-33P] ATP is used to perform the in vitro kinase probing on protein microarrays. Compared to [γ-32P], the radiation from [γ-33P] has less energy and smaller effective angle of radiation scatter, resulting in smaller and sharper exposed spots on the x-ray films than [γ-32P]. The protein spots are printed on the slides at very high density, and smaller and sharper spot with [γ-33P] reduces the interference from neighboring spots. 6. Often the kinase activity of the in vitro assay is quite weak due to little amount of protein used in the assay. We recommend exposing the film up to 96 h to enhance the signal intensity. 7. The appearance of the auto-phosphorylated spot is a sign for the quality of the protein microarray slides and the kinase phosphorylation experimental setup. Appearance of autophosphorylated spot on the scanned film indicates successful probing experiment. 8. Since proteins are printed as duplicated spots on the slides, true positive should include signal from both spots. Signals resulting from only one spot are possible false positives. 9. The scanned image can be cropped with PHOTOSHOP software to reduce the size of the image.
Plant Protein Kinase Substrates Identification Using Protein Microarrays
165
Acknowledgement The protein microarray work in SPD-K lab was supported by National Science Foundation grants DBI-0723722 and DBI1042344 and currently by UC Davis funds. References 1. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983 2. Jonak C, Okresz L, Bogre L, Hirt H (2002) Complexity, cross talk and integration of plant MAP kinase signalling. Curr Opin Plant Biol 5:415–424 3. Wang HC, Ngwenyama N, Liu YD, Walker JC, Zhang SQ (2007) Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19:63–73 4. Nam KH, Li JM (2002) BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell 110:203–212 5. Jeong S, Trotochaud AE, Clark SE (1999) The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase. Plant Cell 11:1925–1933 6. Gomez-Gomez L, Boller T (2000) FLS2: An LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5:1003–1011 7. Popescu SC, Popescu GV, Bachan S, Zhang Z, Gerstein M, Snyder M, Dinesh-Kumar SP (2009) MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes Develop 23:80–92 8. Shiu SH, Bleecker AB (2003) Expansion of the receptor-like kinase/Pelle gene family and
9.
10.
11.
12.
13.
receptor-like proteins in Arabidopsis. Plant Physiol 132:530–543 Popescu SC, Popescu GV, Bachan S, Zhang Z, Seay M, Gerstein M, Snyder M, Dinesh-Kumar SP (2007) Differential binding of calmodulinrelated proteins to their targets revealed through high-density Arabidopsis protein microarrays. Proc Natl Acad Sci U S A 104:4730–4735 Lee HY, Bowen CH, Popescu GV, Kang HG, Kato N, Ma S, Dinesh-Kumar S, Snyder M, Popescu SC (2011) Arabidopsis RTNLB1 and RTNLB2 reticulon-like proteins regulate intracellular trafficking and activity of the FLS2 immune receptor. Plant Cell 23: 3374–3391 Rubio V, Shen Y, Saijo Y, Liu Y, Gusmaroli G, Dinesh-Kumar SP, Deng XW (2005) An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation. Plant J 41:767–778 Dong Y, Burch-Smith TM, Liu Y, Mamillapalli P, Dinesh-Kumar SP (2007) A ligationindependent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development. Plant Physiol 145: 1161–1170 Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8:291–298
