Journal: Molecular & Cellular Proteomics : MCP
Article Title: Identifying Receptor Kinase Substrates Using an 8000 Peptide Kinase Client Library Enriched for Conserved Phosphorylation Sites
doi: 10.1016/j.mcpro.2025.100926
Figure Lengend Snippet: ILK4, which was identified through the KiC assay with the 2k peptide library, interacts with and is phosphorylated by P2K1 kinase domain. A , subcellular localization of ILK4 in transgenic plants expressing a 35S:ILK4 - YFP construct. Fluorescent confocal images displaying the subcellular distribution of ILK4-YFP protein were detected from primary root tissue of seven-day-old seedlings. Plasma membrane was counter-stained by incubation for 1 min in a FM4-64 solution (5 μM). P2K1-YFP and free-YFP were used as controls. B , the split-luciferase assay provides evidence that P2K1 interacts with ILK4. The emitted luminescence from the leaves was captured using a low-light imaging CCD camera (Photek; Photek, Ltd). Dotted circles indicate the regions of interest in N. benthamiana leaves corresponding to the infiltrated areas. ILK5-cLUC and MKK3-cLUC proteins served as positive and negative controls, respectively. C , the signal intensities of the P2K1–ILK4 interaction were quantified. Captured images were analyzed for luciferase signal intensities using C-vision/Im32 software, and further data analysis was performed with GraphPad Prism (version 8). The results are presented as the mean ± SEM of eight biological replicates. Statistical significance was determined by unpaired two-tailed Student’s t test, with significance levels denoted as follows: ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05. D , coimmunoprecipitation (Co-IP) was carried out to investigate the interaction between P2K1 and ILK4 proteins in N. benthamiana leaves. Total protein was utilized for the Co-IP assay with anti-HA and anti-Myc antibodies. ILK5-Myc was employed as a positive control. E , a BiFC assay was conducted in Arabidopsis protoplasts. FM4-64 was employed as a plasma membrane marker, and the chlorophyll autofluorescence signal (Chl) was also observed. The merged image (Merge) represents the overlapping fluorescence signals from YFP and FM4-64. RBOHD-cYFP and ILK6-cYFP were used as positive and negative controls, respectively. Scale bars represent 10 μm. F , GST-P2K1-CD directly phosphorylates ILK4-His, whereas GST-LYK5-CD is not phosphorylated in vitro . In an in vitro kinase assay, bacterial recombinant ILK4-His protein was incubated with GST-P2K1-CD, GST-P2K1 D572N -CD (a kinase-dead version), or GST. G , a schematic diagram of ILK4 depicts its structural features, including the ankyrin repeat (ANK) and serine-threonine or tyrosine kinase (S-T/Y_kinase) domains. The identified phosphopeptide RPVVT(p)CLDS(p)S(p)WRY(p)MAP is situated within the kinase domain. H , mutation of ILK4 at the residues T326, S330, S331, and Y334 resulted in diminished phosphorylation by P2K1 in vitro . The in vitro kinase assay involved the incubation of purified GST or GST-P2K1-CD recombinant proteins with ILK4 WT -His or ILK4 quadruple A -His. I , quantification of phosphorylated ILK4 WT and ILK4 quadruple A proteins was performed. Phosphorylation signal intensities were measured and analyzed using ImageJ and the GraphPad Prism 8 software. The data is presented as mean ± SEM (n = 4) based on measurements from four independent experiments. Significance levels are represented as ∗∗ p < 0.01, with the p value indicating the significance concerning the band intensity of ILK4 WT -His, determined through unpaired two-tailed Student’s t test. In panels ( E and G ), the presence of autophosphorylation and transphosphorylation events was identified by the incorporation of [γ- 32 P] ATP. Asterisk (∗) represents the nonspecific protein. Myelin basic protein (MBP) was used as a universal substrate, while GST-LYK5-CD was used as a negative control. Protein loading was visualized through Coomassie Brilliant Blue (CBB) staining. J , ILK4 phosphorylation is significantly increased in P2K1 -overexpressing plants. ILK4-HA protein was expressed in WT and P2K1 -overexpressing plants protoplasts with/without 250 μM ATP treatment and then subjected to immunoprecipitation and IB using anti-HA and anti-phospho-Ser/Thr antibodies. Protein loading was visualized by CBB staining. All of the above experiments were repeated two times (biological replicates) with similar results. BiFC, bimolecular fluorescence complementation; CD, cytosolic domain; GST, glutathione- S -transferase; IB, immunoblotting; ILK, integrin-linked kinase; KiC, kinase client; RBOHD, respiratory burst oxidase homolog D.
Article Snippet: The luminescence was monitored and captured using a low-light imaging CCD camera (Photek; Photek, Ltd).
Techniques: Transgenic Assay, Expressing, Construct, Clinical Proteomics, Membrane, Staining, Incubation, Luciferase, Imaging, Software, Two Tailed Test, Co-Immunoprecipitation Assay, Positive Control, Bimolecular Fluorescence Complementation Assay, Marker, Fluorescence, In Vitro, Kinase Assay, Recombinant, Phospho-proteomics, Mutagenesis, Purification, Negative Control, Immunoprecipitation, Western Blot
![ILK4, which was identified through the KiC assay with the 2k peptide library, interacts with and is phosphorylated by P2K1 kinase domain. A , subcellular localization of ILK4 in transgenic plants expressing a 35S:ILK4 - YFP construct. Fluorescent confocal images displaying the subcellular distribution of ILK4-YFP protein were detected from primary root tissue of seven-day-old seedlings. Plasma membrane was counter-stained by incubation for 1 min in a FM4-64 solution (5 μM). P2K1-YFP and free-YFP were used as controls. B , the split-luciferase assay provides evidence that P2K1 interacts with ILK4. The emitted luminescence from the leaves was captured using a low-light imaging <t>CCD</t> <t>camera</t> (Photek; Photek, Ltd). Dotted circles indicate the regions of interest in N. benthamiana leaves corresponding to the infiltrated areas. ILK5-cLUC and MKK3-cLUC proteins served as positive and negative controls, respectively. C , the signal intensities of the P2K1–ILK4 interaction were quantified. Captured images were analyzed for luciferase signal intensities using C-vision/Im32 software, and further data analysis was performed with GraphPad Prism (version 8). The results are presented as the mean ± SEM of eight biological replicates. Statistical significance was determined by unpaired two-tailed Student’s t test, with significance levels denoted as follows: ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05. D , coimmunoprecipitation (Co-IP) was carried out to investigate the interaction between P2K1 and ILK4 proteins in N. benthamiana leaves. Total protein was utilized for the Co-IP assay with anti-HA and anti-Myc antibodies. ILK5-Myc was employed as a positive control. E , a BiFC assay was conducted in Arabidopsis protoplasts. FM4-64 was employed as a plasma membrane marker, and the chlorophyll autofluorescence signal (Chl) was also observed. The merged image (Merge) represents the overlapping fluorescence signals from YFP and FM4-64. RBOHD-cYFP and ILK6-cYFP were used as positive and negative controls, respectively. Scale bars represent 10 μm. F , GST-P2K1-CD directly phosphorylates ILK4-His, whereas GST-LYK5-CD is not phosphorylated in vitro . In an in vitro kinase assay, bacterial recombinant ILK4-His protein was incubated with GST-P2K1-CD, GST-P2K1 D572N -CD (a kinase-dead version), or GST. G , a schematic diagram of ILK4 depicts its structural features, including the ankyrin repeat (ANK) and serine-threonine or tyrosine kinase (S-T/Y_kinase) domains. The identified phosphopeptide RPVVT(p)CLDS(p)S(p)WRY(p)MAP is situated within the kinase domain. H , mutation of ILK4 at the residues T326, S330, S331, and Y334 resulted in diminished phosphorylation by P2K1 in vitro . The in vitro kinase assay involved the incubation of purified GST or GST-P2K1-CD recombinant proteins with ILK4 WT -His or ILK4 quadruple A -His. I , quantification of phosphorylated ILK4 WT and ILK4 quadruple A proteins was performed. Phosphorylation signal intensities were measured and analyzed using ImageJ and the GraphPad Prism 8 software. The data is presented as mean ± SEM (n = 4) based on measurements from four independent experiments. Significance levels are represented as ∗∗ p < 0.01, with the p value indicating the significance concerning the band intensity of ILK4 WT -His, determined through unpaired two-tailed Student’s t test. In panels ( E and G ), the presence of autophosphorylation and transphosphorylation events was identified by the incorporation of [γ- 32 P] ATP. Asterisk (∗) represents the nonspecific protein. Myelin basic protein (MBP) was used as a universal substrate, while GST-LYK5-CD was used as a negative control. Protein loading was visualized through Coomassie Brilliant Blue (CBB) staining. J , ILK4 phosphorylation is significantly increased in P2K1 -overexpressing plants. ILK4-HA protein was expressed in WT and P2K1 -overexpressing plants protoplasts with/without 250 μM ATP treatment and then subjected to immunoprecipitation and IB using anti-HA and anti-phospho-Ser/Thr antibodies. Protein loading was visualized by CBB staining. All of the above experiments were repeated two times (biological replicates) with similar results. BiFC, bimolecular fluorescence complementation; CD, cytosolic domain; GST, glutathione- S -transferase; IB, immunoblotting; ILK, integrin-linked kinase; KiC, kinase client; RBOHD, respiratory burst oxidase homolog D.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2801/pmc11952801/pmc11952801__gr1.jpg)