Tyrosine Autophosphorylation Research Articles

Cocrystallization of the Src-Family Kinase Hck with the ATP-Site Inhibitor A-419259 Stabilizes an Extended Activation Loop Conformation.

Hematopoietic cell kinase (Hck) is a member of the Src kinase family and is a promising drug target in myeloid leukemias. Here, we report the crystal structure of human Hck in complex with the pyrrolopyrimidine inhibitor A-419259, determined at a resolution of 1.8 Å. This structure reveals the complete Hck active site in the presence of A-419259, including the αC-helix, the DFG motif, and the activation loop. A-419259 binds at the ATP-site of Hck and induces an overall closed conformation of the kinase with the regulatory SH3 and SH2 domains bound intramolecularly to their respective internal ligands. A-419259 stabilizes the DFG-in/αC-helix-out conformation observed previously with Hck and the pyrazolopyrimidine inhibitor PP1 (PDB: 1QCF). However, the activation loop conformations are distinct, with PP1 inducing a folded loop structure with the tyrosine autophosphorylation site (Tyr416) pointing into the ATP binding site, while A-419259 stabilizes an extended loop conformation with Tyr416 facing out into the solvent. Autophosphorylation also induces activation loop extension and significantly reduces the Hck sensitivity to PP1 but not A-419259. In cancer cells where Hck is constitutively active, the extended autophosphorylation loop may render Hck more sensitive to inhibitors like A-419259 which prefer this kinase conformation. More generally, these results provide additional insight into targeted kinase inhibitor design and how conformational preferences of inhibitors may impact selectivity and potency.

Piezo1 activates noncanonical EGFR endocytosis and signaling.

EGFR-ERK signaling controls cell cycle progression during development, homeostasis, and disease. While EGF ligand and mechanical inputs can activate EGFR-ERK signaling, the molecules linking mechanical force to this axis have remained mysterious. We previously found that stretch promotes mitosis via the stretch-activated ion channel Piezo1 and ERK signaling. Here, we show that Piezo1 provides the missing link between mechanical signals and EGFR-ERK activation. While both EGF- and Piezo1-dependent activation trigger clathrin-mediated EGFR endocytosis and ERK activation, EGF relies on canonical tyrosine autophosphorylation, whereas Piezo1 involves Src-p38 kinase-dependent serine phosphorylation. In addition, unlike EGF, ex vivo lung slices treated with Piezo1 agonist promoted cell cycle re-entry via nuclear ERK, AP-1 (FOS and JUN), and YAP accumulation, typical of regenerative and malignant signaling. Our results suggest that mechanical activation via Piezo1, Src, and p38 may be more relevant to controlling repair, regeneration, and cancer growth than tyrosine kinase signaling via canonical EGF signaling, suggesting an alternative therapeutic approach.

Iruplinalkib (WX‑0593), a novel ALK/ROS1 inhibitor, overcomes crizotinib resistance in preclinical models for non-small cell lung cancer

Despite remarkable initial responses of anaplastic lymphoma kinase (ALK) inhibitors in ALK-positive non-small cell lung cancer (NSCLC) patients, cancers eventually develop resistance within one to two years. This study aimed to compare the properties of iruplinalkib (WX‑0593) with other ALK inhibitors and report the comprehensive characterization of iruplinalkib against the crizotinib resistance. The inhibitory effect of iruplinalkib on kinase activity was detected. A kinase screen was performed to evaluate the selectivity of iruplinalkib. The effect of iruplinalkib on related signal transduction pathways of ALK and c-ros oncogene 1 (ROS1) kinases was examined. The cellular and in vivo activities of ALK inhibitors were compared in engineered cancer-derived cell lines and in mice xenograft models, respectively. Human hepatocytes derived from three donors were used for evaluating hepatic enzyme inducing activity. HEK293 cell lines expressing transportors were used to invesigated the drug interaction potential mediated by several transporters. The results showed iruplinalkib potently inhibited the tyrosine autophosphorylation of wild-type ALK, ALKL1196M, ALKC1156Y and epidermal growth factor receptor (EGFR)L858R/T790M. The inhibitory effects of iruplinalkib in patient-derived xenograft and cell line-derived xenograft models were observed. Moreover, iruplinalkib showed robust antitumor effects in BALB/c nude mice xenograft models with ALK-/ROS1-positive tumors implanted subcutaneously, and the tumor suppressive effects in crizotinib-resistant model was significantly better than that of brigatinib. Iruplinalkib did not induce CYP1A2, CYP2B6 and CYP3A4 at therapeutic concentration, and was also a strong inhibitor of MATE1 and MATE2K transporters, as well as P-gp and BCRP. In conclusion, iruplinalkib, a highly active and selective ALK/ROS1 inhibitor, exhibited strong antitumor effects in vitro and in crizotinib-resistant models.

PIEZO1 activates non-canonical EGFR internalization and signaling.

EGFR-ERK signaling controls cell cycle progression during development, homeostasis, and disease. Both the soluble extracellular ligand, EGF, and mechanical inputs like matrix stiffness, cell adhesion, or stretch can activate EGFR-ERK signaling. However, the molecules transducing mechanical forces into EGFR signaling remain unidentified. We previously found that stretch promotes mitosis by mechanically activating the ion channel Piezo1 to trigger ERK signaling. Here, we show that Piezo1 provides the missing link to mechanical EGFR-ERK activation. Both EGF or the synthetic Piezo1 agonist Yoda1 triggered EGFR internalization and ERK activation, established markers of EGFR signaling. However, while EGF stimulation requires canonical EGFR tyrosine autophosphorylation, activation by Piezo1 requires non-canonical serine EGFR phosphorylation by a Src-p38 kinase axis. Additionally, Yoda1 stimulation of Piezo1 promoted cell cycle re-entry and proliferation via long-term nuclear accumulation of ERK, AP-1 (FOS and JUN), and YAP, typical of regenerative and malignant signaling. Our results suggest two modes of EGFR signaling: basal EGF-dependent signaling, requiring tyrosine autophosphorylation, and mechanically activated EGFR signaling, that results in sustained proliferation, critical to repair and regeneration, and cancer growth.

A first comprehensive look at the order-disorder nature of RTK KIT native and carcinogenic targets

Receptors Tyrosine Kinases (RTKs) act as sensors for extracellular ligands, the binding of which triggers dimerization, activation, and autophosphorylation of specific tyrosine (Y) residues in the Cytoplasmic Domain (CD). This leads to the recruitment and activation of multiple downstream signaling proteins, which regulate various aspects of cellular physiology.

Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interact with Wzd and Wze using bacterial two hybrid assays and fluorescence microscopy. We found that Wzd interacts with Wzg, the putative ligase that attaches capsule to the bacterial cell wall, and recruits it to the septal area. This interaction required residue V56 of Wzd and both the transmembrane regions and DNA-PPF domain of Wzg. When compared to the wild type, Wzd null pneumococci lack capsule at midcell, bind the peptidoglycan hydrolase LytA better and are more susceptible to LytA-induced lysis, and are less virulent in a zebrafish embryo infection model. In this manuscript, we propose that the Wzd/Wze pair guarantees full encapsulation of pneumococcal bacteria by recruiting Wzg to the division septum, ensuring that capsule attachment is coordinated with peptidoglycan synthesis. Impairing the encapsulation process, at localized subcellular sites, may facilitate elimination of bacteria by strategies that target the pneumococcal peptidoglycan.

SH2-Domain Protein Isolation Using Synthetic Phosphorylated Peptides to Study VEGFR2 Signaling.

Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathways are tightly regulated multistep chain reactions that involve a wide range of molecular interactions and enzymatic activities. The first signal induced by VEGF binding to VEGFR2, is the activation of the receptor tyrosine kinase and autophosphorylation of intracellular tyrosine residues of the receptor. In endothelial cells, five tyrosine residues in the VEGFR2 intracellular domain are essential in signal transmission and in the respective regulation of cellular processes. Because of their number and their localization on the receptor, it is challenging to locate the proteins with which these tyrosine residues interact that result in further downstream signaling cascades. In this chapter, we describe a method to precipitate phosphotyrosine binding proteins using phosphotyrosine-containing synthetic peptides immobilized to magnetic beads. The identity of the precipitated proteins is determined by mass spectrometry and the findings validated by Western blot. Using this method, we identified and verified two proteins, growth factor receptor binding-2 (GRB2) and phosphoinositide 3'-kinase (PI3Kp85), binding to the tyrosine 1214 of VEGFR2. Thereby, we can predict the signaling pathways downstream of pY1214.

Diabetic Kinome Inhibitors-A New Opportunity for β-Cells Restoration.

Diabetes, and several diseases related to diabetes, including cancer, cardiovascular diseases and neurological disorders, represent one of the major ongoing threats to human life, becoming a true pandemic of the 21st century. Current treatment strategies for diabetes mainly involve promoting β-cell differentiation, and one of the most widely studied targets for β-cell regeneration is DYRK1A kinase, a member of the DYRK family. DYRK1A has been characterized as a key regulator of cell growth, differentiation, and signal transduction in various organisms, while further roles and substrates are the subjects of extensive investigation. The targets of interest in this review are implicated in the regulation of β-cells through DYRK1A inhibition—through driving their transition from highly inefficient and death-prone populations into efficient and sufficient precursors of islet regeneration. Increasing evidence for the role of DYRK1A in diabetes progression and β-cell proliferation expands the potential for pharmaceutical applications of DYRK1A inhibitors. The variety of new compounds and binding modes, determined by crystal structure and in vitro studies, may lead to new strategies for diabetes treatment. This review provides recent insights into the initial self-activation of DYRK1A by tyrosine autophosphorylation. Moreover, the importance of developing novel DYRK1A inhibitors and their implications for the treatment of diabetes are thoroughly discussed. The evolving understanding of DYRK kinase structure and function and emerging high-throughput screening technologies have been described. As a final point of this work, we intend to promote the term “diabetic kinome” as part of scientific terminology to emphasize the role of the synergistic action of multiple kinases in governing the molecular processes that underlie this particular group of diseases.

Insulin Resistance the Hinge Between Hypertension and Type 2 Diabetes.

Epidemiological studies have documented a high incidence of diabetes in hypertensive patients.Insulin resistance is defined as a less than expected biologic response to a given concentration of the hormone and plays a pivotal role in the pathogenesis of diabetes. However, over the last decades, it became evident that insulin resistance is not merely a metabolic abnormality, but is a complex and multifaceted syndrome that can also affect blood pressure homeostasis. The dysregulation of neuro-humoral and neuro-immune systems is involved in the pathophysiology of both insulin resistance and hypertension. These mechanisms induce a chronic low grade of inflammation that interferes with insulin signalling transduction. Molecular abnormalities associated with insulin resistance include the defects of insulin receptor structure, number, binding affinity, and/or signalling capacity. For instance, hyperglycaemia impairs insulin signalling through the generation of reactive oxygen species, which abrogate insulin-induced tyrosine autophosphorylation of the insulin receptor. Additional mechanisms have been described as responsible for the inhibition of insulin signalling, including proteasome-mediated degradation of insulin receptor substrate 1/2, phosphatase-mediated dephosphorylation and kinase-mediated serine/threonine phosphorylation of both insulin receptor and insulin receptor substrates. Insulin resistance plays a key role also in the pathogenesis and progression of hypertension-induced target organ damage, like left ventricular hypertrophy, atherosclerosis and chronic kidney disease. Altogether these abnormalities significantly contribute to the increase the risk of developing type 2 diabetes.

Two-step release of kinase autoinhibition in discoidin domain receptor 1

Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase with important functions in organogenesis and tissue homeostasis. Aberrant DDR1 activity contributes to the progression of human diseases, including fibrosis and cancer. How DDR1 activity is regulated is poorly understood. We investigated the function of the long intracellular juxtamembrane (JM) region of human DDR1 and found that the kinase-proximal segment, JM4, is an important regulator of kinase activity. Crystal structure analysis revealed that JM4 forms a hairpin that penetrates the kinase active site, reinforcing autoinhibition by the activation loop. Using in vitro enzymology with soluble kinase constructs, we established that release from autoinhibition occurs in two distinct steps: rapid autophosphorylation of the JM4 tyrosines, Tyr569 and Tyr586, followed by slower autophosphorylation of activation loop tyrosines. Mutation of JM4 tyrosines abolished collagen-induced DDR1 activation in cells. The insights may be used to develop allosteric, DDR1-specific, kinase inhibitors.

Proline Hydroxylation Primes Protein Kinases for Autophosphorylation and Activation

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.

The Effects of Insulin Therapy on Fetuin-A Levels in Type-2 Diabetic Patients

Aim: Fetuin-A is a natural inhibitor of tyrosine kinase activity and autophosphorylation of the insulin receptor. It is thought that fetuin-A plays an essential role in adjusting postprandial glucose level plays, insulin sensitivity, weight gain, and fat accumulation. In this study, we investigated the effect of insulin use on fetuin-A level in patients with type-2 diabetes mellitus. Also compared fetuin-a levels between healthy individuals and type 2 diabetes patients. Material and Methods: The cross-sectional study was performed between May 2013 and July 2013. Sixty-nine patients with type 2 diabetes mellitus (37.7% oral antidiabetic agents and 62.3% insulin users) and 20 healthy individuals were included in the study. Diabetic group was divided into two subgroups as insulin group and oral antidiabetic agent group. We studied fetuin-A levels in these individuals. Additionally, we compared other biochemical parameters as ALT, AST, total cholesterol, LDL, HDL, VLDL, triglycerides, urea and creatinine in the diabetic group and control group as well diabetic year in subgroups of the diabetic group. Results: The mean age in the diabetic group was significantly higher than the control group (54.97 ± 6.13 and 49.95 ± 8.82, respectively, p=0.025). ALT levels in the diabetic group were higher than the control group (p=0.018), but there was no statically significant difference between the two groups in terms of other biochemical parameters (p>0.05). Fasting blood glucose and HbA1c levels in the insulin group were significantly higher than the oral antidiabetic agent group (p=0.004 and p<0.001, respectively). Additionally, the diabetic year in insulin group was significantly higher in the insulin group than the oral antidiabetic agent group (p<0.001). The mean fetuin-A level was 65.5 ± 27.8 ng/mL in the control group and 86.3 ± 26.1 ng/mL in the diabetic group, as well fetuin-A levels were significantly lower in the control group than the diabetic group (p=0.003). Besides, the mean fetuin-A level was 88.6 ± 23.3 ng/mL in the oral antidiabetic agent group and 84.8 ± 27.8 ng/mL in the insulin group, but it was not statistically significant (p=0.570). Conclusion: Fetuin-A level was significantly higher in the diabetic group, but there was no statistically significant difference between the oral antidiabetic agent group and the insulin group. Among the other biochemical parameters, only the ALT level was higher in the diabetic group than the control group, but this difference was not related to insulin therapy or oral antidiabetic agent. This is the first study to investigate the effect of treatment on fetuin-a levels in type-2 diabetic patients.

Transcriptome Analysis Identifies LINC00152 as a Biomarker of Early Relapse and Mortality in Acute Lymphoblastic Leukemia.

Evidence showing the role of long non-coding RNAs (lncRNAs) in leukemogenesis have emerged in the last decade. It has been proposed that these genes can be used as diagnosis and/or prognosis biomarkers in childhood acute lymphoblastic leukemia (ALL). To know if lncRNAs are associated with early relapse and early mortality, a microarray-based gene expression analysis in children with B-lineage ALL (B-ALL) was conducted. Cox regression analyses were performed. Hazard ratios (HR) and 95% confidence intervals (95% CI) were calculated. LINC00152 and LINC01013 were among the most differentially expressed genes in patients with early relapse and early mortality. For LINC00152 high expression, the risks of relapse and death were HR: 4.16 (95% CI: 1.46–11.86) and HR: 1.99 (95% CI: 0.66–6.02), respectively; for LINC01013 low expression, the risks of relapse and death were HR: 3.03 (95% CI: 1.14–8.05) and HR: 6.87 (95% CI: 1.50–31.48), respectively. These results were adjusted by NCI risk criteria and chemotherapy regimen. The lncRNA–mRNA co-expression analysis showed that LINC00152 potentially regulates genes involved in cell substrate adhesion and peptidyl–tyrosine autophosphorylation biological processes. The results of the present study point out that LINC00152 could be a potential biomarker of relapse in children with B-ALL.

Peptide-based NTA(Ni)-nanodiscs for studying membrane enhanced FGFR1 kinase activities.

Tyrosine autophosphorylation plays a crucial regulatory role in the kinase activities of fibroblast growth factor receptors (FGFRs), and in the recruitment and activation of downstream intracellular signaling pathways. Biophysical and biochemical investigations of FGFR kinase domains in membrane environments offer key insights into phosphorylation mechanisms. Hence, we constructed nickel chelating nanodiscs based on a 22-residue peptide. The spontaneous anchoring of N-terminal His6-tagged FGFR1c kinase domain (FGFR1K) onto peptide nanodiscs grants FGFR1K orientations occurring on native plasma membranes. Following membrane incorporation, the autophosphorylation of FGFR1K, as exemplified by Y653 and Y654 in the A-loop and the total tyrosine phosphorylation, increase significantly. This in vitro reconstitution system may be applicable to studies of other membrane associated phenomena.

HUMAN UMBILICAL CORD BLOOD DERIVED MESENCHYMAL STEM CELLS REDUCE PHOSPHORYLATION AND AGGREGATION OF TAU IN AN ALZHEIMER’S DISEASE MODEL

In Alzheimer's Disease (AD), administration of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) mitigate hyperphosphorylation and aggregation of tau as well as improve impairment of spatial learning and memory in AD model. hUCB-MSCs were isolated and expanded from umbilical cord blood. The 5xFAD model rapidly develops severe amyloid pathology and appears cognitive impairment at 4 to 6 months-of-age. All assays were qualified or quantified by thioflavin T assay, immunofluorescence, western blot analysis, enzyme-linked immunosorbent assays and T-maze test. hUCB-MSCs have inhibitory effect on tau aggregation through in vitro aggregation assays using K18 fragment of human tau. Remarkably, hUCB-MSCs specifically decreases autophosphorylation of tyrosine 216 on the GSK-3β residue, as a kinase to promote phosphorylation of tau. Moreover, hUCB-MSCs effectively decreased phosphorylated forms of Tau at Thr181, Thr231 and Ser404, whereas effects of total Tau proteins on its stability by hUCB-MSCs were not detected. Strikingly, these phenomena mentioned above lead to results that paired helical filaments (PHF) of tau in the brain of 5XFAD mice diminished by treatment of hUCB-MSCs. Additionally, in T-maze testing, administered mice support that hUCB-MSCs have behavioral improvement of memory tasks most likely to occur in AD model. hUCB-MSCs diminish the phosphorylation and aggregation of tau through regulation of hUCB-MSCs, thus elucidating a therapeutic mechanism for AD.

Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs.

Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the -3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif amplifiers of Plk1 signaling.

Negative feedback regulation of ErbB4 tyrosine kinase activity by ERK-mediated non-canonical phosphorylation

ErbB4 receptor tyrosine kinase has four different isoforms that are classified based on variants in the extracellular juxtamembrane domain (JM-a and JM-b) and the C-terminal region (CYT-1 and CYT-2). Here, we used the JM-b/CYT-1 isoform to investigate the roles of serine/threonine phosphorylation in MEK-ERK-dependent feedback inhibition. TPA as an activator of the ERK pathway markedly induced ErbB4 phosphorylation at Thr-674, the conserved common feedback site in the intracellular JM domain, which resulted in the downregulation of tyrosine autophosphorylation. We also identified Ser-1026 as an ErbB4-specific ERK target site in the CYT-1 region. Moreover, double mutations (Thr-674/Ser-1026 to Ala) significantly upregulated ErbB4 activation, indicating that Thr-674 and Ser-1026 are cooperatively involved in negative feedback regulation. Given the fact that ErbB4 mutation is one of the most common genetic alterations in melanoma cells, we demonstrated that a typical oncogenic ErbB4 mutant was resistant to the negative feedback regulation to maintain a highly active status of tyrosine kinase activity. Together, these findings indicate that feedback mechanisms are key switches determining oncogenic potentials of ErbB receptor kinases.

A high-throughput integrated microfluidics method enables tyrosine autophosphorylation discovery

Autophosphorylation of receptor and non-receptor tyrosine kinases is a common molecular switch with broad implications for pathogeneses and therapy of cancer and other human diseases. Technologies for large-scale discovery and analysis of autophosphorylation are limited by the inherent difficulty to distinguish between phosphorylation and autophosphorylation in vivo and by the complexity associated with functional assays of receptors kinases in vitro. Here, we report a method for the direct detection and analysis of tyrosine autophosphorylation using integrated microfluidics and freshly synthesized protein arrays. We demonstrate the efficacy of our platform in detecting autophosphorylation activity of soluble and transmembrane tyrosine kinases, and the dependency of in vitro autophosphorylation assays on membranes. Our method, Integrated Microfluidics for Autophosphorylation Discovery (IMAD), is high-throughput, requires low reaction volumes and can be applied in basic and translational research settings. To our knowledge, it is the first demonstration of posttranslational modification analysis of membrane protein arrays.

Four tyrosine residues of the rice immune receptor XA21 are not required for interaction with the co-receptor OsSERK2 or resistance to Xanthomonas oryzae pv. oryzae.

Tyrosine phosphorylation has emerged as an important regulator of plasma membrane-localized immune receptors activity. Here, we investigate the role of tyrosine phosphorylation in the regulation of rice XANTHOMONAS RESISTANCE 21 (XA21)-mediated immunity. We demonstrate that the juxtamembrane and kinase domain of Escherichia coli–expressed XA21 (XA21JK) autophosphorylates on tyrosine residues. Directed mutagenesis of four out of the nine tyrosine residues in XA21JK reduced autophosphorylation. These sites include Tyr698 in the juxtamembrane domain, and Tyr786, Tyr907, and Tyr909 in the kinase domain. Rice plants expressing XA21-GFP fusion proteins or proteins with these tyrosine residues individually mutated to phenylalanine (XA21YF-GFP), which prevents phosphorylation at these sites, maintain resistance to Xanthomonas oryzae pv. oryzae. In contrast, plants expressing phosphomimetic XA21 variants with tyrosine mutated to aspartate (XA21YD-GFP) were susceptible. In vitro purified XA21JKY698F, XA21JKY907F, and XA21JKY909F variants are catalytically active, whereas activity was not detected in XA21JKY768F and the four XA21JKYD variants. We previously demonstrated that interaction of XA21 with the co-receptor OsSERK2 is critical for biological function. Four of the XA21JKYF variants maintain interaction with OsSERK2 as well as the XA21 binding (XB) proteins XB3 and XB15 in yeast, suggesting that these four tyrosine residues are not required for their interaction. Taken together, these results suggest that XA21 is capable of tyrosine autophosphorylation, but the identified tyrosine residues are not required for activation of XA21-mediated immunity or interaction with predicted XA21 signaling proteins.

Identification of tyrosine autophosphorylation sites of Arabidopsis MEKK1 and their involvement in the regulation of kinase activity.

The MEKK1 kinase is a key regulator of stress signaling in Arabidopsis; however, little is known about the regulation of its kinase activity. Here, we found that recombinant MEKK1, expressed in both mammalian HEK293 cells and Escherichia coli, shows a mobility shift in SDS-PAGE, and immunoblotting detected phosphorylation of serine, threonine, and tyrosine residues. N-terminal deletions, site-directed mutagenesis, and protein phosphatase treatment revealed that the mobility shift results from autophosphorylation of the kinase domain. We identified the tyrosine autophosphorylation sites in the N-terminal region of MEKK1. Tyrosine to phenylalanine mutations decrease phosphorylation of the substrate MKK1, suggesting the important role of this residue in the regulation of MEKK1 kinase activity. The present study is the first to show that plant MAPKKKs are regulated by tyrosine phosphorylation.