Arabidopsis thaliana Pattern Recognition Receptors for Bacterial Elongation Factor Tu and Flagellin Can Be Combined to Form Functional Chimeric Receptors

The receptor kinase EFR of Arabidopsis thaliana detects the microbe-associated molecular pattern elf18, a peptide that represents the N terminus of bacterial elongation factor Tu. Here, we tested subdomains of EFR for their importance in receptor function. Transient expression of tagged versions of EFR and EFR lacking its cytoplasmic domain in leaves of Nicotiana benthamiana resulted in functional binding sites for elf18. No binding of ligand was found with the ectodomain lacking the transmembrane domain or with EFR lacking the first 5 of its 21 leucine-rich repeats (LRRs). EFR is structurally related to the receptor kinase flagellin-sensing 2 (FLS2) that detects bacterial flagellin. Chimeric receptors with subdomains of FLS2 substituting for corresponding parts of EFR were tested for functionality in ligand binding and receptor activation assays. Substituting the transmembrane domain and the cytoplasmic domain resulted in a fully functional receptor for elf18. Replacing also the outer juxtamembrane domain with that of FLS2 led to a receptor with full affinity for elf18 but with a lower efficiency in response activation. Extending the substitution to encompass also the last two of the LRRs abolished binding and receptor activation. Substitution of the N terminus by the first six LRRs from FLS2 reduced binding affinity and strongly affected receptor activation. In summary, chimeric receptors allow mapping of subdomains relevant for ligand binding and receptor activation. The results also show that modular assembly of chimeras from different receptors can be used to form functional receptors.     

A Calcium/Calmodulin-regulated Member of the Receptor-like Kinase Family Confers Cold Tolerance in Plants

Cold is a limiting environmental factor that adversely affects plant growth and productivity. Calcium/calmodulin-mediated signaling is believed to play a pivotal role in plant response to cold stress, but its exact role is not clearly understood. Here, we report that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase, is crucial for cold tolerance in plants. CRLK1 has two calmodulin-binding sites with different affinities as follows: one located at residues 369–390 with a K_d of 25 nm, and the other located at residues 28–112 with a K_d of 160 nm. Calcium/calmodulin stimulated the kinase activity, but the addition of chlorpromazine, a calmodulin antagonist, blocked its stimulation. CRLK1 is mainly localized in the plasma membrane, and its expression is stimulated by cold and hydrogen peroxide treatments. Under normal growth conditions, there is no noticeable phenotypic difference between wild-type and crlk1 knock-out mutant plants. However, as compared with wild-type plants, the crlk1 knock-out mutants exhibited an increased sensitivity to chilling and freezing temperatures. Northern analysis showed that the induction of cold-responsive genes, including CBF1, RD29A, COR15a, and KIN1 in crlk1 mutants, is delayed as compared with wild-type plants. These results indicate that CRLK1 is a positive regulator of cold tolerance in plants. Furthermore, our results suggest that CRLK1 plays a role in bridging calcium/calmodulin signaling and cold signaling.     

Glycogen Synthase Kinase 3�� Interaction Protein Functions as an A-kinase Anchoring Protein

A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways.     

Reciprocal Negative Regulation of PDK1 and ASK1 Signaling by Direct Interaction and Phosphorylation

Cell survival and death-inducing signals are tightly associated with each other, and the decision as to whether a cell survives or dies is determined by controlling the relationship between these signals. However, the mechanism underlying the reciprocal regulation of such signals remains unclear. In this study, we reveal a functional association between PDK1 (3-phosphoinositide-dependent protein kinase 1), a critical mediator of cell survival, and ASK1 (apoptosis signal-regulating kinase 1), an apoptotic stress-activated MAPKKK. The physical association between PDK1 and ASK1 is mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1 and is decreased by ASK1-activating stimuli, such as H₂O₂, tumor necrosis factor α, thapsigargin, and ionomycin, as well as insulin, a PDK1 stimulator. Wild-type PDK1, but not kinase-dead PDK1, negatively regulates ASK1 activity by phosphorylating Ser⁹⁶⁷, a binding site for 14-3-3 protein, on ASK1. PDK1 functionally suppresses ASK1-mediated AP-1 transactivation and H₂O₂-mediated apoptosis in a kinase-dependent manner. On the other hand, ASK1 has been shown to inhibit PDK1 functions, including PDK1-mediated regulation of apoptosis and cell growth, by phosphorylating PDK1 at Ser³⁹⁴ and Ser³⁹⁸, indicating that these putative phosphorylation sites are involved in the negative regulation of PDK1 activity. These results provide evidence that PDK1 and ASK1 directly interact and phosphorylate each other and act as negative regulators of their respective kinases in resting cells.     

The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate K(m) of 7.5 μm and V(max) of 1800 nmol min(-1) mg(-1) of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg(2+) or Mn(2+). Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains.     

Direct Binding of a Plant LysM Receptor-like Kinase, LysM RLK1/CERK1, to Chitin in Vitro

Plants induce immune responses against fungal pathogens by recognition of chitin, which is a component of the fungal cell wall. Recent studies have revealed that LysM receptor-like kinase 1/chitin elicitor receptor kinase 1 (LysM RLK1/CERK1) is a critical component for the immune responses to chitin in Arabidopsis thaliana. However, the molecular mechanism of the chitin recognition by LysM RLK1 still remains unknown. Here, we present the first evidence for direct binding of LysM RLK1 to chitin. We expressed LysM RLK1 fused with yeast-enhanced green fluorescent protein (LysM RLK1-yEGFP) in yeast cells. Binding studies using the solubilized LysM RLK1-yEGFP and several insoluble polysaccharides having similar structures showed that LysM RLK1-yEGFP specifically binds to chitin. Subsequently, the fluorescence microscopic observation of the solubilized LysM RLK1-yEGFP binding to chitin beads revealed that the binding was saturable and had a high affinity, with a K_d of ∼82 nm. This binding was competed by the addition of soluble glycol chitin or high concentration of chitin oligosaccharides having 4–8 residues of N-acetyl glucosamine. However, the competition of these chitin oligosaccharides is weaker than that of glycol chitin. These data suggest that LysM RLK1 has a higher affinity for chitin having a longer residue of N-acetyl glucosamine. We also found that LysM RLK1-yEGFP was autophosphorylated in vitro and that chitin does not affect the phosphorylation of LysM RLK1-yEGFP. Our results provide a new dimension to chitin elicitor perception in plants.     

Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel

Ca²⁺ signals through store-operated Ca²⁺ (SOC) channels, activated by the depletion of Ca²⁺ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca²⁺ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca²⁺ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCβ both resulted in increased Ca²⁺ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.     

Down-regulation of S1P1 Receptor Surface Expression by Protein Kinase C Inhibition

The sphingosine 1-phosphate receptor type 1 (S1P₁) is important for the maintenance of lymphocyte circulation. S1P₁ receptor surface expression on lymphocytes is critical for their egress from thymus and lymph nodes. Premature activation-induced internalization of the S1P₁ receptor in lymphoid organs, mediated either by pharmacological agonists or by inhibition of the S1P degrading enzyme S1P-lyase, blocks lymphocyte egress and induces lymphopenia in blood and lymph. Regulation of S1P₁ receptor surface expression is therefore a promising way to control adaptive immunity. Hence, we analyzed potential cellular targets for their ability to alter S1P₁ receptor surface expression without stimulation. The initial observation that preincubation of mouse splenocytes with its natural analog sphingosine was sufficient to block Transwell^TM chemotaxis to S1P directed subsequent investigations to the underlying mechanism. Sphingosine is known to inhibit protein kinase C (PKC), and PKC inhibition with nanomolar concentrations of staurosporine, calphostin C, and GF109203X down-regulated surface expression of S1P₁ but not S1P₄ in transfected rat hepatoma HTC₄ cells. The PKC activator phorbol 12-myristate 13-acetate partially rescued FTY720-induced down-regulation of the S1P₁ receptor, linking PKC activation with S1P₁ receptor surface expression. FTY720, but not FTY720 phosphate, efficiently inhibited PKC. Cell-based efficacy was obvious with 10 nm FTY720, and in vivo treatment of mice with 0.3–3 mg/kg/day FTY720 showed increasing concentration-dependent effectiveness. PKC inhibition therefore may contribute to lymphopenia by down-regulating S1P₁ receptor cell surface expression independently from its activation.     

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Enhances Activation of p38 and ERK MAPKs,Increasing Interleukin-6 Synthesis in Airway Epithelial Cells Exposed to Pseudomonas aeruginosa

In cystic fibrosis (CF), the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRΔF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRΔF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRΔF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRΔF508 mutation in airway epithelial cells contributes to increase inflammation of the airways.     

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.     

A Minimal Cysteine Motif Required to Activate the SKOR K+ Channel of Arabidopsis by the Reactive Oxygen Species H2O2

Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca²⁺ and K⁺ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K⁺ channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H₂O₂. We show that H₂O₂ rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H₂O₂ and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H₂O₂-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys¹⁶⁸ located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H₂O₂. The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys¹⁶⁸ as a critical target for H₂O₂, and implicate ROS-mediated control of the K⁺ channel in regulating mineral nutrient partitioning within the plant.     

Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase

In metazoans, the highly conserved MAPK signaling pathway regulates cell fate decision. Aberrant activation of this pathway has been implicated in multiple human cancers and some developmental disorders. KSR1 functions as an essential scaffold that binds the individual components of the cascade and coordinates their assembly into multiprotein signaling platforms. The mechanism of KSR1 regulation is highly complex and not completely understood. In this study, we identified Tyr⁷²⁸ as a novel regulatory phosphorylation site in KSR1. We show that Tyr⁷²⁸ is phosphorylated by LCK, uncovering an additional and unexpected link between Src kinases and MAPK signaling. To understand how phosphorylation of Tyr⁷²⁸ may regulate the role of KSR1 in signal transduction, we integrated structural modeling and biochemical studies. We demonstrate that Tyr⁷²⁸ is involved in maintaining the conformation of the KSR1 kinase domain required for binding to MEK. It also affects phosphorylation and activation of MEK by RAF kinases and consequently influences cell proliferation. Moreover, our studies suggest that phosphorylation of Tyr⁷²⁸ may affect the intrinsic kinase activity of KSR1. Together, we propose that phosphorylation of Tyr⁷²⁸ may regulate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.     

Appl1 Is Dispensable for Mouse Development, and Loss of Appl1 Has Growth Factor-selective Effects on Akt Signaling in Murine Embryonic Fibroblasts

The adaptor protein APPL1 (adaptor protein containing pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motifs) was first identified as a binding protein of AKT2 by yeast two-hybrid screening. APPL1 was subsequently found to bind to several membrane-bound receptors and was implicated in their signal transduction through AKT and/or MAPK pathways. To determine the unambiguous role of Appl1 in vivo, we generated Appl1 knock-out mice. Here we report that Appl1 knock-out mice are viable and fertile. Appl1-null mice were born at expected Mendelian ratios, without obvious phenotypic abnormalities. Moreover, Akt activity in various fetal tissues was unchanged compared with that observed in wild-type littermates. Studies of isolated Appl1^−/− murine embryonic fibroblasts (MEFs) showed that Akt activation by epidermal growth factor, insulin, or fetal bovine serum was similar to that observed in wild-type MEFs, although Akt activation by HGF was diminished in Appl1^−/− MEFs. To rule out a possible redundant role played by the related Appl2, we used small interfering RNA to knock down Appl2 expression in Appl1^−/− MEFs. Unexpectedly, cell survival was unaffected under normal culture conditions, and activation of Akt was unaltered following epidermal growth factor stimulation, although Akt activity did decrease further after HGF stimulation. Furthermore, we found that Appl proteins are required for HGF-induced cell survival and migration via activation of Akt. Our studies suggest that Appl1 is dispensable for development and only participate in Akt signaling under certain conditions.     

Requirement of Calcium Binding,Myristoylation, and Protein-Protein Interaction for the Copine BON1 Function in Arabidopsis

Copines are highly conserved proteins with lipid-binding activities found in animals, plants, and protists. They contain two calcium-dependent phospholipid binding C2 domains at the amino terminus and a VWA domain at the carboxyl terminus. The biological roles of most copines are not understood and the biochemical properties required for their functions are largely unknown. The Arabidopsis copine gene BON1/CPN1 is a negative regulator of cell death and defense responses. Here we probed the potential biochemical activities of BON1 through mutagenic studies. We found that mutations of aspartates in the C2 domains did not alter plasma membrane localization but compromised BON1 activity. Mutation at putative myristoylation residue glycine 2 altered plasma membrane localization of BON1 and rendered BON1 inactive. Mass spectrometry analysis of BON1 further suggests that the N-peptide of BON1 is modified. Furthermore, mutations that affect the interaction between BON1 and its functional partner BAP1 abolished BON1 function. This analysis reveals an unanticipated regulation of copine protein localization and function by calcium and lipid modification and suggests an important role in protein-protein interaction for the VWA domain of copines.     

LKB1 Suppresses p21-activated Kinase-1 (PAK1) by Phosphorylation of Thr109 in the p21-binding Domain

The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr¹⁰⁹ in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr¹⁰⁹ by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/−) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.     

Regulation of the Tyrosine Kinase Pyk2 by Calcium Is through Production of Reactive Oxygen Species in Cytotoxic T Lymphocytes

Pyk2 was identified as a Ca²⁺-dependent kinase, however, the regulation of Pyk2 by Ca²⁺ in T cells remains controversial. We found that Ca²⁺ mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca²⁺ dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the calmodulin inhibitor W7, and we detected no Ca²⁺-regulated association between Pyk2 and calmodulin. Ca²⁺-stimulated Pyk2 phosphorylation was dependent on Src-family kinase activity, even at the Pyk2 autophosphorylation site. We sought to identify a Ca²⁺-regulated pathway that could trigger Pyk2 phosphorylation in T cells and found that ionomycin stimulated the production of reactive oxygen species and an H₂O₂ scavenger inhibited ionomycin-induced Pyk2 phosphorylation. Additionally, H₂O₂ induced strong Erk activation and ionomycin-stimulated Pyk2 phosphorylation was Erk dependent. These data support the conclusion that Ca²⁺ mobilization induces the production of reactive oxygen species, which in turn activate the Erk pathway, leading to Src-family kinase-dependent Pyk2 phosphorylation. Our data demonstrate that Pyk2 is not a Ca²⁺-dependent kinase in T cells but instead, increased intracellular Ca²⁺ induces Pyk2 phosphorylation through production of reactive oxygen species. These findings are consistent with the possibility that Pyk2 acts as an early sensor of numerous extracellular signals that trigger a Ca²⁺ flux and/or reactive oxygen species to amplify tyrosine phosphorylation signaling events.     

Crystal Structure of the cGMP-dependent Protein Kinase II Leucine Zipper and Rab11b Protein Complex Reveals Molecular Details of G-kinase-specific Interactions

cGMP-dependent protein kinase (PKG)-interacting proteins (GKIPs) mediate cellular targeting of PKG isoforms by interacting with their leucine zipper (LZ) domains. These interactions prevent aberrant signaling cross-talk between different PKG isotypes. To gain detailed insight into isotype-specific GKIP recognition by PKG, we analyzed the type II PKG leucine zipper domain and found that residues 40–83 dimerized and specifically interacted with Rab11b. Next, we determined a crystal structure of the PKG II LZ-Rab11b complex. The PKG II LZ domain presents a mostly nonpolar surface onto which Rab11b docks, through van der Waals interactions. Contact surfaces in Rab11b are found in switch I and II, interswitch, and the β1/N-terminal regions. This binding surface dramatically differs from that seen in the Rab11 family of interacting protein complex structures. Structural comparison with PKG Iα and Iβ LZs combined with mutagenic analysis reveals that GKIP recognition is mediated through surface charge interactions.     

Major Role of Epidermal Growth Factor Receptor and Src Kinases in Promoting Oxidative Stress-dependent Loss of Adhesion and Apoptosis in Epithelial Cells

A growing body of evidence suggests that reactive oxygen species are critical components of cell signaling pathways, in particular regulating protein phosphorylation events. Here, we show that oxidative stress in response to hydrogen peroxide treatment of human epithelial cells induces robust tyrosine phosphorylation on multiple proteins. Using an anti-phosphotyrosine purification and liquid chromatography-tandem mass spectrometry approach, we have identified many of these H₂O₂-induced tyrosine-phosphorylated proteins. Importantly, we show that epidermal growth factor receptor (EGFR) and Src are the primary upstream kinases mediating these events through their redox activation. The finding that many of the identified proteins have functions in cell adhesion, cell-cell junctions, and the actin cytoskeleton prompted us to examine stress-induced changes in adhesion. Immunofluorescence analysis showed that H₂O₂ alters cell adhesion structures and the actin cytoskeleton causing loss of adhesion and apoptosis. Remarkably, these cellular changes could be attenuated by inhibition of EGFR and Src, identifying these kinases as targets to block oxidative damage. In summary, our data demonstrate that EGFR and Src together play a central role in oxidative stress-induced phosphorylation, which in turn results in loss of adhesion, morphological changes, and cell damage in epithelial cells. These data also provide a general model for redox signaling in other cell systems.