Impact of MET targeting on tumor-associated angiogenesis and growth of MET mutations-driven models of liver cancer

Deregulated expression of the MET receptor tyrosine kinase has been reported in up to 50% of patients with hepatocellular carcinoma, the most abundant form of liver cancers, and is associated with decreased survival. Consequently, MET is considered as a molecular target in this malignancy, whose progression is highly dependent on extensive angiogenesis. Here we studied the impact of MET small molecule inhibitors on angiogenesis-associated parameters and growth of xenograft liver models consisting of cells expressing MET-mutated variants M1268T and Y1248H, which exhibit constitutive kinase activity. We demonstrate that MET mutations expression is associated with significantly increased production of vascular endothelial growth factor, which is blocked by MET targeting only in cells expressing the M1268T inhibitor-sensitive but not in the Y1248H inhibitor-resistant variant. Decrease in vascular endothelial growth factor production is also associated with reduction of tyrosine phopshorylation of the vascular endothelial growth factor receptor 2 expressed on primary liver sinusoidal endothelial cells and with inhibition of vessel formation. Furthermore, MET inhibition demonstrated an efficient anti-tumor activity and considerable reduction in microvessel density only against the M1268T-derived intrahepatic tumors. Collectively, our data support the role of targeting MET-associated angiogenesis as a major biological determinant for liver tumor growth control.

Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes

BACKGROUND Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues. RESULTS HERE, WE USE AN ASSAY THAT ALLOWS TO BIOCHEMICALLY PURIFY EXTENDING PROTRUSIONS OF CELLS MIGRATING IN RESPONSE TO THREE PROTOTYPICAL RECEPTORS: integrins, recepor tyrosine kinases and G-coupled protein receptors. Using quantitative proteomics and phospho-proteomics approaches, we provide evidence for the existence of cue-specific, spatially distinct protein networks in the different cell migration modes. CONCLUSIONS The integrated analysis of the large-scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration. This provides the cell migration community with a large-scale view of the distribution of proteins and phospho-proteins regulating directed cell migration.

Novel mechanisms for PKC family enzymes to regulate PI3K signaling pathway

Phosphatidylinositol 3-kinase (PI3K) generates membrane phospholipids that serve as second messengers to recruit signaling proteins to plasma membrane consequently regulating cell growth and survival. PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit. Association of the p85 with other signal proteins is critical for induced PI3K activation. Activated PI3K, in turn, leads to signal flows through a variety of PI3K effectors including PDK1, AKT, GSK3, BAD, p70 S6K and NFκB. The PI3K pathway is under regulation by multiple signal proteins representing cross-talk between different signaling cascades. In this study, we have evaluated the role of protein kinase C family kinases on signaling through PI3K at multiple levels. Firstly, we observed that the action of PKC specific inhibitors like Ro-31-8220 and GF109203X was associated with an increased AKT phosphorylation and activity, suggesting that PKC kinases might play a negative role in the regulation of PI3K pathway. Then, we demonstrated the stimulation of AKT by PKC inhibition was dependent on functional PI3K enzyme and able to be transmitted to the AKT effector p70 S6K. Furthermore, we showed an inducible physical association between the PKCζ isotype and AKT, which was accompanied by an attenuated AKT activity. However, a kinase-dead form of PKC failed to affect AKT. In the second part of our research we revealed the ability of a different PKC family member, PKCδ to bind to the p85 subunit of PI3K in response to oxidative stress, a process requiring the activity of src tyrosine kinases. The interaction was demonstrated to be a direct and specific contact between the carboxyl terminal SH2 domain of p85 and tyrosine phosphorylated PKCδ. Several different types of agonists were capable to induce this association including tyrosine kinases and phorbol esters with PKCδ tyrosine phosphorylation being integral components. Finally, the PKCδ-PI3K complex was related to a reduction in the AKT phosphorylation induced by src. A kinase-deficient mutant of PKCδ was equally able to inhibit AKT signal as the wild type, indicative of a process independent of PKCδ catalytic activity. Altogether, our data illustrate different PKC isoforms regulating PI3K pathway at multiple levels, suggesting a mechanism to control signal flows through PI3K for normal cell activities. Although further investigation is required for full understanding of the regulatory mechanism, we propose that complex formation of signal proteins in PI3K pathway and specific PKC isoforms plays important role in their functional linkage. ^

Transcriptional upregulation of the p21Cip1 promoter by STAT3 and nuclear ErbB2

Over-expression of the receptor tyrosine kinase ErbB2 is prevalent in approximately 30% of human breast carcinomas and confers Taxol resistance. In breast cancer cells, Taxol induces tubulin polymerization and hyperstable microtubule formation. This in turn prematurely activates Cdc2 kinase allowing early entry into the G2/M phase of the cell cycle resultant in mitotic catastrophe followed by apoptosis. Over-expression of ErbB2 upregulates p21Cip1, which inhibits Cdc2 activation, and leads to Taxol resistance in patients. However, the mechanism of ErbB2-mediated p21 Cip1 upregulation is unclear. Here in this study, we investigated the mechanism of ErbB2 downstream signaling events leading to upregulation. The CDKN1A (p21Cip1) gene promoter contains numerous cis-elements including a Signal transducer and activator of transcription (STAT) Inducable Element (SIE) located at -679 kb. Our studies showed ErbB2 overexpressing cells had increased activated levels of STAT3, and therefore we hypothesized that STAT3 is responsible for the upregulation of the p21Cip1 promoter by ErbB2. EMSA and ChIP assays confirmed the binding of STAT3 to the p21Cip1 promoter and luciferase assays showed higher p21 Cip1 promoter activity in ErbB2 over-expressing transfectants when compared to parental cells, in a STAT3 binding site dependant manner. Additionally, reduced level of STAT3 led to reduced p21Cip1 protein expression and promoter activity indicating that both the STAT3 binding site and STAT3 protein are required for ErbB2-mediated p21Cip1 upregulation. Further investigation of ErbB2 downstream signaling showed increased Src kinase activity in ErbB2 over-expressing cells which was required for ErbB2-mediated STAT3 activation and p21Cip1 increase. Treatment of ErbB2 over-expressing resistant cells with STAT3 inhibitor peptides sensitized the cells to Taxol. In addition to classical signal transduction pathways, I identified a novel ErbB2 mediated regulatory mechanism of p21Cip1. I found that a nuclear ErbB2 and STAT3 complex binds directly to the p21Cip1 promoter offering a non-classical mechanism of p21Cip1 promoter regulation. These data suggest that ErbB2 over-expression can confer Taxol resistance of breast cancer cells by transcriptional upregulation of p21 Cip1 via activation of STAT3 by Src kinase and also by cooperation with nuclear ErbB2. The data suggest a potential clinical mechanism for STAT3 inhibitors in sensitizing ErbB2 over-expressing breast cancers to Taxol. ^

EphA2 expression is regulated by EGFR and KRAS and promotes non-small cell lung cancer progression

Lung cancer is the leading cause of cancer deaths worldwide. The development of improved systemic therapy is needed for the most common form of the disease, non-small cell lung cancer (NSCLC). This will depend on the identification of valid molecular targets. Recent studies point to the receptor tyrosine kinase EphA2 as a novel therapeutic target. Overexpression of EphA2 has been demonstrated in a number of epithelial cancers, and its expression has been associated with more severe disease. Regulation of EphA2 in cancer is poorly understood. Recently, regulation of EphA2 by EGFR and KRAS has been reported in a number of in vitro models, but no examination of this relationship has been undertaken in patient tumors. Because of the established importance of EGFR and KRAS in NSCLC, we have investigated the relationship between these mutations and EphA2 in NSCLC patient tissues and cell lines. The significance of Epha2 expression was further examined by testing for correlation with survival, metastases, histology, and smoking status in patient tissues, and tumor cell proliferation and migration in vitro. EphA2 expression was analyzed in by immunohistochemistry in tissue microarray (TMA) format utilizing surgically resected lung cancer specimens. EGFR and KRAS mutation status was determined for the majority of specimens. EphA2 expression was detected in >90% of NSCLC tumors. High EphA2 expression was associated with decreased time to recurrence and metastases, and predicted poorer progression free and overall survival. Expression of EphA2 was positively correlated with activated EGFR and with KRAS mutation. Expression of EphA2 was also positively correlated with a history of smoking. There was no association between gender or histology and EphA2 expression. In H322 cells, activation of EGFR or KRAS resulted in an increase in EphA2 protein expression. Downregulation of EphA2 resulted in decreased proliferation in a clonal growth assay, and inhibited migration in a wound healing assay, in a panel of cell lines. The decrease in proliferation correlated with a transient decrease in the levels of phospho-ERK, a downstream effector of EGFR and KRAS. Based on these data, the potential of EphA2 as a therapeutic target for NSCLC should be further investigated. ^

The survival role and the potential therapeutic targeting of MET in multiple myeloma

Multiple myeloma (MM) is a debilitating and incurable B-cell malignancy. Previous studies have documented that the hepatocyte growth factor (HGF) plays a role in the pathobiology of MM. The receptor tyrosine kinase MET induced signaling initiates when its ligand HGF binds to the MET receptor. However, the direct importance of MET in MM has not been elucidated. The present work used three different but complementary approaches to reduce MET protein levels or its activity to demonstrate the importance of MET in MM. ^ In the first approach, MET transcript and protein levels were reduced by directly targeting the cellular MET transcripts using shRNA retroviral infection techniques. This direct reduction of MET mRNA leads to a reduction of MET protein levels, which caused an inhibition of growth and induction of cell death. ^ In the second approach, a global transcription inhibitor flavopiridol was used as a potential pharmacological tool to reduce MET levels. MET has a short half-life of 30 min for mRNA and 4 hours for protein; therefore using a RNA pol II inhibitor such as flavopiridol would be a viable option to reduce MET levels. When using flavopiridol in MM cell lines, there was a reduction of MET transcript and protein levels, which was associated with the induction of cell death. ^ Finally in the last strategy, MET kinase activity was suppressed by MP470, a small molecule inhibitor that binds to the ATP binding pocket in the kinase domain. At concentrations where phosphorylation of MET was inhibited there was induction of cell death in MM cell lines and primary cells from patients. In addition, in MM cell lines there was a decrease in phosphorylation of AKT (ser473) and caspase-9 (ser196); downstream of MET, suggesting that the mechanism of action for survival may be through these cascade of events. ^ Overall, this study provides a proof-of-principle that MET is important for the survival of MM cell lines as well as primary plasma cells obtained from patients. Therefore, targeting MET therapeutically may be a possible strategy to treat patients with this debilitating disease of MM. ^

TRANSCRIPTIONAL REGULATION OF PROFILIN IS REQUIRED FOR DROSOPHILA LARVAL WOUND CLOSURE

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways (the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway) and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound- induced cell migrations. Here we show that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. We found that JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, we show that chic deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. Our results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized.

Transformation studies of a nontumorigenic rat prostatic epithelial cell line with activated {\it myc\/} and {\it neu\/} oncogenes

A cloned nontumorigenic prostatic epithelial cell line, NbE-1.4, isolated from Noble (nbl/crx) rat ventral prostate, was used to examine the potential role of activated myc and neu oncogenes in prostate carcinogenesis. Transfection of SV40 promoter/enhancer driven constructs containing either v-myc, truncated c-myc, or neu-T (activated neu) oncogenes was accomplished using calcium phosphate-mediated DNA transfer. Cells were cotransfected, as necessary, with pSV2neo, allowing for selection of positive clones using the antibiotic geneticin (G418). G418 resistant colonies were pooled in some cases or limiting dilution exclusion cloned in others as described. Transfection of NbE-1.4 cells with activated myc oncogenes resulted only in the partial transformation. These cells display an altered morphology and decreased dependence on serum factors in vitro; however, saturation density, soft agar colony formation and growth assay in male athymic nude mice were all negative. Transfection and overexpression of NbE-1.4 cells with an activated neu oncogene alone resulted in tumorigenic conversion. Cell transformation was evident following an examination of the altered cellular morphology, an increased soft agar colony formation, and an acquisition of a tumorigenic potential when injected s.c. into male athymic nude mice. neu-transformed NbE-1.4 cells displayed elevated activity of the neu receptor tyrosine kinase. Furthermore, qualitative changes in tyrosine phosphorylated proteins were found in neu transformed cell clones. These changes were associated with elevated expression of mRNAs for laminin $\beta$1, $\beta$2, and procollagen type IV. The expression of fibronectin and E-cadherin, which are often lost during tumorigenesis, did not correlate with the tumorigenic phenotype. Therefore, it appears that neu oncogene overexpression has been found to be associated with the transformation of rat prostatic epithelial cells, presumably through alterations in gene expression that regulate extracellular matrix. The possible interrelationship and functional significance between neu oncogene expression and the elevated extracellular matrix gene expression is discussed. ^

Biological effects of PEA3 expression in {\it HER-2\/}/{\it neu\/}-overexpressing human cancer cells and the molecular mechanisms of PEA3-mediated transcriptional repression on {\it HER-2\/}/{\it neu\/}

HER-2/neu is a receptor tyrosine kinase highly homologous with epidermal growth factor receptor. Overexpression and/or amplification of HER-2/neu has been implicated in the genesis of a number of human cancers, especially breast and ovarian cancers. Transcriptional upregulation has been shown to contribute significantly to the overexpression of this gene. Studies on the transcriptional regulation of HER-2/neu gene are important for understanding the mechanism of cell transformation and developing the therapeutic strategies to block HER-2/neu-mediated cancers. PEA3 is a DNA binding transcriptional factor and its consensus sequence exists on the HER-2/neu promoter. To examine the role of PEA3 in HER-2/neu expression and cell transformation, we transfected PEA3 into the human breast and ovarian cancer cells that overexpress HER-2/neu and showed that PEA3 dramatically represses HER-2/neu transcription. PEA3 suppresses the oncogenic neu-mediated transformation in mouse fibroblast NIH 3T3 cells. Expression of PEA3 selectively blocks the growth of human cancer cells that overexpress HER-2/neu and inhibits their colony formation. It does not occur in the cancer cells expressing basal level of HER-2/neu. Further studies in the orthotopic ovarian cancer model demonstrated that expression of PEA3 preferentially inhibits growth and tumor development of human cancer cells that overexpress HER-2/neu, the tumor-bearing mice survived significantly longer if treated by injection of the PEA3-liposome complex intraperitoneally. Immunoblotting and immunohistochemical analysis of the tumor tissues indicated that PEA3 mediates the tumor suppression activity through targeting HER-2/neu-p185. Thus, PEA3 is a negative regulator of HER-2/neu gene expression and functions as a tumor suppressor gene in the HER-2/neu-overexpressing human cancer cells.^ The molecular mechanisms of PEA3 mediated transcriptional repression were investigated. PEA3 binds specifically at the PEA3 site on HER-2/neu promoter and this promoter-binding is required for the PEA3 mediated transcriptional repression. Mutation of the PEA3 binding site on HER-2/neu promoter causes decreased transcriptional activity, indicating that the PEA3 binding site is an enhancer-like element in the HER-2/neu-overexpressing cells. We therefore hypothesized that in the HER-2/neu-overexpressing cells, PEA3 competes with a transactivator for binding to the PEA3 site, preventing the putative factor from activating the transcription of HER-2/neu. This hypothesis was supported by the data which demonstrate that PEA3 competes with another nuclear protein for binding to the HER-2/neu promoter in vitro, and expression of a truncated protein which encodes the DNA binding domain of PEA3 is sufficient to repress HER-2/neu transcription in the HER-2/neu-overexpressing human cancer cells. ^

The antiapoptotic effect of overexpression c -erBb2 in breast cancer

Overexpression of the receptor tyrosine kinase p185ErbB2 confers taxol resistance in breast cancers and activation of p34Cdc2 is required for taxol-induced apoptosis and cytotoxicity. Here, we investigated the underlying mechanisms and found that overexpression of p185 ErbB2 inhibits taxol-induced apoptosis through two branches to inhibit activation of p34Cdc2. ^ Overexpression of p185ErbB2 in MDA-MB-435 cells by transfection transcriptionally upregulated p21Cip1, which associates with p34Cdc2, inhibits taxol-mediated p34Cdc2 activation, delays cell entrance to G2/M phase, and thereby inhibits taxol-induced apoptosis. In p21Cip1 antisense-transfected MDA-MB-435 cells or in p21−/− MEF cells, p185ErbB2 was unable to inhibit taxol-induced apoptosis. Therefore, p21Cip1 participates in the regulation of a G2/M checkpoint that contributes to resistance to taxol-induced apoptosis in p185ErbB2-overexpressing breast cancer cells. ^ Direct phosphorylation on Tyrosine-15 of p34Cdc2 by p185 ErbB2 receptor tyrosine kinase inhibits p34Cdc2 activation. The wild-type p185ErbB2 but not the kinase-defective mutant, when overexpressed in breast cancer cells, can phosphorylate p34Cdc2 on tyrosine (Tyr)15, an inhibitory phosphorylation site of p34 Cdc2. The kinase domain of the ErbB2 receptor was sufficient for binding to p34Cdc2 and directly phosphorylating the recombinant Cdc2. Phosphospecific Cdc2-Tyr15 immunoblot analyses, immunocomplex kinase assays, and phospho-amino acid analyses revealed that p185ErbB2 specifically phosphorylates Cdc2 on Tyr15. Phosphorylation of Cdc2-Tyr15 by ErbB2 is modulated during cell cycle and corresponded with delayed cell entry into G2/M phase. The kinase-defective p185ErbB2, which incapable of phosphorylating Cdc2-Tyr15, failed to inhibit taxol-induced activation and apoptosis, whereas the wild-type and the constitutive-active p185ErbB2 did. Increased Cdc2-Tyr15 phosphorylation was found in Erb132-overexpressing tumors from breast cancer patients. Thus, direct phosphorylation of Cdc2-Tyr15 by p185 ErbB2 RTK in breast cancer cells inhibits taxol-induced p34 Cdc2 activation and apoptosis, thereby conferring taxol resistance. ^

YODA MAPK kinase kinase regulates a novel immunity pathway conferring broad-spectrum resistance to pathogens

Plant mitogen-activated protein kinase (MAPK) casca des transduce environmental molecular signals and developmental cues into cellular responses. Among these signals are the pathogen-associated molecular patterns (PAMPs) that upon recognition by plant pattern recognition receptors (PRR), including Receptor-Like Kinases (RLKs), activate MAPK cascades that regulate PAMP-triggered immunity responses (PTI).

Functional characterization of YODA, a mitogen-activated protein kinase kinase kinase (MAP3K) that regulates a novel innate immunity pathway in Arabidopsis thaliana

Las cascadas de señalización mediadas por proteína quinasas activadas por mitógeno (MAP quinasas) son capaces de integrar y transducir señales ambientales en respuestas celulares. Entre estas señales se encuentran los PAMPs/MAMPs (Pathogen/Microbe-Associated Molecular Patterns), que son moléculas de patógenos o microorganismos, o los DAMPs (Damaged-Associated Molecular Patterns), que son moléculas derivadas de las plantas producidas en respuesta a daño celular. Tras el reconocimiento de los PAMPs/DAMPs por receptores de membrana denominados PRRs (Pattern Recognition Receptors), como los receptores con dominio quinasa (RLKs) o los receptores sin dominio quinasa (RLPs), se activan respuestas moleculares, incluidas cascadas de MAP quinasas, que regulan la puesta en marcha de la inmunidad activada por PAMPs (PTI). Esta Tesis describe la caracterización funcional de la MAP quinasa quinasa quinasa (MAP3K) YODA (YDA), que actúa como un regulador clave de la PTI en Arabidopsis. Se ha descrito previamente que YDA controla varios procesos de desarrollo, como la regulación del patrón estomático, la elongación del zigoto y la arquitectura floral. Hemos caracterizado un alelo mutante hipomórfico de YDA (elk2 o yda11) que presenta una elevada susceptibilidad a patógenos biótrofos y necrótrofos. Notablemente, plantas que expresan una forma constitutivamente activa de YDA (CA-YDA), con una deleción en el dominio N-terminal, presentan una resistencia de amplio espectro frente a diferentes tipos de patógenos, incluyendo hongos, oomicetos y bacterias, lo que indica que YDA juega un papel importante en la regulación de la resistencia de las plantas a patógenos. Nuestros datos indican que esta función es independiente de las respuestas inmunes mediadas por los receptores previamente caracterizados FLS2 y CERK1, que reconocen los PAMPs flg22 y quitina, respectivamente, y que están implicados en la resistencia de Arabidopsis frente a bacterias y hongos. Hemos demostrado que YDA controla la resistencia frente al hongo necrótrofo Plectosphaerella cucumerina y el patrón estomático mediante su interacción genética con la RLK ERECTA (ER), un PRR implicado en la regulación de estos procesos. Por el contrario, la interacción genética entre ER y YDA en la regulación de otros procesos de desarrollo es aditiva en lugar de epistática. Análisis genéticos indicaron que MPK3, una MAP quinasa que funciona aguas abajo de YDA en el desarrollo estomático, es un componente de la ruta de señalización mediada por YDA para la resistencia frente a P. cucumerina, lo que sugiere que el desarrollo de las plantas y la PTI comparten el módulo de transducción de MAP quinasas asociado a YDA. Nuestros experimentos han revelado que la resistencia mediada por YDA es independiente de las rutas de señalización reguladas por las hormonas de defensa ácido salicílico, ácido jasmónico, ácido abscísico o etileno, y también es independiente de la ruta de metabolitos secundarios derivados del triptófano, que están implicados en inmunidad vegetal. Además, hemos demostrado que respuestas asociadas a PTI, como el aumento en la concentración de calcio citoplásmico, la producción de especies reactivas de oxígeno, la fosforilación de MAP quinasas y la expresión de genes de defensa, no están afectadas en el mutante yda11. La expresión constitutiva de la proteína CA-YDA en plantas de Arabidopsis no provoca un aumento de las respuestas PTI, lo que sugiere la existencia de mecanismos de resistencia adicionales regulados por YDA que son diferentes de los regulados por FLS2 y CERK1. En línea con estos resultados, nuestros datos transcriptómicos revelan una sobre-representación en plantas CA-YDA de genes de defensa que codifican, por ejemplo, péptidos antimicrobianos o reguladores de muerte celular, o proteínas implicadas en la biogénesis de la pared celular, lo que sugiere una conexión potencial entre la composición e integridad de la pared celular y la resistencia de amplio espectro mediada por YDA. Además, análisis de fosfoproteómica indican la fosforilación diferencial de proteínas relacionadas con la pared celular en plantas CA-YDA en comparación con plantas silvestres. El posible papel de la ruta ER-YDA en la regulación de la integridad de la pared celular está apoyado por análisis bioquímicos y glicómicos de las paredes celulares de plantas er, yda11 y CA-YDA, que revelaron cambios significativos en la composición de la pared celular de estos genotipos en comparación con la de plantas silvestres. En resumen, nuestros datos indican que ER y YDA forman parte de una nueva ruta de inmunidad que regula la integridad de la pared celular y respuestas defensivas, confiriendo una resistencia de amplio espectro frente a patógenos. ABSTRACT Plant mitogen-activated protein kinase (MAPK) cascades transduce environmental signals and developmental cues into cellular responses. Among these signals are the pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) and the damage-associated molecular patterns (DAMPs). These PAMPs/DAMPs, upon recognition by plant pattern recognition receptors (PRRs), such as Receptor-Like Kinases (RLKs) and Receptor-Like Proteins (RLPs), activate molecular responses, including MAPK cascades, which regulate the onset of PAMP-triggered immunity (PTI). This Thesis describes the functional characterization of the MAPK kinase kinase (MAP3K) YODA (YDA) as a key regulator of Arabidopsis PTI. YDA has been previously described to control several developmental processes, such as stomatal patterning, zygote elongation and inflorescence architecture. We characterized a hypomorphic, non-embryo lethal mutant allele of YDA (elk2 or yda11) that was found to be highly susceptible to biotrophic and necrotrophic pathogens. Remarkably, plants expressing a constitutive active form of YDA (CA-YDA), with a deletion in the N-terminal domain, showed broad-spectrum resistance to different types of pathogens, including fungi, oomycetes and bacteria, indicating that YDA plays a relevant function in plant resistance to pathogens. Our data indicated that this function is independent of the immune responses regulated by the well characterized FLS2 and CERK1 RLKs, which are the PRRs recognizing flg22 and chitin PAMPs, respectively, and are required for Arabidopsis resistance to bacteria and fungi. We demonstrate that YDA controls resistance to the necrotrophic fungus Plectosphaerella cucumerina and stomatal patterning by genetically interacting with ERECTA (ER) RLK, a PRR involved in regulating these processes. In contrast, the genetic interaction between ER and YDA in the regulation of other ER-associated developmental processes was additive, rather than epistatic. Genetic analyses indicated that MPK3, a MAP kinase that functions downstream of YDA in stomatal development, also regulates plant resistance to P. cucumerina in a YDA-dependent manner, suggesting that the YDA-associated MAPK transduction module is shared in plant development and PTI. Our experiments revealed that YDA-mediated resistance was independent of signalling pathways regulated by defensive hormones like salicylic acid, jasmonic acid, abscisic acid or ethylene, and of the tryptophan-derived metabolites pathway, which are involved in plant immunity. In addition, we showed that PAMP-mediated PTI responses, such as the increase of cytoplasmic Ca2+ concentration, reactive oxygen species (ROS) burst, MAPK phosphorylation, and expression of defense-related genes are not impaired in the yda11 mutant. Furthermore, the expression of CA-YDA protein does not result in enhanced PTI responses, further suggesting the existence of additional mechanisms of resistance regulated by YDA that differ from those regulated by the PTI receptors FLS2 and CERK1. In line with these observations, our transcriptomic data revealed the over-representation in CA-YDA plants of defensive genes, such as those encoding antimicrobial peptides and cell death regulators, and genes encoding cell wall-related proteins, suggesting a potential link between plant cell wall composition and integrity and broad spectrum resistance mediated by YDA. In addition, phosphoproteomic data revealed an over-representation of genes encoding wall-related proteins in CA-YDA plants in comparison with wild-type plants. The putative role of the ER-YDA pathway in regulating cell wall integrity was further supported by biochemical and glycomics analyses of er, yda11 and CA-YDA cell walls, which revealed significant changes in the cell wall composition of these genotypes compared with that of wild-type plants. In summary, our data indicate that ER and YDA are components of a novel immune pathway that regulates cell wall integrity and defensive responses, which confer broad-spectrum resistance to pathogens.

Two-domain reconstitution of a functional protein histidine kinase

In prokaryotes, in the absence of protein serine/threonine/tyrosine kinases, protein histidine kinases play a major role in signal transduction involved in cellular adaptation to various environmental changes and stresses. Histidine kinases phosphorylate their cognate response regulators at a specific aspartic acid residue with ATP in response to particular environmental signals. In this His-Asp phosphorelay signal transduction system, it is still unknown how the histidine kinase exerts its enzymatic function. Here we demonstrate that the cytoplasmic kinase domain of EnvZ, a transmembrane osmosensor of Escherichia coli can be further divided into two distinct functional subdomains: subdomain A [EnvZ(C)⋅(223–289); 67 residues] and subdomain B [EnvZ(C)⋅(290–450); 161 residues]. Subdomain A, with a high helical content, contains the autophosphorylation site, H–243, and forms a stable dimer having the recognition site for OmpR, the cognate response regulator of EnvZ. Subdomain B, an α/β-protein, exists as a monomer. When mixed, the two subdomains reconstitute the kinase function to phosphorylate subdomain A at His-243 in the presence of ATP. Subsequently, the phosphorylated subdomain A is able to transfer its phosphate group to OmpR. The two-domain structure of this histidine kinase provides an insight into the structural arrangement of the enzyme and its transphosphorylation mechanism.

P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase

Protein tyrosine phosphatases (PTPs) have long been thought to play a role in tumor suppression due to their ability to antagonize the growth promoting protein tyrosine kinases. Recently, a candidate tumor suppressor from 10q23, termed P-TEN, was isolated, and sequence homology was demonstrated with members of the PTP family, as well as the cytoskeletal protein tensin. Here we show that recombinant P-TEN dephosphorylated protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibited a high degree of substrate specificity, showing selectivity for extremely acidic substrates in vitro. Furthermore, we demonstrate that mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan–Zonana syndrome, resulted in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is necessary for its ability to function as a tumor suppressor.

Identification of genes required for Drosophila eye development using a phenotypic enhancer-trap

A novel method of P-element mutagenesis is described for the isolation of mutants affecting the development of the Drosophila compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase that triggers the formation of the UV-sensitive photoreceptor neuron, R7. Transposition of a boss cDNA transgene, in an otherwise boss mutant background, was used as a “phenotypic trap” in live flies to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Some 1,800 R7-containing flies were identified. Among these, 21 independent insertions with expression of the boss reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the boss reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the marbles, glass, gap1, and fasciclin II genes were isolated. This phenotypic enhancer-trap facilitates (i) the isolation of enhancer-traps with a specific expression pattern, and (ii) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages.