Background:Production of chimeric antigen receptor T cell (CAR-T) therapies depends on antibody reagents to label, isolate, and expand T cell products. We sought to create antibody tools specific for the variable domain of heavy-chain only antibodies (VHHs), also known as nanobodies, used in some CARs. Methods:We immunized a mouse with VHH and selected two murine monoclonal antibodies (mAbs) that bind to distinct epitopes in conserved framework regions of llama-derived VHHs, and not to human VH domains. Anti-VHH mAbs were characterized by enzyme-linked immunosorbent assay, surface plasmon resonance, and hydrogen-deuterium exchange mass spectrometry; were then tested for cell/tissue labeling and for modulating cellular activity in VHH-CAR-T cells. Results:We produced a high-quality dual-clonal anti-VHH antibody product and confirmed reactivity to over 98% of VHH proteins regardless of their antigenic specificity, with no reactivity to human or mouse IgG and reduced reactivity to conventional llama or alpaca IgG. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus was appropriate for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies (Abs) potently blocked VHH-CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH Abs induced strong activation and expansion of VHH CAR-T cells; with 730-fold mean expansion, >94% CAR purity, and retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen-specific activity without functional exhaustion. Conclusions:Overall, these data identify useful anti-VHH mAbs that can be applied to better understand and manipulate VHH-based CAR-T cells or other VHH-based immunotherapies.

An attractive feature of human VHs over camelid VHHs as immunotherapeutics is their perceived lower risk of immunogenicity. While human VHs can readily be obtained from synthetic phage display libraries, they often suffer from low affinity and poor solubility compared to VHHs derived from immune libraries. Using SARS-CoV-2 spike protein as a model antigen, we screened a synthetic human VH phage display library and identified a diverse set of antigen-specific VHs. However, the VHs exhibited low affinity, and many had low solubility; that is, they were prone to aggregation. To explore the feasibility of improving the affinity, we subjected a representative VH to in vitro affinity maturation. We created a yeast surface display library of VH variants employing a site-saturated mutagenesis approach targeting complementarity-determining regions and selected against the target antigen. Next-generation sequencing of the selected variants, combined with structural modeling, identified a set of VHs as potentially improved candidates. Characterization of these candidates revealed several VHs with improved affinities of up to 100-fold (KDs as low as 3 nM) and potent neutralization capabilities; however, they still showed significant aggregation. By introducing as few as two camelid residues into the framework region 2 of a high-affinity VH (a process referred to as camelization), we were able to completely solubilize the VH without compromising its affinity and other important attributes, including thermostability and protein A binding. This study demonstrates the feasibility of generating high-affinity, -solubility, and -stability human VHs from synthetic libraries through a combination of in vitro affinity maturation and minimal camelization.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single- domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAbCARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. . Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

Carbonic anhydrase (CA)-IX is an extracellular enzyme that is essential in the adaptation of tumor cells to their increasingly more hypoxic and acidic microenvironment. Within the family of carbonic anhydrases, CA-IX is unique in that it is the only CA with an N-terminal intrinsically disordered region (IDR) containing a proteoglycan (PG)-like domain. This PG-like IDR has been described to be instrumental in CA-IX's enzyme activity, as well as tumor cell motility and invasion. We have characterized the antibody-epitope interactions of two novel and unique antibodies (11H9 and 12H8) that are specific for the human CA-IX's IDR. Binding interactions of these antibodies to the intact IDR were studied by surface plasmon resonance and high-resolution nuclear magnetic resonance (NMR) spectroscopy, while the specific epitopes were determined by both NMR and yeast surface display (YSD). Our data show that 12H8 binds to the N-terminus of CA-IX, while 11H9 has a high affinity for an epitope located in the central region of the IDR containing three GEEDLP repeats in a manner that is different from the previously described M75 antibody. Titration NMR spectroscopy using CA-IX's entire IDR in addition identified a secondary epitope of 11H9 at the beginning of the PG-like domain that remains exposed and available for further binding events after the engagement at its primary epitope at the center of the PG-like domain. Transverse relaxation optimized NMR spectroscopy of 11H9-F(Ab) in complex with the CA-IX IDR outlines structural rigidification of a linear epitope, while the rest of the IDR remains largely unstructured upon complex formation. This study illustrates how high-resolution NMR and YSD are used as complementary tools for a comprehensive characterization of antibody-epitope interactions involving intrinsically unstructured antigen domains with highly repetitive sequences.

In response to pheromone, many proteins localize on the plasma membrane of yeast cell to reform it into a polarized shmoo structure. The adaptor protein Ste50p, known as a pheromone signal enhancer critical for shmoo polarization, has never been explored systematically for its localization and function in the polarization process. Time-lapse single-cell imaging and quantitation shown here characterizes Ste50p involvement in the establishment of cell polarity. We found that Ste50p patches on the cell cortex mark the point of shmoo initiation, these patches could move, and remain associated with the growing shmoo tip in a pheromone concentration time-dependent manner until shmoo maturation. A Ste50p mutant impaired in patch localization suffers a delay in polarization. By quantitative analysis we show that polarization correlates with the rising levels of Ste50p, enabling rapid cell responses to pheromone that correspond to a critical level of Ste50p at the initial G1 phase. We exploited the quantitative differences in the pattern of Ste50p expression to correlate with the cell-cell phenotypic heterogeneity, showing Ste50p involvement in the cellular differentiation choice. Taken together, these findings present Ste50p to be part of the early shmoo development phase, suggesting that Ste50p may be involved with the polarisome in the initiation of polarization, and plays a role in regulating the polarized growth of shmoo during pheromone response.

Abstract Poor vascularization of solid tumors leads to inadequate nutrient and oxygen supplies which forces tumor cells to reprogram their metabolism. Consequently, the tumor cell's environment becomes acidic and hypoxic. This triggers signaling cascades involving e.g. heterodimeric hypoxia-inducible factor (HIF). Activation of this hypoxia-induced transcriptional program is crucial for the tumor cell to survive its hostile microenvironment and its ability to metastasize. One of the genes HIF upregulated is carbonic anhydrase (CA)-IX (CAIX, gene G250/MN-encoded transmembrane protein). CA-IX catalyzes carbon dioxide (CO2) thereby generating a proton (H+) and bicarbonate (HCO3−), the latter of which is transported back into the cell and utilized to help safeguard intracellular pH (pHi) stability. Except for the stomach and the gallbladder, CA-IX expression is negligible in normal tissues. In contrast, a broad range of tumors express high levels of CA-IX, where the protein can serve as a biomarker for the early stages of tumor development but also as tumor marker of hypoxia associated with resistance to chemotherapy and radiotherapy. Preclinical and clinical studies have shown that CA-IX is a promising therapeutic target for detection and therapy for several cancer types. To date only a limited number of ant-CAIX monoclonal antibodies (mAbs) have been available for clinical testing as therapeutic and imaging agents. In the current study, we generated and functionally categorized a panel of 51 mouse mAbs that specifically bind to human CA-IX. Characterization of the mAbs revealed that of the mAbs with the best biophysical characteristics, three (3) mAbs are suitable as an antibody-drug conjugate (ADC), two (2) mAbs inhibit the CA-IX enzyme activity, and one (1) mAb that is suitable for CA-IX imaging purposes. These preliminary data presented here could thus form the basis for the development of novel CA-IX targeted immunotherapies and diagnostic tools for the treatment of cancer. Citation Format: Anne E. G. Lenferink, Jason Baardsnes, Traian Sulea, Cunle Wu, Maurizio Acchione, Maria L. Jaramillo, Paul C. McDonald, Francois Benard, Shoukat Dedhar. Identification and functional evaluation of monoclonal antibodies specifically targeting human Carbonic Anhydrase IX [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P038.

Overexpression of Axl, a TAM-family receptor tyrosine kinase, plays key roles in the formation, growth, and spread of tumors as well as resistance to targeted therapies and chemotherapies. We identified novel llama VHHs against human Axl using multiple complementary phage display selection strategies and characterized a subset of high-affinity VHHs. The VHHs targeted multiple sites in Ig-like domains 1 and 2 of the Axl extracellular domain, including an immunodominant epitope overlapping the site of Gas6 interaction and two additional non-Gas6 competitive epitopes recognized by murine monoclonal antibodies. Only a subset of VHHs cross-reacted with cynomolgus monkey Axl and none recognized mouse Axl. As fusions to human IgG1 Fc, VHH-Fcs bound Axl+ tumor cell lines and mertansine-loaded VHH-Fcs were cytotoxic in vitro against Axl+ cells in proportion to their binding affinities. Engineered biparatopic VHH-VHH heterodimers bound Axl avidly, and a subset of molecules showed dramatically enhanced association rates indicative of intramolecular binding. These VHHs may have applications as modular elements of biologic drugs such as antibody-drug conjugates.

The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these proteins is carbonic anhydrase (CA)IX, a zinc-containing extracellular membrane bound enzyme that has been shown to play a critical role in the maintenance of a neutral intracellular pH (pHi), allowing tumor cells to survive and thrive in these harsh conditions. Although CAIX has been considered a promising cancer target, only two antibody-based therapeutics have been clinically tested so far. To fill this gap, we generated a series of novel monoclonal antibodies (mAbs) that specifically recognize the extracellular domain (ECD) of human CAIX. Here we describe the biophysical and functional properties of a set of antibodies against the CAIX ECD domain and their applicability as: 1) suitable for development as an antibody-drug-conjugate, 2) an inhibitor of CAIX enzyme activity, or 3) an imaging/detection antibody. The results presented here demonstrate the potential of these specific hCAIX mAbs for further development as novel cancer therapeutic and/or diagnostic tools.

Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO2, is often overexpressed in solid tumors. This enzyme is instrumental in maintaining the survival of cancer cells in a hypoxic and acidic tumor microenvironment. Absent in most normal tissues, hCAIX is a promising therapeutic target for detection and treatment of solid tumors. Screening of a library of anti-hCAIX monoclonal antibodies (mAbs) previously identified three therapeutic candidates (mAb c2C7, m4A2 and m9B6) with distinct biophysical and functional characteristics. Selective binding to the catalytic domain was confirmed by yeast surface display and isothermal calorimetry, and deeper insight into the dynamic binding profiles of these mAbs upon binding were highlighted by bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS). Here, a conformational and allosterically silent epitope was identified for the antibody-drug conjugate candidate c2C7. Unique binding profiles are described for both inhibitory antibodies, m4A2 and m9B6. M4A2 reduces the ability of the enzyme to hydrate CO2 by steric gating at the entrance of the catalytic cavity. Conversely, m9B6 disrupts the secondary structure that is necessary for substrate binding and hydration. The synergy of these two inhibitory mechanisms is demonstrated in in vitro activity assays and HDX-MS. Finally, the ability of m4A2 to modulate extracellular pH and intracellular metabolism is reported. By highlighting three unique modes by which hCAIX can be targeted, this study demonstrates both the utility of HDX-MS as an important tool in the characterization of anti-cancer biotherapeutics, and the underlying value of CAIX as a therapeutic target.

Discriminating among diverse environmental stimuli is critical for organisms to ensure their proper development, homeostasis, and survival. Saccharomyces cerevisiae regulates mating, osmoregulation, and filamentous growth using three different MAPK signaling pathways that share common components and therefore must ensure specificity. The adaptor protein Ste50 activates Ste11p, the MAP3K of all three modules. Its Ras association (RA) domain acts in both hyperosmolar and filamentous growth pathways, but its connection to the mating pathway is unknown. Genetically probing the domain, we found mutants that specifically disrupted mating or HOG-signaling pathways or both. Structurally these residues clustered on the RA domain, forming distinct surfaces with a propensity for protein-protein interactions. GFP fusions of wild-type (WT) and mutant Ste50p show that WT is localized to the shmoo structure and accumulates at the growing shmoo tip. The specifically pheromone response-defective mutants are severely impaired in shmoo formation and fail to localize ste50p, suggesting a failure of association and function of Ste50 mutants in the pheromone-signaling complex. Our results suggest that yeast cells can use differential protein interactions with the Ste50p RA domain to provide specificity of signaling during MAPK pathway activation.

EGFRvIII is a naturally occurring variant of epidermal growth factor receptor (EGFR) that results from an in-frame deletion that removes 267 amino acids from the extracellular domain. EGFRvIII is a tumor-specific receptor that is amplified and present in 25-64% of glioblastoma multiforme and in 20-36% of breast cancers. Numerous studies show that normal tissues are devoid of EGFRvIII, thus making this target ideal for development as an antibody drug conjugate (ADC) for the treatment of cancer,including glioblastoma. At the National Research Council (NRC) of Canada, we have selected mouse monoclonal mAbs that are selective for the extracellular domain of EGFRvIII and which have been shown to be internalized into EGFRvIII expressing cells for ADC development . A panel of EGFRvIII internalizing antibodies was directly conjugated to maytansine, the microtubule disrupting agent through a noncleavable linker. EGFRvIII -DM1 ADCs were shown to efficiently induce growth inhibition in vitro, in EGFRvIII expressing U87MG and DKMG glioblastoma cells. When tested in vivo, EGFRvIII -DM1 ADCs were shown to have potent anti-tumor activity in the U87MG-EGFRIII expressing tumor xenograft model. Citation Format: Maria Luz Jaramillo, Anne Marcil, Alma Robert, Normand Jolicoeur, Cunle Wu, Yves Fortin, Yuneivy Cepero Donates, Mauro Acchione, Jacqueline Slinn, Binbing Ling, Maria Moreno. Preclinical development of antibody drug conjugates targetting EGFRvIII [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1556.

Abstract AXL is a member of the Tyro3- AXL -Mer (TAM) receptor tyrosine kinase subfamily which has been associated with several cellular functions including growth, migration, aggregation, and anti-inflammation (Li Y et al., 2009) via activation through its ligand, Gas6. Originally identified as an oncogene in chronic myelogenous leukemia, expression of Axl has been reported to be upregulated in a variety of cancers including breast, gastric, prostate, ovarian, and lung. AXL expression has been shown to be negatively associated with patient survival and implicated in epithelial-to-mesenchymal transition, a process closely linked with invasive motility and metastasis of malignant cells. Several recent studies have reported that AXL is overexpressed during resistance to chemotherapy and other molecularly targeted therapies, as well as during hypoxia (Schoumacher M et al 2017). Consequently, Axl is promising therapeutic target for development into ADCs for the first or second line treatment of cancer. At the National Research Council (NRC) of Canada, we have selected mouse monoclonal (mAbs) or llama derived single domain antibodies (sdAbs) that are selective for the extracellular domain of human Axl and which have been shown to be internalized into Axl expressing cells for antibody drug conjugate (ADC) development using a surrogate ADC screen. These antibodies were also characterized based on their binding domains and ability to compete with Gas6, the extracellular ligand for Axl. A panel of Axl-specific ADCs was subsequently directly conjugated to maytansine, the microtubule disrupting agent through a noncleavable linker. Axl-DM1 ADCs were shown to efficiently induce cytotoxicity in vitro, in Axl expressing cells including breast (MDA-MB 231), lung (NCI-H292) and ovarian (SKOV3) cell lines with sub nM potencies achieved with both sdAb and conventional mAb-based ADCs. When tested in vivo using ovarian (SKOV3) and breast cancer (MDA-MB 231) xenograft models, Axl-DM1 ADCs were shown to have potent anti-tumor activity. Biparatopic antibodies (i.e. those which target two non-overlapping epitopes) directed against HER2 have been previously shown to induce target clustering and promote robust internalization, lysosomal trafficking, and degradation (LI JY et al 2016). Since these properties are important in the mechanism of action of ADCs, we have further tested anti-Axl antibodies in various configurations and have identified potent biparatopic antibodies for further development into ADCs. Citation Format: Maria L. Jaramillo, Myriam Banville, Alma Robert, Luc Meury, Maurizio Acchione, Anne Marcil, Christine Gadoury, Cunle Wu, Yves Fortin, Kevin Henry, Joseph Schrag, The-Minh Tu, Binbing (Erica) Ling, Jacqueline Slinn, Maria Moreno. Development of novel formats of anti-AXL ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 746.

Studying Candida biology requires access to genomic sequence data in conjunction with experimental information that provides functional context to genes and proteins. The Candida Genome Database (CGD) integrates functional information about Candida genes and their products with a set of analysis tools that facilitate searching for sets of genes and exploring their biological roles. This chapter describes how the various types of information available at CGD can be searched, retrieved, and analyzed. Starting with the guided tour of the CGD Home page and Locus Summary page, this unit shows how to navigate the various assemblies of the C. albicans genome, how to use Gene Ontology tools to make sense of largescale data, and how to access the microarray data archived at CGD.

Many biological processes are regulated by protein-protein interactions, and the analysis of these interactions has been a productive endeavor contributing to our understanding of cellular organization and function. The yeast two-hybrid technique is a widely used, powerful method of analyzing protein-protein interactions. The currently used formats, however, have inherent limitations, providing an opportunity to develop new alternatives that extend our ability to detect protein-protein interactions of biological relevance. Here we present a two-hybrid system named SRYTH (Ste11p/Ste50p related yeast two-hybrid) based on the Ste11p/Ste50p interaction that uses the activation of the HOG pathway of Saccharomyces cerevisiae as a reporter for interactions. The system is suitable for detecting cytoplasmic protein interactions in their natural subcellular environment, and has been successfully used to investigate protein-protein interactions, including transcription factor associations, in Candida albicans.

The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source.

The Ste5 protein forms a scaffold that associates and regulates the components of the mitogen-activated protein (MAP) kinase cascade that controls mating-pheromone-mediated signaling in the yeast Saccharomyces cerevisiae. Although it is known that the MEK kinase of the pathway, Ste11, associates with Ste5, details of this interaction have not been established. We identified a Ras-binding-domain-like (RBL) region in the Ste11 protein that is required specifically for the kinase to function in the mating pathway. This module is structurally related to domains in other proteins that mediate Ras-MAP kinase kinase kinase associations; however, this RBL module does not interact with Ras, but instead binds the PH domain of the Ste5 scaffold. Structural and functional studies suggest that the key role of this PH domain is to mediate the Ste5-Ste11 interaction. Overall these two evolutionarily conserved modules interact with each other through a unique interface, and thus in the pheromone pathway the structural context of the RBL domain contribution to kinase activation has been shifted through a change of its interaction partner from Ras to a PH domain.

Scaffold proteins play central roles in the function of many signaling pathways. Among the best-studied examples are the Ste5 and Far1 proteins of the yeast Saccharomyces cerevisiae. These proteins contain three conserved modules, the RING and PH domains, characteristic of some ubiquitin-ligating enzymes, and a vWA domain implicated in protein-protein interactions. In yeast, Ste5p regulates the mating pathway kinases while Far1p coordinates the cellular polarity machinery. Within the fungal lineage, the Basidiomycetes and the Pezizomycetes contain a single Far1-like protein, while several Saccharomycotina species, belonging to the CTG (Candida) clade, contain both a classic Far1-like protein and a Ste5-like protein that lacks the vWA domain. We analyzed the function of C. albicans Ste5p (Cst5p), a member of this class of structurally distinct Ste5 proteins. CST5 is essential for mating and still coordinates the mitogen-activated protein (MAP) kinase (MAPK) cascade elements in the absence of the vWA domain; Cst5p interacts with the MEK kinase (MEKK) C. albicans Ste11p (CaSte11p) and the MAPK Cek1 as well as with the MEK Hst7 in a vWA domain-independent manner. Cst5p can homodimerize, similar to Ste5p, but can also heterodimerize with Far1p, potentially forming heteromeric signaling scaffolds. We found direct binding between the MEKK CaSte11p and the MEK Hst7p that depends on a mobile acidic loop absent from S. cerevisiae Ste11p but related to the Ste7-binding region within the vWA domain of Ste5p. Thus, the fungal lineage has restructured specific scaffolding modules to coordinate the proteins required to direct the gene expression, polarity, and cell cycle regulation essential for mating.

Activation of the high-osmolarity glycerol (HOG) pathway for osmoregulation in the yeast Saccharomyces cerevisiae involves interaction of the adaptor Ste50p with the cytoplasmic tail of single-transmembrane protein Opy2p. We have determined the solution structure of the Ste50p-RA (Ras association) domain, and it shows an atypical RA fold lacking the beta 1 and beta 2 strands of the canonical motif. Although the core of the RA domain is fully functional in the pheromone response, an additional region is required for the HOG pathway activation. Two peptide motifs within the intrinsically disordered cytoplasmic tail of Opy2p defined by NMR spectroscopy physically interact with the Step50p-RA domain. These Opy2p-derived peptides bind overlapping regions of the Step50p-RA domain with similarly weak affinities, suggesting a multivalent interaction of these proteins as a crucial point of control of the HOG pathway. As well, overall selection of signaling pathways depends on functionally distinct regions of the Ste50p-RA domain, implicating this element in the control of global regulatory decisions.

Small GTPases of the Rho family act as molecular switches, and modulation of the GTP-bound state of Rho proteins is a well-characterized means of regulating their signaling activity in vivo. In contrast, the regulation of Rho-type GTPases by posttranslational modifications is poorly understood. Here, we present evidence of the control of the Saccharomyces cerevisiae Rho-type GTPase Rho5p by phosphorylation and ubiquitination. Rho5p binds to Ste50p, and the expression of the activated RHO5( Q91H) allele in an Delta ste50 strain is lethal under conditions of osmotic stress. An overexpression screen identified RGD2 and MSI1 as being high-copy suppressors of the osmotic sensitivity of this lethality. Rgd2p had been identified as being a possible Rho5p GTPase-activating protein based on an in vitro assay; this result supports its function as a regulator of Rho5p activity in vivo. MSI1 was previously identified as being a suppressor of hyperactive Ras/cyclic AMP signaling, where it antagonizes Npr1p kinase activity and promotes ubiquitination. Here, we show that Msi1p also acts via Npr1p to suppress activated Rho5p signaling. Rho5p is ubiquitinated, and its expression is lethal in a strain that is compromised for proteasome activity. These data identify Rho5p as being a target of Msi1p/Npr1p regulation and describe a regulatory circuit involving phosphorylation and ubiquitination.

In a variety of yeast cellular pathways, the Ste50p protein regulates the kinase function of the mitogen extracellular signal-regulated kinase kinase (MEKK) Ste11p. Both Ste11p and Ste50p contain sterile alpha motif (SAM) domains; these are interchangeable, and can be replaced by other protein-interacting modules. Furthermore, the function of the Ras association (RA)-like domain of Ste50p can be mimicked by a plasma membrane recruiting signal, and direct plasma membrane targeting of Ste11p bypasses the requirement of Ste50p for Ste11p function. Thus the regulatory role of Ste50p requires both the N-terminal SAM domain to bind Ste11p and the C-terminal RA-like domain to direct kinase localization. We have identified Opy2p, an integral membrane protein that can interact with Ste50p, as a new component in the Sho1p-Ste11p/Ste50p signaling branch of the high-osmolarity glycerol (HOG) pathway. We propose that Opy2p can serve as a membrane anchor for the Ste50p/Ste11p module in the activation of the HOG pathway.