Robust and scalable single-molecule protein sequencing with fluorosequencing.

The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications.

Estimating error rates for single molecule protein sequencing experiments.

The practical application of new single molecule protein sequencing (SMPS) technologies requires accurate estimates of their associated sequencing error rates. Here, we describe the development and application of two distinct parameter estimation methods for analyzing SMPS reads produced by fluorosequencing. A Hidden Markov Model (HMM) based approach, extends whatprot, where we previously used HMMs for SMPS peptide-read matching. This extension offers a principled approach for estimating key parameters for fluorosequencing experiments, including missed amino acid cleavages, dye loss, and peptide detachment. Specifically, we adapted the Baum-Welch algorithm, a standard technique to estimate transition probabilities for an HMM using expectation maximization, but modified here to estimate a small number of parameter values directly rather than estimating every transition probability independently, which should help prevent overfitting. We demonstrate a high degree of accuracy on simulated data, but on experimental datasets, we observed that the model needed to be augmented with an additional error type, N-terminal blocking. This, in combination with data pre-processing, results in reasonable parameterizations of experimental datasets that agree with controlled experimental perturbations. A second independent implementation using a hybrid of DIRECT and Powell's method to reduce the root mean squared error (RMSE) between simulations and the real dataset was also developed. We compare these methods on both simulated and real data, finding that our Baum-Welch based approach outperforms DIRECT and Powell's method by most, but not all, criteria. Although some discrepancies between the results exist, we also find that both approaches provide similar error rate estimates from experimental single molecule fluorosequencing datasets.

Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics.

Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which labeling chemistry is designed. In practice, only two functional groups are present in every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative alkylation for peptide mass spectrometry and single-molecule protein sequencing that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino-acid residues for decarboxylative labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid-labeling technology in tandem to establish a new platform for fluorosequencing.

Cryo-EM structure of OSCA1.2 from Oryza sativa elucidates the mechanical basis of potential membrane hyperosmolality gating.

Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (Oryza sativa; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.

Effect of a Smac Mimetic (TL32711, Birinapant) on the Apoptotic Program and Apoptosis Biomarkers Examined with Validated Multiplex Immunoassays Fit for Clinical Use.

PURPOSE: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors. EXPERIMENTAL DESIGN: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax-Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL-Bak heterodimer, Mcl1-Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis. RESULTS: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies. CONCLUSIONS: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials.

Caspase-mediated activation of Caenorhabditis elegans CED-8 promotes apoptosis and phosphatidylserine externalization.

During apoptosis, phosphatidylserine (PS), normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and serves as an 'eat-me' signal to trigger phagocytosis. It is poorly understood how PS exposure is activated in apoptotic cells. Here we report that CED-8, a Caenorhabditis elegans protein implicated in controlling the kinetics of apoptosis and a homologue of the XK family proteins, is a substrate of the CED-3 caspase. Cleavage of CED-8 by CED-3 activates its proapoptotic function and generates a carboxyl-terminal cleavage product, acCED-8, that promotes PS externalization in apoptotic cells and can induce ectopic PS exposure in living cells. Consistent with its role in promoting PS externalization in apoptotic cells, ced-8 is important for cell corpse engulfment in C. elegans. Our finding identifies a crucial link between caspase activation and PS externalization, which triggers phagocytosis of apoptotic cells.

SMA-MAP: a plasma protein panel for spinal muscular atrophy.

OBJECTIVES: Spinal Muscular Atrophy (SMA) presents challenges in (i) monitoring disease activity and predicting progression, (ii) designing trials that allow rapid assessment of candidate therapies, and (iii) understanding molecular causes and consequences of the disease. Validated biomarkers of SMA motor and non-motor function would offer utility in addressing these challenges. Our objectives were (i) to discover additional markers from the Biomarkers for SMA (BforSMA) study using an immunoassay platform, and (ii) to validate the putative biomarkers in an independent cohort of SMA patients collected from a multi-site natural history study (NHS). METHODS: BforSMA study plasma samples (N = 129) were analyzed by immunoassay to identify new analytes correlating to SMA motor function. These immunoassays included the strongest candidate biomarkers identified previously by chromatography. We selected 35 biomarkers to validate in an independent cohort SMA type 1, 2, and 3 samples (N = 158) from an SMA NHS. The putative biomarkers were tested for association to multiple motor scales and to pulmonary function, neurophysiology, strength, and quality of life measures. We implemented a Tobit model to predict SMA motor function scores. RESULTS: 12 of the 35 putative SMA biomarkers were significantly associated (p<0.05) with motor function, with a 13(th) analyte being nearly significant. Several other analytes associated with non-motor SMA outcome measures. From these 35 biomarkers, 27 analytes were selected for inclusion in a commercial panel (SMA-MAP) for association with motor and other functional measures. CONCLUSIONS: Discovery and validation using independent cohorts yielded a set of SMA biomarkers significantly associated with motor function and other measures of SMA disease activity. A commercial SMA-MAP biomarker panel was generated for further testing in other SMA collections and interventional trials. Future work includes evaluating the panel in other neuromuscular diseases, for pharmacodynamic responsiveness to experimental SMA therapies, and for predicting functional changes over time in SMA patients.

CED-1, CED-7, and TTR-52 regulate surface phosphatidylserine expression on apoptotic and phagocytic cells.

BACKGROUND: Phosphatidylserine (PS) normally confined to the cytoplasmic leaflet of plasma membrane (PM) is externalized to the exoplasmic leaflet (exPS) during apoptosis, where it serves as an "eat-me" signal to phagocytes. In addition, some living cells such as macrophages also express exPS. RESULTS: A secreted Annexin V (sAnxV::GFP) PS sensor reveals that exPS appears early on apoptotic cells in C. elegans embryos and decreases in older or unengulfed apoptotic cells. This decrease in exPS expression is blocked by loss of CED-7, an ATP binding cassette (ABC) transporter, or TTR-52, a secreted PS binding protein. Phagocytic cells also express exPS, which is dependent on the activity of CED-7, TTR-52, and TTR-52-interacting phagocyte receptor CED-1. Interestingly, a secreted lactadherin PS sensor (sGFP::Lact(C1C2)) labels apoptotic cells but not phagocytes, prevents sAnxV::GFP from labeling phagocytes, and compromises phagocytosis. Immuno-electron micrographs of embryos expressing sAnxV::GFP or sGFP::Lact(C1C2) reveal the presence of extracellular PS-containing vesicles between the apoptotic cell and neighboring cells, which are absent or greatly reduced in the ced-7 and ttr-52 mutants, respectively, indicating that CED-7 and TTR-52 promote the generation of extracellular PS vesicles. Loss of the tat-1 gene, which maintains PS asymmetry in the PM, restores phagocyte exPS expression in ced-1, ced-7, and ttr-52 mutants and partially rescues their engulfment defects. CONCLUSIONS: CED-7 and TTR-52 may promote the efflux of PS from apoptotic cells through the generation of extracellular PS vesicles, which lead to exPS expression on phagocytes via TTR-52 and CED-1 to facilitate cell corpse clearance.

Evidence for an antagonist form of the chemokine CXCL10 in patients chronically infected with HCV.

Chronic infection with hepatitis C virus (HCV) is a major public health problem, with nearly 170 million infected individuals worldwide. Current treatment for chronic infection is a combination of pegylated IFN-α2 and ribavirin (RBV); however, this treatment is effective in fewer than 50% of patients infected with HCV genotype 1 or 4. Recent studies identified the chemokine CXCL10 (also known as IP-10) as an important negative prognostic biomarker. Given that CXCL10 mediates chemoattraction of activated lymphocytes, it is counterintuitive that this chemokine correlates with therapeutic nonresponsiveness. Herein, we offer new insight into this paradox and provide evidence that CXCL10 in the plasma of patients chronically infected with HCV exists in an antagonist form, due to in situ amino-terminal truncation of the protein. We further demonstrated that dipeptidyl peptidase IV (DPP4; also known as CD26), possibly in combination with other proteases, mediates the generation of the antagonist form(s) of CXCL10. These data offer what we believe to be the first evidence for CXCL10 antagonism in human disease and identify a possible factor contributing to the inability of patients to clear HCV.

Comprehensive serum profiling for the discovery of epithelial ovarian cancer biomarkers.

FDA-cleared ovarian cancer biomarkers are limited to CA-125 and HE4 for monitoring and recurrence and OVA1, a multivariate panel consisting of CA-125 and four additional biomarkers, for referring patients to a specialist. Due to relatively poor performance of these tests, more accurate and broadly applicable biomarkers are needed. We evaluated the dysregulation of 259 candidate cancer markers in serum samples from 499 patients. Sera were collected prospectively at 11 monitored sites under a single well-defined protocol. All stages of ovarian cancer and common benign gynecological conditions were represented. To ensure consistency and comparability of biomarker comparisons, all measurements were performed on a single platform, at a single site, using a panel of rigorously calibrated, qualified, high-throughput, multiplexed immunoassays and all analyses were conducted using the same software. Each marker was evaluated independently for its ability to differentiate ovarian cancer from benign conditions. A total of 175 markers were dysregulated in the cancer samples. HE4 (AUC=0.933) and CA-125 (AUC=0.907) were the most informative biomarkers, followed by IL-2 receptor α, α1-antitrypsin, C-reactive protein, YKL-40, cellular fibronectin, CA-72-4 and prostasin (AUC>0.800). To improve the discrimination between cancer and benign conditions, a simple multivariate combination of markers was explored using logistic regression. When combined into a single panel, the nine most informative individual biomarkers yielded an AUC value of 0.950, significantly higher than obtained when combining the markers in the OVA1 panel (AUC 0.912). Additionally, at a threshold sensitivity of 90%, the combination of the top 9 markers gave 88.9% specificity compared to 63.4% specificity for the OVA1 markers. Although a blinded validation study has not yet been performed, these results indicate that alternative biomarker combinations might lead to significant improvements in the detection of ovarian cancer.

Somatic sex determination in Caenorhabditis elegans is modulated by SUP-26 repression of tra-2 translation.

Translational repression mediated by RNA-binding proteins or micro RNAs has emerged as a major regulatory mechanism for fine-tuning important biological processes. In Caenorhabditis elegans, translational repression of the key sex-determination gene tra-2 (tra, transformer) is controlled by a 28-nucleotide repeat element, the TRA-2/GLI element (TGE), located in its 3' untranslated region (UTR). Mutations that disrupt TGE or the germline-specific TGE-binding factor GLD-1 increase TRA-2 protein expression and inhibit sperm production in hermaphrodites. Here we report the characterization of the sup-26 gene, which regulates sex determination in the soma and encodes an RNA recognition motif (RRM)-containing protein. We show that SUP-26 regulates the level of the TRA-2 protein through TGE in vivo and binds directly to TGE in vitro through its RRM domain. Interestingly, SUP-26 associates with poly(A)-binding protein 1 (PAB-1) in vivo and may repress tra-2 expression by inhibiting the translation-stimulating activity of PAB-1. Taken together, our results provide further insight into how mRNA-binding factors repress translation and modulate sexual development in different tissues of C. elegans.

Live imaging of apoptotic cells in zebrafish.

Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.

Validation of a blood-based laboratory test to aid in the confirmation of a diagnosis of schizophrenia.

We describe the validation of a serum-based test developed by Rules-Based Medicine which can be used to help confirm the diagnosis of schizophrenia. In preliminary studies using multiplex immunoassay profiling technology, we identified a disease signature comprised of 51 analytes which could distinguish schizophrenia (n = 250) from control (n = 230) subjects. In the next stage, these analytes were developed as a refined 51-plex immunoassay panel for validation using a large independent cohort of schizophrenia (n = 577) and control (n = 229) subjects. The resulting test yielded an overall sensitivity of 83% and specificity of 83% with a receiver operating characteristic area under the curve (ROC-AUC) of 89%. These 51 immunoassays and the associated decision rule delivered a sensitive and specific prediction for the presence of schizophrenia in patients compared to matched healthy controls.

Caenorhabditis elegans myotubularin MTM-1 negatively regulates the engulfment of apoptotic cells.

During programmed cell death, apoptotic cells are recognized and rapidly engulfed by phagocytes. Although a number of genes have been identified that promote cell corpse engulfment, it is not well understood how phagocytosis of apoptotic cells is negatively regulated. Here we have identified Caenorhabditis elegans myotubularin MTM-1 as a negative regulator of cell corpse engulfment. Myotubularins (MTMs) constitute a large, highly conserved family of lipid phosphatases. MTM gene mutations are associated with various human diseases, but the cellular functions of MTM proteins are not clearly defined. We found that inactivation of MTM-1 caused significant reduction in cell corpses in strong loss-of-function mutants of ced-1, ced-6, ced-7, and ced-2, but not in animals deficient in the ced-5, ced-12, or ced-10 genes. In contrast, overexpression of MTM-1 resulted in accumulation of cell corpses. This effect is dependent on the lipid phosphatase activity of MTM-1. We show that loss of mtm-1 function accelerates the clearance of cell corpses by promoting their internalization. Importantly, the reduction of cell corpses caused by mtm-1 RNAi not only requires the activities of CED-5, CED-12, and CED-10, but also needs the functions of the phosphatidylinositol 3-kinases (PI3Ks) VPS-34 and PIKI-1. We found that MTM-1 localizes to the plasma membrane in several known engulfing cell types and may modulate the level of phosphatidylinositol 3-phosphate (PtdIns(3)P) in vivo. We propose that MTM-1 negatively regulates cell corpse engulfment through the CED-5/CED-12/CED-10 module by dephosphorylating PtdIns(3)P on the plasma membrane.

Molecular analyte profiling of the early events and tissue conditioning following intravesical bacillus calmette-guerin therapy in patients with superficial bladder cancer.

PURPOSE: We characterized the innate immune response to intravesical bacillus Calmette-Guerin therapy using a systems approach based on proteomic and cytometric screens. MATERIALS AND METHODS: Blood and urine were collected from patients receiving intravesical bacillus Calmette-Guerin therapy before, and 2 and 4 hours after bacillus Calmette-Guerin treatment, at the first and third instillation. Proteomic and cytometry based screens were performed. RESULTS: Molecular analyte profiling revealed a prime/boost pattern to the innate response to intravesical bacillus Calmette-Guerin. We identified 36 statistically significant changes in the proteins induced during the third instillation compared to the initial treatment. These analytes were classified into 3 categories of 1) plasma proteins that leaked into the urine, 2) cytokines/chemokines produced locally during the first hours of inflammation and 3) other innate molecules that modulate the bladder microenvironment. To characterize the marked increase in the inflammatory response after multiple treatments we evaluated the cells present in the urine and again a prime/boost response was revealed. For the locally produced analytes it was possible to define the cell source(s) and, thus, provide a first generation map of what occurs during the initial phase of bacillus Calmette-Guerin therapy. CONCLUSIONS: This study provides in vivo information concerning the ability of bacillus Calmette-Guerin to sensitize the tissue microenvironment to enhance innate responses and establishes a framework for improving vaccination strategies while decreasing adverse events.

Multianalyte profiling of serum antigens and autoimmune and infectious disease molecules to identify biomarkers dysregulated in epithelial ovarian cancer.

Ovarian cancer is the deadliest gynecologic cancer in the United States. When detected early, the 5-year survival rate is 92%, although most cases remain undetected until the late stages where 5-year survival rates are 30%. Serum biomarkers may hold promise. Although many markers have been proposed and multivariate diagnostic models were built to fit the data on small, disparate sample sets, there has been no systematic evaluation of these markers on a single, large, well-defined sample set. To address this, we evaluated the dysregulation of 204 molecules in a sample set consisting of serum from 294 patients, collected from multiple collection sites, under a well-defined Gynecologic Oncology Group protocol. The population, weighted with early-stage cancers to assess biomarker value for early detection, contained all stages of ovarian cancer and common benign gynecologic conditions. The panel of serum molecules was assayed using rigorously qualified, high-throughput, multiplexed immunoassays and evaluated for their independent ovarian cancer diagnostic potential. Seventy-seven biomarkers were dysregulated in the ovarian cancer samples, although cancer antigen 125, C-reactive protein, epidermal growth factor receptor, interleukin 10, interleukin 8, connective tissue growth factor, haptoglobin, and tissue inhibitor of metalloproteinase 1 stood out as the most informative. When analyzed by cancer subtype and stage, there were differences in the relative value of biomarkers. In this study, using a large sample cohort, we show that some of the reported ovarian cancer biomarkers are more robust than others, and we identify additional informative candidates. These findings may guide the development of multivariate diagnostic models, which should be tested on additional, prospectively collected samples.

Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry.

The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.

Targeting of PP2C in budding yeast.

Type 2C Ser/Thr phosphatases or PP2Cs are monomeric metal-requiring protein phosphatases that are present in prokaryotes and eukaryotes. In the yeast Saccharomyces cerevisiae, there are seven PP2Cs called PTCs (phosphatase 2C). Molecular genetic studies have implicated PTCs in many different functions, including RNA splicing, the unfolded protein response, mitogen-activated protein kinase (MAPK) pathway, and cell-cycle regulation. We have shown that three PTCs (Ptc1, Ptc2, and Ptc3), regulate the stress-activated high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway. Proteomics studies have provided additional possible functions for these phosphatases by identifying interacting proteins. These studies have also provided the possible means by which these phosphatases are targeted to their substrates. For example, Nbp2-Ptc1 was identified as an interacting pair in yeast two-hybrid studies, and Nbp2 was found together with Ptc1 and HOG pathway kinases. We have shown that Nbp2 is an adapter in this pathway, mediating interaction between Ptc1 and the Pbs2 MAP/ERK kinase in the HOG pathway.

Nbp2 targets the Ptc1-type 2C Ser/Thr phosphatase to the HOG MAPK pathway.

The yeast high osmolarity glycerol (HOG) pathway signals via the Pbs2 MEK and the Hog1 MAPK, whose activity requires phosphorylation of Thr and Tyr in the activation loop. The Ptc1-type 2C Ser/Thr phosphatase (PP2C) inactivates Hog1 by dephosphorylating phospho-Thr, while the Ptp2 and Ptp3 protein tyrosine phosphatases dephosphorylate phospho-Tyr. In this work, we show that the SH3 domain-containing protein Nbp2 negatively regulates Hog1 by recruiting Ptc1 to the Pbs2-Hog1 complex. Consistent with this role, NBP2 acted as a negative regulator similar to PTC1 in phenotypic assays. Biochemical analysis showed that Nbp2, like Ptc1, was required to inactivate Hog1 during adaptation. As predicted for an adapter, deletion of NBP2 disrupted Ptc1-Pbs2 complex formation. Furthermore, Nbp2 contained separate binding sites for Ptc1 and Pbs2: the novel N-terminal domain bound Ptc1, while the SH3 domain bound Pbs2. In addition, the Pbs2 scaffold bound the Nbp2 SH3 via a Pro-rich motif distinct from that which binds the SH3 domain of the positive regulator Sho1. Thus, Nbp2 recruits Ptc1 to Pbs2, a scaffold for both negative and positive regulators.