Discovery of a Hidden Proinflammatory Signaling Proteome Using a Large-Scale, Targeted Antibody Microarray Platform.

Dynamic post-translational processes regulate protein expression in eukaryotic cells. However, the processes are difficult to assess on a proteomic scale because protein levels actually reflect the sum of individual biosynthesis and degradation rates. These rates are presently hidden from the conventional proteomic technologies. We present here a novel and dynamic, antibody microarray-based time-resolved approach to simultaneously measure not only the total protein changes but also the rates of biosynthesis of low abundance proteins in the proteome of lung epithelial cells. In this chapter, we describe the feasibility of this technique by investigating the complete proteomic kinetics of 507 low abundance proteins in cultured cystic fibrosis (CF) lung epithelial cells using 35[S] methionine or 32[P] and the consequences of repair by gene therapy with [wildtype] CFTR. This novel antibody microarray-based technology identifies relevant, hidden proteins whose regulation by the CF genotype would never have been detected by simple measurements of total proteomic masses.

A Dominant-Negative Mutant of ANXA7 Impairs Calcium Signaling and Enhances the Proliferation of Prostate Cancer Cells by Downregulating the IP3 Receptor and the PI3K/mTOR Pathway.

Annexin A7/ANXA7 is a calcium-dependent membrane fusion protein with tumor suppressor gene (TSG) properties, which is located on chromosome 10q21 and is thought to function in the regulation of calcium homeostasis and tumorigenesis. However, whether the molecular mechanisms for tumor suppression are also involved in the calcium- and phospholipid-binding properties of ANXA7 remain to be elucidated. We hypothesized that the 4 C-terminal endonexin-fold repeats in ANXA7 (GX(X)GT), which are contained within each of the 4 annexin repeats with 70 amino acids, are responsible for both calcium- and GTP-dependent membrane fusion and the tumor suppressor function. Here, we identified a dominant-negative triple mutant (DNTM/DN-ANXA7J) that dramatically suppressed the ability of ANXA7 to fuse with artificial membranes while also inhibiting tumor cell proliferation and sensitizing cells to cell death. We also found that the [DNTM]ANA7 mutation altered the membrane fusion rate and the ability to bind calcium and phospholipids. In addition, in prostate cancer cells, our data revealed that variations in phosphatidylserine exposure, membrane permeabilization, and cellular apoptosis were associated with differential IP3 receptor expression and PI3K/AKT/mTOR modulation. In conclusion, we discovered a triple mutant of ANXA7, associated with calcium and phospholipid binding, which leads to the loss of several essential functions of ANXA7 pertinent to tumor protection and highlights the importance of the calcium signaling and membrane fusion functions of ANXA7 for preventing tumorigenesis.

ANXA7-GTPase as Tumor Suppressor: Mechanisms and Therapeutic Opportunities.

Chromosomal abnormalities, including homozygous deletions and loss of heterozygosity at 10q, are commonly observed in most human tumors, including prostate, breast, and kidney cancers. The ANXA7-GTPase is a tumor suppressor, which is frequently inactivated by genomic alterations at 10q21. In the last few years, considerable amounts of data have accumulated describing inactivation of ANXA7-GTPase in a variety of human malignancies and demonstrating the tumor suppressor potential of ANXA7-GTPase. ANXA7-GTPase contains a calcium binding domain that classifies it as a member of the annexin family. The cancer-specific expression of ANXA7-GTPase, coupled with its importance in regulating cell death, cell motility, and invasion, makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Recently, emerging evidence suggests that ANXA7-GTPase is a critical factor associated with the metastatic state of several cancers and can be used as a risk biomarker for HER2 negative breast cancer patients. Cross talk between ANXA7, PTEN, and EGFR leads to constitutive activation of PI3K-AKT signaling, a central pathway of tumor cell survival and proliferation. This review focuses on the recent progress in understanding the tumor suppressor functions of ANXA7-GTPase emphasizing the role of this gene in Ca2+ metabolism, and exploring opportunities for function as an example of a calcium binding GTPase acting as a tumor suppressor and opportunities for ANXA7-GTPase gene cancer therapy.

Validation of Biomarker Proteins Using Reverse Capture Protein Microarrays.

Genomics has revolutionized large-scale and high-throughput sequencing and has led to the discovery of thousands of new proteins. Protein chip technology is emerging as a miniaturized and highly parallel platform that is suited to rapid, simultaneous screening of large numbers of proteins and the analysis of various protein-binding activities, enzyme substrate relationships, and posttranslational modifications. Specifically, reverse capture protein microarrays provide the most appropriate platform for identifying low-abundance, disease-specific biomarker proteins in a sea of high-abundance proteins from biological fluids such as blood, serum, plasma, saliva, urine, and cerebrospinal fluid as well as tissues and cells obtained by biopsy. Samples from hundreds of patients can be spotted in serial dilutions on many replicate glass slides. Each slide can then be probed with one specific antibody to the biomarker of interest. That antibody's titer can then be determined quantitatively for each patient, allowing for the statistical assessment and validation of the diagnostic or prognostic utility of that particular antigen. As the technology matures and the availability of validated, platform-compatible antibodies increases, the platform will move further into the desirable realm of discovery science for detecting and quantitating low-abundance signaling proteins. In this chapter, we describe methods for the successful application of the reverse capture protein microarray platform for which we have made substantial contributions to the development and application of this method, particularly in the use of body fluids other than serum/plasma.

Reduced PARP1 as a Serum Biomarker for Graft Rejection in Kidney Transplantation.

A serum proteomics platform enabling expression Profiling in transplantation-associated clinical subsets gives an opportunity to identify non-invasive biomarkers that can accurately predict transplant outcome. In this study, we attempted to identify candidate serum biomarkers that could predict kidney allograft rejection/injury, regardless of its etiological and therapeutic heterogeneity. Using serum samples collected from kidney transplantation patients and healthy controls, we first employed Clontech-500 Ab microarrays to Profile acute rejection (AR) and chronic graft injury (CGI) versus stable graft function (SF) and normal kidneys (NK). Using GenePattern analysis of duplicate arrays on pooled samples, we identified gender-independent biomarkers PARP1, MAPK1, SRP54, DP1, and p57 (FDR ≈ 25%), the concordant downregulation of which represented a detrimental Profile common for both rejection/ injury types (AR-CGI). The reverse phase arrays qualified a 2-fold upregulation of PARP1 with an ROC of 0.87 in individual samples from patients with SF vs. AR-CGI rendering serum PARP1 as a biomarker for early prognosis. Ingenuity Pathways Analysis (IPA) connected PARP1 to some other markers (MAPK1), elucidating their possible interactions and connections to the immune response and graft-versus-host disease signaling. The downregulation of serum PARP1 in the damaged graft tissues, represents a perspective non-invasive marker, predicting the failing kidney graft, regardless of rejection/injury causes or gender. Thus, the successful identification of PARP1 as a bio-marker in limited patient cohorts demonstrates that serum proteomics platform empowered by the GenePattern- and IPA-based Bioinformatics algorithm can guarantee a successful development of the clinically applicable prognostic biomarker panel.

Personalized Radioproteomics: Identification of a Protein Biomarker Signature for Preemptive Rescue by Tocopherol Succinate in CD34+ Irradiated Progenitor Cells Isolated from a Healthy Control Donor.

Tocopherol succinate (TS) has been shown to protect mice against acute radiation syndrome, however, its exact mechanism of action and its possible use in humans has not yet been evaluated. Our approach has been to test the radioprotectant properties of TS on CD34-positive stem cells from healthy volunteers. We hypothesize that a radioproteomics strategy can identify a drug-dependent, personalized proteomics signature for radioprotection. To directly test the radioproteomics hypothesis, we treated human CD34-positive stem cells with 20 µM TS for 24 h, and then exposed the cells to 2 Gy of cobalt-60 gamma-radiation. We isolated protein from all cultures and used a high throughput Antibody Microarray (AbMA) platform to measure concentrations of 725 low abundance proteins. As an in vivo control, we also tested mouse CD34-positive stem cells using the same preemptive TS paradigm on progenitor colony forming units. TS pretreatment of in vitro or in vivo CD34-positive stem cells rescued radiation-induced loss of colony-forming potential of progenitors. We identified 50 of 725 proteins that could be preemptively rescued from radiation-induced reduction by pretreatment with TS. Ingenuity Pathway Analysis (IPA) reveals that the modified proteins fall into categories dominated by epigenetic regulation, DNA repair, and inflammation. Our results suggest that radioproteomics can be used to develop personalized medicine for radioprotection using protein signatures from primary CD34-positive progenitors derived from the patient or victim prior to radiation exposure. The protective effect of TS may be due to its ability to preemptively activate epigenetic mechanisms relevant to radioprotection and to preemptively activate the programs for DNA repair and inflammation leading to cell survival.

Activation of 3-phosphoinositide-dependent kinase 1 (PDK1) and serum- and glucocorticoid-induced protein kinase 1 (SGK1) by short-chain sphingolipid C4-ceramide rescues the trafficking defect of ΔF508-cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR).

Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser(422). SGK1[Ser(P)(422)] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser(241). Then PDK1[Ser(P)(241)] phosphorylates SGK1[Ser(P)(422)] at Thr(256) to generate fully activated SGK1[Ser(422), Thr(P)(256)]. SGK1[Ser(P)(422),Thr(P)(256)] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day.

Digitoxin induces apoptosis in cancer cells by inhibiting nuclear factor of activated T-cells-driven c-MYC expression.

BACKGROUND: Cardiac glycosides such as digitoxin have been shown to directly cause apoptotic death of cancer cells both in vitro, and in vivo. However, the mechanism connecting cardiac glycoside action to apoptosis is not known. It has been reported that compounds resembling digitoxin are able to reduce c-MYC expression. Furthermore, it has been previously shown that the transcription of c-MYC depends on nuclear factor of activated T-cells (NFAT) binding sites in the c-MYC promoter. We have therefore hypothesized that NFAT might mediate digitoxin effects on c-MYC mRNA message. MATERIALS AND METHODS: We have chosen to study this process in HeLa cells where structurally intact c-MYC genes in 8q24 co-localize with human papilloma virus 18 at all integration sites. RESULTS: Here we show that within the 1(st) h following treatment with digitoxin, a significant reduction in c-MYC mRNA occurs. This is followed by a precipitous loss of c-MYC protein, activation of caspase 3, and subsequent apoptotic cell death. To test the NFAT-dependence mechanism, we analyzed the effects of digitoxin on NFAT isoform-dependent auto-activation of a NFAT-luciferase expression system. Drug dependent effects on expression varied according to each of the four canonical NFAT isoforms (1, 2, 3 or 4). The most digitoxin-sensitive NFAT isoform was NFAT1. Using c-MYC chromatin immune precipitation, we find that digitoxin inhibits interaction of NFAT1 with the proximal c-MYC promoter. CONCLUSIONS: These results suggest that the carcinotoxic activity of digitoxin includes suppression of NFAT-driven c-MYC expression.

Antibody microarrays: analysis of cystic fibrosis.

Cystic fibrosis (CF) is the most common autosomal recessive disease in the USA and Europe, whose life-limiting phenotype is manifest on epithelial cells throughout the body. The principal cause of morbidity and mortality is a massively proinflammatory condition in the lung. The mutation responsible for most cases of CF is [ΔF508]CFTR. However, the penetrance of the disease is quite variable, and adverse events leading to hospitalization cannot be easily predicted. Thus, there is a strong need for prognostic endpoints that might serve to identify impending clinical problems long before they happen. Our approach has been to search for proteomic signatures in easily accessed biological fluids that might identify the molecular basis for adverse events. We describe here a workflow that begins with patient-derived bronchial brush biopsies and progresses to analysis of serum and plasma from patients on antibody microarrays.

Elevated expression levels of ANXA11, integrins ß3 and α3, and TNF-α contribute to a candidate proteomic signature in urine for kidney allograft rejection.

PURPOSE: Kidney transplantation is the treatment of choice for end stage renal disease, with long-term allograft loss being the major obstacle, and for which potential treatments are based on a histological diagnosis. The problem is that markers for predicting graft rejection are limited in number, are invasive, and are quite non-specific. We have hypothesized that protein biomarkers might be discovered in the urine of patients when acute or chronic rejection might be occurring. EXPERIMENTAL DESIGN: We have established a workflow in which initial screening for candidate biomarkers is first performed using urine samples on large-scale antibody microarrays. This approach generated several dozen candidates. The next step is to qualify some of the strongest signals using the high-throughput Reverse Capture Protein Microarray platform. RESULTS: Four top candidates including ANXA11, Integrin α3, Integrin ß3 and TNF-α, initially identified by the antibody microarray platform, were all qualified using Reverse Capture Protein Microarrays. We also used receiver operating condition (ROC) curves to independently quantify the specificity and sensitivity of these four analytes. CONCLUSIONS AND CLINICAL RELEVANCE: The present data suggest that these novel four analytes in the urine, together or independently, may contribute to a robust and quantitative urine proteomic signature for diagnosing acute or chronic rejection of renal allografts.

Plasma Proteomic Signature in Overweight Girls Closely Correlates with Homeostasis Model Assessment (HOMA), an Objective Measure of Insulin Resistance.

Obesity is known to be associated with a large number of long-term morbidities, and while in some cases the relationship of obesity and the consequences is clear (for example, excess weight and lower extremity orthopedic problems) in others the mechanism is not as clear. One common system of categorizing overweight in terms of the likelihood of negative consequences involves using the concept of "metabolic syndrome". We hypothesized that the development of a plasma protein profile of overweight adolescents with and without the metabolic syndrome might give a more precise and informative picture of the disease process than the current clinical categorization and permit early targeted intervention. For this paper, we used antibody microarrays to analyze the plasma proteome of a group of 15 overweight female adolescent patients. Upon analysis of the proteome, the overweight patients diverged from the nonoverweight female controls. Furthermore, the overweight patients were divided by the analysis into two population clusters, each with distinctive protein expression patterns. Interestingly, the clusters were characterized by differences in insulin resistance, as measured by HOMA. Categorization according to the presence or absence of the metabolic syndrome did not yield such clusters.

Gender dependence for a subset of the low-abundance signaling proteome in human platelets.

The incidence of cardiovascular diseases is ten-times higher in males than females, although the biological basis for this gender disparity is not known. However, based on the fact that antiplatelet drugs are the mainstay for prevention and therapy, we hypothesized that the signaling proteomes in platelets from normal male donors might be more activated than platelets from normal female donors. We report here that platelets from male donors express significantly higher levels of signaling cascade proteins than platelets from female donors. In silico connectivity analysis shows that the 24 major hubs in platelets from male donors focus on pathways associated with megakaryocytic expansion and platelet activation. By contrast, the 11 major hubs in platelets from female donors were found to be either negative or neutral for platelet-relevant processes. The difference may suggest a biological mechanism for gender discrimination in cardiovascular disease.

Rescue of DeltaF508-CFTR by the SGK1/Nedd4-2 signaling pathway.

The most common mutation in cystic fibrosis (CF) is DeltaF508, which is associated with failure of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) to traffic to the plasma membrane. By a still unknown mechanism, the loss of correctly trafficked DeltaF508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane. ENaC trafficking is known to be regulated by a signaling pathway involving the glucocorticoid receptor, the serum- and glucocorticoid-regulated kinase SGK1, and the ubiquitin E3 ligase Nedd4-2. We show here that dexamethasone rescues functional expression of DeltaF508-CFTR. The half-life of DeltaF508-CFTR is also dramatically enhanced. Dexamethasone-activated DeltaF508-CFTR rescue is blocked either by the glucocorticoid receptor antagonist RU38486 or by the phosphatidylinositol 3-kinase inhibitor LY294002. Co-immunoprecipitation studies indicate that Nedd4-2 binds to both wild-type- and DeltaF508-CFTR. These complexes are inhibited by dexamethasone treatment, and CFTR ubiquitination is concomitantly decreased. We further show that knockdown of Nedd4-2 by small interfering RNA also corrects DeltaF508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated DeltaF508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells.

Tristetraprolin regulates IL-8 mRNA stability in cystic fibrosis lung epithelial cells.

Cystic fibrosis (CF) is due to mutations in the CFTR gene and is characterized by hypersecretion of the proinflammatory chemokine IL-8 into the airway lumen. Consequently, this induces the highly inflammatory cellular phenotype typical of CF. Our initial studies revealed that IL-8 mRNA is relatively stable in CF cells compared with those that had been repaired with [WT]CFTR (wild-type CFTR). Relevantly, the 3'-UTR of IL-8 mRNA contains AU-rich sequences (AREs) that have been shown to mediate posttranscriptional regulation of proinflammatory genes upon binding to ARE-binding proteins including Tristetraprolin (TTP). We therefore hypothesized that very low endogenous levels of TTP in CF cells might be responsible for the relative stability of IL-8 mRNA. As predicted, increased expression of TTP in CF cells resulted in reduced stability of IL-8 mRNA. An in vitro analysis of IL-8 mRNA stability in CF cells also revealed a TTP-induced enhancement of deadenylation causing reduction of IL-8 mRNA stability. We conclude that enhanced stability of IL-8 mRNA in TTP-deficient CF lung epithelial cells serve to drive the proinflammatory cellular phenotype in the CF lung.

Immunohistochemical localization of Phosphoglycerate mutase in capillary endothelium of the brain and periphery.

To elucidate the cellular localization of Phosphoglycerate mutase (PGAM1) type B in the brain and periphery, immunocytochemical studies were performed. The purified antigen used to generate the antiserum to PGAM1 was run on an SDS-PAGE gel, stained with coomassie blue, which yielded one sharp band at 29 kDa. Immunocytochemistry of formalin perfused rats revealed distinct localization of PGAM1 in the endothelium of the capillaries and arteries of the brain, liver and kidneys. Since enhanced glycogenesis is a well-known characteristic of cancer cells, it is of interest that sustained angiogenesis is a hallmark that distinguishes cancer cells from their normal counterparts. In view of the fact that PGAM1 increases in a variety of tumors, we suggest that PGAM1 may have a pathological role of vascular invasion into cancerous tissue.

Protein microarray platforms for clinical proteomics.

Proteomics for clinical applications is presently in a state of transition. It has become clear that the classical approaches based on 2-DE and/or MS need to be complemented by different kinds of technologies. The well-known problems include sample complexity, sensitivity, quantitation, reproducibility, and analysis time. We suggest that the new technologies for clinical proteomics can be supported by antibody-centric protein microarray platforms. These platforms presently include antibody microarrays and lysate, or reverse capture/reverse phase protein microarrays. Other forms of these arrays are in less mature developmental stages, including ORF and self assembling protein microarrays. Bioinformatic support for interpreting these arrays is becoming more available as the whole field of systems biology begins to mature. The present set of applications for these platforms is profoundly focused on certain common cancers, immunology, and cystic fibrosis. However, we predict that many more disease entities will become studied as knowledge of the power and availability of these platforms becomes more widely established. We anticipate that these platforms will eventually evolve to accommodate label-free detection technologies, human genome-scale numbers of analytes, and increases in analytic and bioinformatic speeds.

De novo biosynthetic profiling of high abundance proteins in cystic fibrosis lung epithelial cells.

In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry (Pollard, H. B., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210-2226). In the present work we compared the IB3-1 cells with IB3-1/S9 daughter cells repaired by gene transfer with AAV-(wild type)CFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [(35)S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFkappaB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that approximately 30% of the 51-member hybrid high abundance CF proteome interacts with the NFkappaB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine.

Serum proteomic signature for cystic fibrosis using an antibody microarray platform.

Antibody microarrays are a new proteomic technology, which we have developed as a platform for identifying a cystic fibrosis (CF)-specific serum proteomic signature. Serum samples from CF patients have been pooled and compared with equivalent pools of control sera in order to identify patterns of protein expression unique to CF. We find that the set of significantly differentially expressed proteins is enriched in protein mediators of inflammation from the NFkappaB signaling pathway, and in proteins that may be selectively expressed in CF-affected tissues such as lung and intestine. In several instances, we validate the data from the antibody microarrays by quantitative analysis with Reverse Capture Protein Microarrays. We conclude that antibody microarray technology is sensitive, quantitative, and robust, and can be useful as a proteomic platform to discriminate between sera from CF and control patients.

Pooled ORF expression technology (POET): using proteomics to screen pools of open reading frames for protein expression.

We have developed a pooled ORF expression technology, POET, that uses recombinational cloning and proteomic methods (two-dimensional gel electrophoresis and mass spectrometry) to identify ORFs that when expressed are likely to yield high levels of soluble, purified protein. Because the method works on pools of ORFs, the procedures needed to subclone, express, purify, and assay protein expression for hundreds of clones are greatly simplified. Small scale expression and purification of 12 positive clones identified by POET from a pool of 688 Caenorhabditis elegans ORFs expressed in Escherichia coli yielded on average 6 times as much protein as 12 negative clones. Larger scale expression and purification of six of the positive clones yielded 47-374 mg of purified protein/liter. Using POET, pools of ORFs can be constructed, and the pools of the resulting proteins can be analyzed and manipulated to rapidly acquire information about the attributes of hundreds of proteins simultaneously.

Cardiac glycosides inhibit TNF-alpha/NF-kappaB signaling by blocking recruitment of TNF receptor-associated death domain to the TNF receptor.

Digitoxin and structurally related cardiac glycoside drugs potently block activation of the TNF-alpha/NF-kappaB signaling pathway. We have hypothesized that the mechanism might be discovered by searching systematically for selective inhibitory action through the entire pathway. We report that the common action of these drugs is to block the TNF-alpha-dependent binding of TNF receptor 1 to TNF receptor-associated death domain. This drug action can be observed with native cells, such as HeLa, and reconstituted systems prepared in HEK293 cells. All other antiinflammatory effects of digitoxin on NF-kappaB and c-Jun N-terminal kinase pathways appear to follow from the blockade of this initial upstream signaling event.