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Supplementary Material from A Robust High-Content Imaging Approach for Probing the Mechanism of Action and Phenotypic Outcomes of Cell-Cycle Modulators

Supplementary Materials and Methods

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC50 = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ–stimulated macrophages (IC50 = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)70 = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non–small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer. Mol Cancer Ther; 13(2); 364–74. ©2013 AACR.

High-content screening is increasingly used to elucidate changes in cellular biology arising from treatment with small molecules and biological probes. We describe a cell classifier for automated analysis of multiparametric data from immunofluorescence microscopy and characterize the phenotypes of 41 cell-cycle modulators, including several protein kinase inhibitors in preclinical and clinical development. This method produces a consistent assessment of treatment-induced phenotypes across experiments done by different biologists and highlights the prevalence of nonuniform and concentration-dependent cellular response to treatment. Contrasting cell phenotypes from high-content screening to kinase selectivity profiles from cell-free assays highlights the limited utility of enzyme potency ratios in understanding the mechanism of action for cell-cycle kinase inhibitors. Our cell-level approach for assessing phenotypic outcomes is reliable, reproducible and capable of supporting medium throughput analyses of a wide range of cellular perturbations. Mol Cancer Ther; 10(2); 242–54. ©2011 AACR.

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform–selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A–selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition–associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A–selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

Supplementary Tables

PDF file - 43K, Effect of LY2228820 on MK2 phosphorylation in mouse peripheral blood mononuclear cells (PBMC) and from patients with multiple myeloma.

PDF file - 86K, Supplemental Table 1. Kinases where LY2228820 shows >1000-fold selectivity in vitro (p38 MAPK vs. other kinase).

PDF file - 90K, shRNA silencing of p38a MAPK in U-87MG glioma (Westerns and xenograft tumor growth data).

Supplementary Figures

Table S1:Summary of baseline pathological diagnosis; Table S2:Noncompartmental Pharmacokinetic Summary Following Oral Administration of ralimetinib on Day 1 and on Day 14 (Cycle 1) - Capsule Formulation; Table S3: Noncompartmental Pharmacokinetic Summary Following Oral Administration of ralimetinib on Day 1 and on Day 14 (Cycle 1) - Tablet Formulation

Trial Profile

Purpose:Checkpoint kinase 1 (CHK1) inhibitors potentiate the DNA-damaging effects of cytotoxic therapies and/or promote elevated levels of replication stress, leading to tumor cell death. Prexasertib (LY2606368) is a CHK1 small-molecule inhibitor under clinical evaluation in multiple adult and pediatric cancers. In this study, prexasertib was tested in a large panel of preclinical models of pediatric solid malignancies alone or in combination with chemotherapy.Experimental Design:DNA damage and changes in cell signaling following in vitro prexasertib treatment in pediatric sarcoma cell lines were analyzed by Western blot and high content imaging. Antitumor activity of prexasertib as a single agent or in combination with different chemotherapies was explored in cell line–derived (CDX) and patient-derived xenograft (PDX) mouse models representing nine different pediatric cancer histologies.Results:Pediatric sarcoma cell lines were highly sensitive to prexasertib treatment in vitro, resulting in activation of the DNA damage response. Two PDX models of desmoplastic small round cell tumor and one malignant rhabdoid tumor CDX model responded to prexasertib with complete regression. Prexasertib monotherapy also elicited robust responses in mouse models of rhabdomyosarcoma. Concurrent administration with chemotherapy was sufficient to overcome innate resistance or prevent acquired resistance to prexasertib in preclinical models of neuroblastoma, osteosarcoma, and Ewing sarcoma, or alveolar rhabdomyosarcoma, respectively.Conclusions:Prexasertib has significant antitumor effects as a monotherapy or in combination with chemotherapy in multiple preclinical models of pediatric cancer. These findings support further investigation of prexasertib in pediatric malignancies.

Purpose: p38 MAPK regulates the production of cytokines in the tumor microenvironment and enables cancer cells to survive despite oncogenic stress, radiotherapy, chemotherapy, and targeted therapies. Ralimetinib (LY2228820 dimesylate) is a selective small-molecule inhibitor of p38 MAPK. This phase I study aimed to evaluate the safety and tolerability of ralimetinib, as a single agent and in combination with tamoxifen, when administered orally to patients with advanced cancer.Experimental Design: The study design consisted of a dose-escalation phase performed in a 3+3 design (Part A; n = 54), two dose-confirmation phases [Part B at 420 mg (n = 18) and Part C at 300 mg (n = 8)], and a tumor-specific expansion phase in combination with tamoxifen for women with hormone receptor–positive metastatic breast cancer refractory to aromatase inhibitors (Part D; n = 9). Ralimetinib was administered orally every 12 hours on days 1 to 14 of a 28-day cycle.Results: Eighty-nine patients received ralimetinib at 11 dose levels (10, 20, 40, 65, 90, 120, 160, 200, 300, 420, and 560 mg). Plasma exposure of ralimetinib (Cmax and AUC) increased in a dose-dependent manner. After a single dose, ralimetinib inhibited p38 MAPK–induced phosphorylation of MAPKAP-K2 in peripheral blood mononuclear cells. The most common adverse events, possibly drug-related, included rash, fatigue, nausea, constipation, pruritus, and vomiting. The recommended phase II dose was 300 mg every 12 hours as monotherapy or in combination with tamoxifen. Although no patients achieved a complete response or partial response,19 patients (21.3%) achieved stable disease with a median duration of 3.7 months, with 9 of these patients on study for ≥6 cycles.Conclusions: Ralimetinib demonstrated acceptable safety, tolerability, and pharmacokinetics for patients with advanced cancer. Clin Cancer Res; 22(5); 1095–102. ©2015 AACR.

Supplemental methodology, specifically the in vitro cord formation assay and the enzyme-linked immunosorbent assay.

Supplementary Methods, Supplementary Tables 1-6, Supplementary Figures 1-9

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G2–M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma.Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line–derived xenograft mouse models of neuroblastoma.Results: Within 24 hours, single-agent prexasertib promoted γH2AX–positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA–PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature.Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354–63. ©2017 AACR.

Elevated eukaryotic translation initiation factor 4E (eIF4E) function induces malignancy in experimental models by selectively enhancing translation of key malignancy-related mRNAs (c-myc and BCL-2). eIF4E activation may reflect increased eIF4E expression or phosphorylation of its inhibitory binding proteins (4E-BP). By immunohistochemical analyses of 148 tissues from 89 prostate cancer patients, we now show that both eIF4E expression and 4E-BP1 phosphorylation (p4E-BP1) are increased significantly, particularly in advanced prostate cancer versus benign prostatic hyperplasia tissues. Further, increased eIF4E and p4E-BP1 levels are significantly related to reduced patient survival, whereas uniform 4E-BP1 expression is significantly related to better patient survival. Both immunohistochemistry and Western blotting reveal that elevated eIF4E and p4E-BP1 are evident in the same prostate cancer tissues. In two distinct prostate cancer cell models, the progression to androgen independence also involves increased eIF4E activation. In these prostate cancer cells, reducing eIF4E expression with an eIF4E-specific antisense oligonucleotide currently in phase I clinical trials robustly induces apoptosis, regardless of cell cycle phase, and reduces expression of the eIF4E-regulated proteins BCL-2 and c-myc. Collectively, these data implicate eIF4E activation in prostate cancer and suggest that targeting eIF4E may be attractive for prostate cancer therapy. [Cancer Res 2009;69(9):3866–73]

Supplementary Table 1 from eIF4E Activation Is Commonly Elevated in Advanced Human Prostate Cancers and Significantly Related to Reduced Patient Survival

Advancements in state-of-the-art molecular profiling techniques have resulted in better understanding of pediatric cancers and driver events. It has become apparent that pediatric cancers are significantly more heterogeneous than previously thought as evidenced by the number of novel entities and subtypes that have been identified with distinct molecular and clinical characteristics. For most of these newly recognized entities there are extremely limited treatment options available. The ITCC-P4 consortium is an international collaboration between several European academic centers and pharmaceutical companies, with the overall aim to establish a sustainable platform of >400 molecularly well-characterized PDX models of high-risk pediatric cancers, their tumors and matching controls and to use the PDX models for in vivo testing of novel mechanism-of-action based treatments. Currently, 251 models are fully characterized, including 182 brain and 69 non-brain PDX models, representing 112 primary models, 92 relapse, 42 metastasis and 4 progressions under treatment models. Using low coverage whole-genome and whole exome sequencing, somatic mutation calling, DNA copy number and methylation analysis we aim to define genetic features in our PDX models and estimate the molecular fidelity of PDX models compared to their patient tumor. Based on DNA methylation profiling we identified 43 different tumor subgroups within 18 cancer entities. Mutational landscape analysis identified key somatic and germline oncogenic drivers. Ependymoma PDX models displayed the C11orf95-RELA fusion event, YAP1, C11orf95 and RELA structural variants. Medulloblastoma models were driven by MYCN, TP53, GLI2, SUFU and PTEN. High-grade glioma samples showed TP53, ATRX, MYCN and PIK3CA somatic SNVs, along with focal deletions in CDKN2A in chromosome 9. Neuroblastoma models were enriched for ALK SNVs and/or MYCN focal amplification, ATRX SNVs and CDKN2A/B deletions. Tumor mutational burden across entities and copy number analysis was performed to identify allele-specific copy number detection in tumor-normal pairs. Large chromosomal aberrations (deletions, duplications) detected in the PDX models were concurrent with molecular alterations frequently observed in each tumor type -isochromosome 17 was detected in 5 medulloblastoma models, while deletion of chromosome arm 1p or gain of parts of 17q in neuroblastomas which correlate with tumor progression. We observe clonal evolution of somatic variants not only in certain PDX-tumor pairs but also between disease states. The multi-omics approach in this study provides insight into the mutational landscape and patterns of the PDX models thus providing an overview of molecular mechanisms facilitating the identification and prioritization of oncogenic drivers and potential biomarkers for optimal treatment therapies. Citation Format: Apurva Gopisetty, Aniello Federico, Didier Surdez, Yasmine Iddir, Sakina Zaidi, Alexandra Saint-Charles, Joshua Waterfall, Elnaz Saberi-Ansari, Justyna Wierzbinska, Andreas Schlicker, Norman Mack, Benjamin Schwalm, Christopher Previti, Lena Weiser, Ivo Buchhalter, Anna-Lisa Böttcher, Martin Sill, Robert Autry, Frank Estermann, David Jones, Richard Volckmann, Danny Zwijnenburg, Angelika Eggert, Olaf Heidenreich, Fatima Iradier, Irmela Jeremias, Heinrich Kovar, Jan-Henning Klusmann, Klaus-Michael Debatin, Simon Bomken, Petra Hamerlik, Maureen Hattersley, Olaf Witt, Louis Chesler, Alan Mackay, Johannes Gojo, Stefano Cairo, Julia Schueler, Johannes Schulte, Birgit Geoerger, Jan J. Molenaar, David J. Shields, Hubert N. Caron, Gilles Vassal, Louis F. Stancato, Stefan M. Pfister, Natalie Jaeger, Jan Koster, Marcel Kool, Gudrun Schleiermacher. ITCC-P4: Genomic profiling and analyses of pediatric patient tumor and patient-derived xenograft (PDX) models for high throughput in vivo testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 234.

This article describes the development of micro-opioid receptor (MOR) binding and GTPgammaS functional SPAs as improved screening tools for the identification of MOR antagonists. Opioid receptors are members of the seven-transmembrane G protein-coupled receptor (GPCR) family and are involved in the control of various aspects of human physiology, including pain, stress, reward, addiction, respiration, gastric motility, and pituitary hormone secretion. Activation of the MOR initiates intracellular signaling pathways leading to a reduction in intracellular cyclic AMP levels, inhibition of calcium channels, and activation of potassium channels resulting in a reduction of the excitability of neurons. Characterization of opioid receptor ligand binding has traditionally been accomplished through the use of low throughput filtration-based binding assays, whereas functional activity has been based upon cyclic AMP measurements or filtration-based GTPgammaS functional assays. This report describes the development of a MOR displacement binding SPA using the radiolabeled antagonist [(3)H]diprenorphine ((3)H-DPN). The assay was optimized using statistical experimental design and demonstrates the stability and robustness necessary for HTS. The assay was biased toward the identification of MOR antagonists through the addition of Na(+). Our assay conditions also minimized the phenomenon of ligand depletion, a problem commonly observed in low-volume assays using high receptor-expressing cell lines. The optimized procedure revealed (3)H-DPN affinity constants at the MOR that were consistent with results obtained using filtration methods (K(D) (SPA) = 1.89 +/- 0.24 nM, K(D) (filtration) = 1.88 +/- 0.35 nM). The binding SPA identified known opioid receptor modulators contained within the Library of Pharmacological Active Compounds (LOPAC) cassette, and the GTPgammaS scintillation proximity assay (SPA) was used to confirm the functional activity of the LOPAC antagonists acting at the MOR. Conversion of the ligand binding and GTPgammaS functional assays to a homogeneous SPA generated a simple assay with dramatically increased throughput. Data from the development and implementation of the displacement binding and GTPgammaS functional SPAs are presented.

The Rho-associated coiled-coil-containing protein serine/threonine kinases ROCK-I and ROCK-II are thought to play a major role in cytoskeletal dynamics by serving as downstream effectors of the Rho/Rac family of cytokine- and growth factor-activated small GTPases. As such, the ROCK family members are attractive intervention targets for a variety of pathologies, including cancer and cardiovascular disease. The authors developed a high-throughput screen to identify ROCK-II inhibitors and report results from a direct comparison of 2 screening campaigns for ROCK-II inhibitors using fluorescence polarization (FP) and filter binding (FB). Screening protocols to identify inhibitors of ROCK-II were developed in FB and FP formats under similar assay and kinetic conditions. A 30,000-member compound library was screened using FB ((33)P) and FP detection systems, and compounds that were active in either assay were retested in 5-point curve confirmation assays. Analysis of these data showed an approximate 95% agreement of compounds identified as active in both assay formats. Also, compound potency determinations from FB and FP had a high degree of correlation and were considered equivalent. These data suggest that the assay methodology has little impact on the quality and productivity of the screen, provided that the assays are developed to standardize kinetic conditions.

Virtually all known cellular processes involve modulation of cellular signaling pathways via changes in protein phosphorylation. With genomics efforts more than doubling the number of proteins available for analysis, a major challenge will be to identify unknown phosphoproteins as they exist in the normal or diseased intracellular environment. Recent advances in proteomic technology have made it possible to examine changes in protein expression with much greater resolution than was previously possible. In this report, we describe a rapid and reproducible method for identifying phosphoproteins upregulated in response to activation of cell surface receptors. Phosphotyrosine-containing proteins were immunoprecipitated from IFNalpha- or IL2-treated primary human lymphocyte extracts using a novel anti-phosphotyrosine immunoprecipitation technique. This technique takes advantage of differing antibody affinities for epitopes on native versus denatured proteins. Following separation from the immunopellets, phosphoproteins are resolved by two-dimensional polyacrylamide gel electrophoresis. With this method, we identified known proteins phosphorylated in response to IL2 or IFNalpha using both silver staining and Western blotting for protein detection/identification. The silver-stained immunoprecipitation profile serves as a fingerprint for phosphorylation events that occur in response to cytokine treatment. By merging these techniques with mass spectrometric microsequencing, new capabilities are achieved. It will then be possible to identify novel signaling proteins that are activated in response to a variety of stimuli, including receptor activation, disease progression, etc.

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from cell surface receptors to the nucleus. In vertebrates, Raf signaling has been implicated in the progression of mouse embryos through the two-cell stage and in the induction of posterior mesoderm. However, mouse embryos mutant for each of the Raf genes exhibit no developmental defects before mid-gestation. Here we describe the phenotype of mouse mutants with different combinations of mutant Craf-1 and Braf alleles. Our results show that Raf signaling is indeed indispensable for normal development beyond the blastocyst stage. However, due to a significant redundancy between Craf-1 and Braf, either gene is sufficient for normal development until mid-gestation. The molecular and developmental mechanisms for this redundancy were investigated by monitoring the expression of Raf genes throughout embryogenesis and by biochemical studies in mutant cell lines.

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from a variety of cell surface receptors to the nucleus. We report here that Craf-1 is essential for mouse development, as its mutation results in embryonic lethality. Developmental defects are found in mutant placentas as well as in the skin and in the lungs of mutant embryos. Craf-1 mutants also display a generalized growth retardation which is consistent with the ubiquitous expression of Craf-1 and which could be due to the reduced proliferation of mutant cells. Interestingly, the time-point of embryonal death varies depending on the genetic background. This suggests that Craf-1-mediated signaling is affected by genetic background-specific alleles of other genes.

Signal transduction through both cytokine and lymphocyte antigen receptors shares some common pathways by which they initiate cellular responses, such as activation of mitogen-activated protein kinase(s)1,2. However, other signalling components appear to be uniquely coupled to each receptor. For example, the interferon receptors transduce regulatory signals through the JAK/STAT pathway, resulting in an inhibition of growth and of antiviral effects, whereas this pathway apparently plays no role in T-cell-receptor (TCR)-dependent gene expression3,4. Conversely, signal transduction through the TCR requires the tyrosine kinases Lck and ZAP-70 and the tyrosine phosphatase CD45 (ref. 5). Here we show that, unexpectedly, transmission of growth-inhibitory signals by interferon-α (IFN-α) in T cells requires the expression and association of CD45, Lck and ZAP-70 with the IFN-α-receptor signalling complex.

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor·hsp90 heterocomplexes. We have reconstituted a minimal receptor·hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833–12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR·hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997)J. Biol. Chem. 272, 13047–13054). In this work, we show that addition of p23 to native GR·hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR·hsp90 heterocomplexes prepared with the minimal (hsp90·p60·hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR·hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90·p60·hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR·hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90·p60·hsp70 forms a GR·hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR·hsp90 heterocomplex to inactivation and disassembly.

Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways.

We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras.Raf, Ras.Src, Raf.Mek, and Raf.Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf.hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.

Aggregation of the high affinity receptor for IgE (FcϵRI) on the mucosal mast cell line, RBL-2H3, results in the rapid and persistent tyrosine phosphorylation of Vav. Immunoprecipitation of Vav from activated cells revealed co-immunoprecipitated phosphoproteins of molecular weights identical to the FcϵRI β and γ chains, and the former was reactive with antibody to the FcϵRI β chain. Conversely, Western blots revealed the presence of p95 Vav in FcϵRI immunoprecipitates. The association of Vav and of Grb2 with the receptor was found to be regulated by aggregation of the receptor, and the interaction of Vav with the FcϵRI was localized to the γ chain. To gain insight on the signaling pathway in which Vav participates, we investigated the in vivo associations of Vav with other molecules. A reducible chemical cross-linking agent was used to covalently maintain protein interactions under nonreducing conditions. A fraction of Vav increased in mass to form a complex of >300 kDa in molecular mass. Under reducing conditions the cross-linked Vav immunoprecipitates showed the presence of Grb2, Raf-1, and p42mapk (ERK2). In vitro kinase assays of Raf-1 activity associated with Vav revealed that this complex had an activity greater than that of Raf-1 derived from nonactivated cells, and aggregation of the FcϵRI did not modulate this activity. In contrast, aggregation of the FcϵRI increased the total Raf-1 activity by 2-5-fold. These results demonstrate that Vav associates constitutively with components of the mitogen-activated protein kinase pathway to form an active multimeric signaling complex whose in vivo activity and associations may be directed by aggregation of the FcϵRI. The findings of this study may also be relevant to other members of the immune recognition receptor family that share the T-cell antigen receptor ζ/γ chains.

The hormone binding domain (HBD) of the glucocorticoid receptor (GR) contains five cysteine residues, with three of them being spaced close to one another in the steroid binding pocket. The HBD also contains the contact region for the chaperone protein hsp90, which must be bound to the GR for it to have a steroid binding conformation. Binding of hsp90 to the receptor through its HBD inactivates the DNA binding domain (DBD). The DBD contains a number of cysteines essential to its DNA binding activity. Here, we assess the effects of hsp90 binding on the accessibility of cysteine residues in both the HBD and DBD to derivatization by a thiol-specific reagent. We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding activity of the immunopurified, untransformed GRhsp90 complex in a manner that is prevented by the sulfhydryl reagents cysteine and dithiothreitol but is not reversed by them. The I-labeled IAT derivative N-iodoacetyl-3-[I]iodotyrosine ([I]IAIT) covalently labels the immunopurified, hsp90-bound receptor in a thiol-specific manner. Dissociation of hsp90 leads to an 2-fold increase in [I]IAIT labeling of the full-length, 100-kDa GR. The increase in thiol labeling is related to the presence of hsp90 because it is blocked by molybdate, which prevents hsp90 dissociation. Cleavage of the [I]IAIT-labeled receptor with trypsin yields a 15-kDa labeled fragment containing the DBD and a 30-kDa labeled fragment containing all of the cysteines in the HBD and the contact region for hsp90. Dissociation of hsp90 from the GR results in a 2.3-fold increase in [I]IAIT labeling of the 15-kDa fragment and a 50% decrease in labeling of the 30-kDa fragment. These data are consistent with the proposal that dissociation of hsp90 from the GR produces a conformational change in the HBD such that some of the thiols that are exposed in the GRhsp90 complex become buried and are no longer accessible to the [I]IAIT probe. In contrast, binding of the GR to hsp90 restricts access of cysteines in the DBD to this small thiol-derivatizing agent, a restriction that is relieved as a result of unmasking or conformational change accompanying hsp90 dissociation.

A variety of cytokines and growth factors act through an induction of gene expression mediated by a family of latent transcription factors called STAT (signal transducers and activators of transcription) proteins. Ligand-induced tyrosine phosphorylation of the STATs promotes their homodimer and heterodimer formation and subsequent nuclear translocation. We demonstrate here that STAT protein heterocomplexes exist prior to cytokine treatment. When unstimulated HeLa cells are ruptured in hypotonic buffer without salt or detergent, immunoadsorption of either STAT1 or STAT2 from the resulting cytosol yields coimmunoadsorption of the other STAT protein. Similarly, STAT1-STAT3 heterocomplexes are coimmunoadsorbed from hypotonic cytosol. STAT1 and STAT2 or STAT1 and STAT3 translated in reticulocyte lysate spontaneously form heterocomplexes when the translation lysates are mixed at 0 degrees C. Our data suggest that interferon-alpha /beta-induced tyrosine phosphorylation increases the stability of a preexisting, latent, STAT1-STAT2 signaling complex. Newly translated STAT1 binds in equilibrium fashion to STAT2 and STAT3, but we show that STAT2 and STAT3 exist in separate heterocomplexes with STAT1, consistent with a model in which STAT1 contains a common binding site for other STAT proteins.

The hormone-binding domain of the glucocorticoid receptor must be bound to heat shock protein (hsp) 90 for it to have a high-affinity steroid-binding conformation. Cell-free assembly of a glucocorticoid receptor-hsp90 heterocomplex is brought about in reticulocyte lysate by a preformed protein-folding complex containing hsp90, hsp70, and other proteins [Hutchison, K.A., Dittmar, K. D.,Pratt, W.B. (1994) J. Biol. Chem. 269, 27894-27899]. In this "foldosome" system, hsp70 is required for assembly of the receptor-hsp90 complex and concomitant activation of steroid-binding activity [Hutchison, K.A., Dittmar, K.D., Czar, M.J.,Pratt, W.B. (1994) J. Biol. Chem. 269, 22157-22161]. All previous experiments involving cell-free assembly of both receptor-hsp90 and protein kinase-hsp90 heterocomplexes have been carried out with the protein-folding system in rabbit reticulocyte lysate. In this work, we show that concentrated lysates of receptor-free mouse (L cells) and insect (Sf9) cells and also a plant (wheat germ) lysate fold the immunopurified glucocorticoid receptor into a functional (i.e., steroid binding) heterocomplex with hsp90. Receptor heterocomplex formation in animal lysates and in the plant lysate are not identical in that the dynamics of complex assembly are different, but both systems produce a functional complex that binds steroid. Also, in contrast to animal and insect complexes, receptor-plant hsp90 complexes are not stabilized by molybdate. When added to the other lysate, purified plant and animal hsp90s show partial complementarity, in that a receptor-hsp90 complex is formed but the receptor is not converted to the steroid-binding conformation. When added to rabbit reticulocyte lysate that has been depleted of endogenous hsp70, purified wheat germ and mouse hsp70's are equally active in promoting both assembly of receptor-hsp90 heterocomplexes and conversion of receptor to the steroid-binding conformation. Thus, hsp70 from the plant kingdom has conserved the ability to interact functionally with chaperone proteins of the animal kingdom to cooperate in protein folding as evidenced by formation of a functional receptor-hsp90 heterocomplex.

In animal cell lysates, multiprotein complexes containing hsp90, hsp70, p60, p23, and several immunophilins can assemble steroid receptors and oncogenic protein kinases, such as v-Src and v-Raf, into heterocomplexes that contain hsp90 and either immunophilins or, in the case of protein kinases, p50. The complexes with hsp90 are required for the proper functioning of these signal transduction systems. Wheat germ lysate contains a similar protein folding activity that forms functional steroid receptor complexes with hsp90, but not all the components of this system have been identified. The plant chaperone system has conserved interactions with animal chaperones in that wheat hsp70 functions in the rabbit reticulocyte lysate heterocomplex assembly system and human p23 functions in the wheat germ lysate. Here, we ask if wheat germ lysate also contains immunophilins of the FK506-binding class (FKBPs) that bind to the hsp90 component of the chaperone complex via tetratricopeptide repeat (TPR) domains. To demonstrate the plant heterocomplex, we add purified mammalian p23, preadsorbed with the JJ3 antibody to protein A-Sepharose, to wheat germ lysate and allow ATP-dependent formation of an animal p23. plant hsp90 complex. The complex is then washed and incubated with the radiolabeled immunosuppressant drug [3H]FK506, which binds in a specific manner to a coimmunoadsorbed plant FKBP. Binding of the plant FKBP to plant hsp90 is prevented by adding to wheat germ lysate a purified fragment containing the TPR domains of human cyclophilin-40. Geldanamycin, a benzoquinone ansamycin that binds to animal hsp90s and prevents their chaperone activity, binds in a temperature-dependent manner to wheat hsp90 to block formation of the p23.hsp90.FKBP heterocomplex. These data show that immunophilin binding to hsp90 via TPR domains is conserved in the plant kingdom as well as in the animal kingdom and that geldanamycin will be an important tool for the study of hsp90-mediated protein chaperoning in plant cells.

We have recently reported that v-Raf forms a native heterocomplex with rat heat shock protein (hsp) 90 and a 50-kDa phosphoprotein (p50) in stably transfected 3Y1 fibroblasts (Stancato, L. F., Chow, Y-H., Hutchison, K. A., Perdew, G. H., Jove, R., and Pratt, W. B. (1993) J. Biol. Chem. 268, 21711-21716). Several members of the nuclear receptor family exist in heterocomplexes containing hsp90 and various members of the immunophilin protein family, including hsp56, an immunophilin of the FK506 binding class (Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458). In this work, we have asked if Raf is also associated with an immunophilin. We have immunoadsorbed v-Raf from stably transfected rat 3Y1 fibroblasts and show that the immunoadsorbed v-Raf.hsp90.p50 heterocomplex binds the immunosuppressant drug [3H]FK506. The binding is of high affinity (KD 82 nM) and specific in the sense that it is competed by nonradioactive FK506 and rapamycin but not by cyclosporin A. The [3H]FK506 binding activity is eliminated when the heterocomplex proteins are dissociated from v-Raf. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for the immunophilin association. We have shown that hsp90 and p50 do not bind FK506, and the v-Raf heterocomplex does not contain any of the established FK506-binding proteins. Thus, we propose that Raf is associated with a novel immunophilin.

Recently, we have demonstrated that the tyrosine kinase pp60v-src can undergo cell-free assembly into a heterocomplex with rabbit hsp90 and p50 when the immunoadsorbed protein is incubated with rabbit reticulocyte lysate (Hutchison, K. A., Brott, B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem 267, 2902-2908). Using a baculovirus system to express a high level of human c-Raf serine/threonine kinase in Sf9 insect cells, we show here that immunoadsorbed c-Raf undergoes similar lysate-mediated assembly into a heterocomplex with hsp90 and p50. As with pp60v-src and steroid receptors, binding of c-Raf to hsp90 occurs in an ATP-dependent and K(+)-dependent manner and the resulting heterocomplex is stabilized by molybdate. With a very rapid and gentle procedure of Sf9 cell cytosol preparation and c-Raf immunoadsorption, we show coimmunoadsorption of the insect homologue of hsp90. The same procedures permit detection of a native complex of v-Raf with rat hsp90 and p50 in stably transfected rat 3Y1 fibroblasts, and v-Raf is also assembled into a heterocomplex with rabbit hsp90 and p50 by reticulocyte lysate. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for both formation of the native heterocomplex in mouse NIH 3T3 cells and cell-free reconstitution of the heterocomplex by rabbit reticulocyte lysate. Although the native Raf-heat shock protein heterocomplex is less stable than native pp60v-src and glucocorticoid receptor heterocomplexes, by analogy with these proteins its detection may have important implications regarding the mechanism of Raf trafficking through the cytoplasm.

The hormone binding domain of the glucocorticoid receptor contains a unique vicinally spaced dithiol, and when it is bound by arsenite under conditions that are specific for reaction with vicinally spaced dithiols versus monothiols, steroid binding activity is eliminated [Simons, S. S., Jr., Chakraborti, P. K., & Cavanaugh, A. H. (1990) J. Biol. Chem. 265, 1938-1945]. The vicinally spaced dithiol lies in a region of the receptor that appears to be a contact site for hsp90, which is required for the high-affinity steroid binding conformation of the glucocorticoid receptor [Dalman, F. C., Scherrer, L. C., Taylor, L. P., Akil, H., & Pratt, W. B. (1991) J. Biol. Chem. 266, 3482-3490]. As part of a long-term project to develop a vicinal dithiol-specific agent that will permit studies of ligand-induced conformational changes in this region of the receptor, we have examined here the differential effects of two reversible thiol-reactive agents, arsenite and MMTS. At low concentration, arsenite inactivates the steroid binding activity of the unliganded receptor in a vicinal dithiol-specific manner, whereas dissociation of steroid from untransformed, transformed, or DNA-bound transformed receptors occurs only at concentrations typical of monothiol interactions. MMTS produces a unique bimodal effect on the steroid binding capacity of the unliganded receptor at pH 9 that is pH-dependent and becomes essentially unimodal at physiological pH. Whereas arsenite disrupts the dexamethasone-receptor complex more readily than the triamcinolone acetonide-receptor complex, MMTS has the opposite effect. During treatment for 1 h at 0 degree C, neither reagent causes dissociation of hsp90 from the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

We have designed and synthesized two radioiodinated analogs of hexadecylphosphocholine in order to evaluate their tumor imaging potential. 12-(m[125I]iodophenyl)dodecyl phosphocholine (NM-324) and hexadecyl-2-[N,N-dimethyl-N-(m[125I]iodobenzyl-ammonium]ethyl phosphate (NM-326) demonstrated the ability of such compounds to localize in and thereby visualize the Walker 256 tumor in rats. However, the tumor avidity of NM-324 was far superior to NM-326. In addition, NM-324 showed excellent tumor localization in athymic mice bearing subcutaneous human tumors.

Action of protein kinases and phosphatases contributes to myocardial hypertrophy. PRL-3, a protein tyrosine phosphatase, was identified in a cDNA library from an explanted human heart obtained from a patient with idiopathic cardiomyopathy. PRL-3 is expressed in heart and skeletal muscle, exhibiting approximately 76% identity to the ubiquitous tyrosine phosphatase PRL-1, which was reported to increase cell proliferation. PRL-3 was cloned into E. coli and purified using affinity chromatography. PRL-3 activity was determined using the substrate 6,8-difluoro-4-methylumbelliferyl phosphate, and was inhibited by vanadate and analogs. HEK293 cells expressing PRL-3 demonstrated increased growth rates versus nontransfected cells or cells transfected with the catalytically inactive C104S PRL-3 mutant. The tyrosine phosphatase inhibitor, potassium bisperoxo (bipyridine) oxovanadate V, normalizes the growth rate of PRL-3 expressing cells to that of parental HEK293 cells in a concentration-dependent manner. Using FLIPR analysis, parental HEK293 cells mobilize calcium when stimulated with angiotensin-II (AngII). However, calcium mobilization is inhibited in cells expressing wild-type PRL-3 when stimulated with AngII, while cells expressing the inactive mutant of PRL-3 mobilize calcium to the same extent as parental HEK293 cells. Western blots comparing PRL-3 transfected cells to parental HEK293 cells showed dephosphorylation of p130 cas in response to AngII. These data suggest a role for PRL-3 in the modulation of intracellular calcium transients induced by AngII.

A primary signaling cascade responsible for the expression of cytokine-stimulated immediate early genes involves the activation of the Jak/Stat pathway. In addition to being tyrosine-phosphorylated, several signal transducers and activators of transcription (Stats), including Stat1alpha, Stat3, and Stat4, are phosphorylated on a conserved serine residue, which is a consensus phosphorylation site for mitogen-activated protein kinases (MAPKs). Serine phosphorylation of Stat1alpha is required for maximal transcriptional activation of early response genes by interferon gamma (IFNgamma) as well as the antiviral and antigrowth actions of this cytokine. Incubation of cells with either IFNgamma or oncostatin M (OSM) activates Raf-1, a serine/threonine kinase responsible for the ultimate activation of p42 MAPK. To examine whether any of the signaling components that are required for activation of the Jak/Stat pathway are also necessary for activation of Raf-1 by IFNs and OSM, we examined activation of Raf-1 in cell lines that are deficient in either Stat1alpha or Stat2. Unexpectedly, incubation of Stat1-deficient, but not Stat2-deficient cells with IFNgamma or OSM for 5 min displayed no increase in Raf-1 activity. In peripheral blood lymphocytes Raf-1 was associated with Stat1, and this interaction was disrupted after incubation of cells with IFNgamma. Stat1-negative cells reconstituted with either Stat1alpha or Stat1alpha with a point mutation in the site where it is serine-phosphorylated displayed normal activation of Raf-1 by IFNgamma and OSM. However, activation of Raf-1 was not observed in lines that expressed Stat1alpha containing a mutation in its tyrosine phosphorylation site or in its SH2 domain. These results provide the first example of a novel role of Stat1alpha not as a transcription factor, but as a protein which may function to scaffold signaling components required for activation of the distinct Raf/MEK/MAPK signaling cascade.

Signal transduction through the interferongamma (IFNgamma) receptor involves the formation of a ligand-dependent multimolecular association of receptor chains (alpha and beta), Janus tyrosine kinases (Jak1 and Jak2), and the transcription factor (signal transducers and activators of transcription 1alpha (STAT1alpha)) in addition to activation of mitogen-activated protein kinases (MAPK). Interactions between components of the Jak/STAT cascade and the p21(ras)/Raf-1/MAPK cascade are unexplored. Treatment of HeLa cells with IFNgamma resulted in the rapid and transient activation of Raf-1 and MAPK. Parallel activation of cells resulted in essentially no enhancement of p21(ras) activation despite marked enhancement after treatment with epidermal growth factor. In HeLa (E1C3) and fibrosarcoma (U4A) cell lines, both of which are deficient in Jak1 kinase, Raf-1 activation by IFNgamma was absent. Reconstitution of Raf-1 activity was observed only with kinase active Jak1 in both cell lines. In COS cells, transient expression of wild type or kinase-inactive Jak1 coimmunoprecipitated with Raf-1, but activation of Raf-1 activity was only observed in cells expressing kinase-active Jak1. These observations suggest that a kinase-active Jak1 is required for IFNgamma activation of Raf-1 that is p21(ras)-independent.

We determined if heat stress induction of heat shock protein (HSP) 70 modulates complement activation in an experimental model of xenograft rejection. Male New Zealand White rabbits were heat stressed (core body temperature to 42 degrees C for 15 min; n = 9). Control rabbits (n = 13) were not exposed to heat stress. Hearts were removed 18 h later and perfused by the Langendorff method. After equilibration, human plasma (source of human complement) was added to the perfusion medium. Hemodynamic variables recorded during perfusion with human plasma were improved in hearts from heat-stressed animals compared with control hearts. Assembly of the soluble membrane attack complex was reduced in the interstitial fluid effluent from the heat-stressed hearts. Electron microscopic evidence of ultrastructural changes was attenuated in the hearts from heat-stressed rabbits. Myocardial tissue from heat-stressed animals exhibited an increase in inducible HSP 70 that was virtually absent in the hearts of control rabbits. Previous whole body hyperthermia protects the rabbit heart against the detrimental effects of heterologous plasma, suggesting that heat-stress induction of HSP 70 limits the extent of complement activation by a discordant vascularized tissue (xenograft). Induction of heat stress proteins by the donor organ might be an important mechanism affecting the outcome of xenograft transplants.

To be in a conformation that binds steroid, the hormone-binding domain of the glucocorticoid receptor (GR) must be bound to the 90 kDa heat shock protein (hsp90). Rabbit reticulocyte lysate contains a protein chaperone system that assembles the receptor into a heterocomplex with hsp90 and converts it from a non-steroid-binding to a steroid-binding form. Assembly of the GR-hsp90 heterocomplex requires hsp70, and in this work we examine the activities of four members of the hsp70 protein family in GR-hsp90 heterocomplex assembly. Rabbit reticulocyte lysate was depleted of hsp70 by passing it through a column of ATP agarose, resulting in the inactivation of its GR-hsp90 heterocomplex assembly activity. Addition of purified animal (mouse) or plant (wheat germ) hsp70 to the hsp70-depleted lysate permits assembly of a GR-hsp90 heterocomplex with a high affinity steroid binding site. However, purified hsp70 homologues from bacteria (DnaK) or the endoplasmic reticulum (BiP) do not promote heterocomplex formation, despite the fact that both DnaK and BiP bind to the GR in the assay system. When added to whole (i.e. hsp70-containing) reticulocyte lysate, DnaK and BiP inhibit GR·hsp90 heterocomplex assembly. Wheat germ lysate forms a heterocomplex between mouse GR and plant hsp90, but the addition of purified rabbit hsp70 to the wheat germ lysate does not increase the amount of receptor-wheat hsp90 complex produced, despite the fact that the rabbit hsp70 binds to the GR when it is added to the wheat chaperone system. The conclusion is that binding of hsp70 to receptors does not necessarily reflect a physiologically meaningful interaction. When native receptor heterocomplexes isolated from cytosols contain hsp70, it is likely that the hsp70-bound receptors represent a minority of receptors that have not yet proceeded fully through the receptor heterocomplex assembly process, which includes the dissociation of hsp70 after the binding of hsp90.

The heat shock proteins hsp90 and hsp70 have been immunopurified from rabbit reticulocyte lysate in a multiprotein complex that acts as a self-sufficient protein folding machine. This immunopurified “foldosome” directs the assembly of the glucocorticoid receptor-hsp90 complex and refolds the receptor to the steroid binding state (Hutchison, K. A., Dittmar, K. D., and Pratt, W. B.(1994) J. Biol. Chem. 269, 27894-27899). Extensive washing of the immunoadsorbed foldosome eliminates a weakly bound component required for receptor heterocomplex assembly and folding. This protein factor is contained in a Centricon C-100 filtrate of lysate which reconstitutes the receptor activating activity of the washed foldosome. This hsp90-associated protein folding system is present in both animal and plant cells, and the Centricon C-100 fraction of rabbit reticulocyte lysate potentiates receptor folding directed by wheat germ lysate. We have used this ability to stimulate wheat germ lysate-directed folding of the glucocorticoid receptor as a rapid assay for the factor. We demonstrate that the activity segregates with the 23-kDa acidic protein component of the hsp90 foldosome when rabbit reticulocyte lysate is fractionated by ammonium sulfate precipitation and ion exchange chromatography. Immunoadsorption of the Centricon C-100 filtrate with a monoclonal antibody against p23 eliminates its ability to stimulate the wheat germ heterocomplex assembly/receptor folding system, and the activity is replaced by purified, bacterially expressed p23. Immunodepletion of p23 also eliminates the ability of the Centricon C-100 filtrate to reconstitute receptor activating activity of the washed foldosome and addition of purified, bacterially expressed p23 restores its activity, confirming that p23 is the weakly bound component of the foldosome complex required for refolding of the receptor to the steroid binding conformation.

In many cells, the glucocorticoid receptor undergoes rapid steroid-mediated translocation from the cytoplasm to the nucleus, and this receptor is an excellent model for studying the mechanism of targeted protein movement through the cytoplasm. For such unidirectional movement to occur, the receptor must attach to a retrograde movement system in a manner that involves the nuclear localization signal. It is improbable that such attachment occurs via a direct protein-protein interaction between the receptor and the movement system; rather, one or more linker proteins are likely to be involved. As with other steroid receptors, the glucocorticoid receptor is associated with several other proteins in a heterocomplex. Two of these receptor-associated proteins are the heat shock proteins hsp90 and hsp56, and a third heat shock protein, hsp70, is required for assembly of the receptor heterocomplex. The hormone binding domain of the steroid receptors determines the interaction with both hsp90 and hsp70. Hsp56 is known to bind to hsp90, but its potential site, or sites, of interaction with the receptor are undefined. Hsp56 has recently been cloned and demonstrated to be an immunophilin of the FK506/rapamycin binding class. The immunophilins have peptidyl-prolyl isomerase activity but their cellular functions are unknown. Herein, we review the literature on the hsp56 immunophilin component of the receptor heterocomplex and present a rationale for hsp56 being the protein that determines the direction of receptor movement via a direct protein-protein interaction with the nuclear localization signal.

Incubation of immunopurified, hormone-free mouse glucocorticoid receptors with rabbit reticulocyte lysate results in ATP-dependent and monovalent cation-dependent assembly of the GR into a heterocomplex with hsp90, hsp70, and hsp56. Heterocomplex assembly is accompanied by conversion of the receptor from a form that does not bind steroid to a high affinity steroid-binding conformation. Reticulocyte lysate also promotes ATP-dependent dissociation of unliganded receptors from a prebound receptor-DNA complex. Receptor released from DNA has been reconstituted into the heat shock protein heterocomplex and converted to the non-DNA-binding state. The reticulocyte lysate also reconstitutes pp60v-src into a heterocomplex containing hsp90 and p50, both of which are components of the native heterocomplex form of the tyrosine kinase in cytoplasm. Although the c-Raf-1 serine/threonine kinase has never been found in native association with hsp90, it can be assembled into a heat shock protein heterocomplex by the ATP-dependent system in reticulocyte lysate.

A ubiquitous, low molecular weight, heat-stable component of cytosol stabilizes the glucocorticoid receptor in its untransformed state in association with hsp90. This heat-stable factor mimics molybdate in its effects on receptor function, and it has the heat stability, charge, and chelation properties of a metal oxyanion [Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H.,Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817]. In this paper, we describe the further purification of the endogenous factor from rat liver cytosol by anion-exchange HPLC (Ion-110) after prepurification by molecular sieving, cation absorption, and charcoal absorption. Elution of the factor with an isocratic gradient of ammonium bicarbonate results in recovery of all of the bioactivity in a single peak which coelutes with inorganic phosphate and contains all of the endogenous molybdenum. The bioactivity can be separated from inorganic phosphate by chromatography of the partially purified endogenous factor on a metal-chelating column of Chelex-100. The chelating procedure results in complete loss of bioactivity with recovery of 98% of the inorganic phosphate in both the column drop-through and a subsequent 1 M NaCl wash. The factor preparation purified through the Ion-110 HPLC step inhibits temperature-mediated dissociation of the immunopurified glucocorticoid receptor-hsp90 complex, but it is considerably more effective at stabilizing the unpurified receptor-hsp90 complex in a Chelex-treated cytosol system that has been depleted of metal components. These observations support the proposal that an endogenous metal can stabilize the binding of hsp90 to the receptor but it is likely that other cytosolic components that are not present in the immunopurified complex must contribute to the stability of the soluble protein-protein complex in cytosol.