Bradley T. Scroggins

Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.

Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1, which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining cochaperones.

Effects of Geldanamycin, a Heat-Shock Protein 90-Binding Agent, on T Cell Function and T Cell Nonreceptor Protein Tyrosine Kinases

The benzoquinoid ansamycins geldanamycin (GA), herbimycin, and their derivatives are emerging as novel therapeutic agents that act by inhibiting the 90-kDa heat-shock protein hsp90. We report that GA inhibits the proliferation of mitogen-activated T cells. GA is actively toxic to both resting and activated T cells; activated T cells appear to be especially vulnerable. The mechanism by which GA acts is reflected by its effects on an essential hsp90-dependent protein, the T cell-specific nonreceptor tyrosine kinase lck. GA treatment depletes lck levels in cultured T cells by a kinetically slow dose-dependent process. Pulse-chase analyses indicate that GA induces the very rapid degradation of newly synthesized lck molecules. GA also induces a slower degradation of mature lck populations. These results correlate with global losses in protein tyrosine kinase activity and an inability to respond to TCR stimuli, but the activity of mature lck is not immediately compromised. Although the specific proteasome inhibitor lactacystin provides marginal protection against GA-induced lck depletion, proteasome inhibition also induces changes in lck detergent solubility independent of GA application. There is no other evidence for the involvement of the proteosome. Lysosome inhibition provides quantitatively superior protection against degradation. These results indicate that pharmacologic inhibition of hsp90 chaperone function may represent a novel immunosuppressant strategy, and elaborate on the appropriate context in which to interpret losses of lck as a reporter for the pharmacology of GA in whole organisms.

Hsp90 charged-linker truncation reverses the functional consequences of weakened hydrophobic contacts in the N domain

Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes, as it regulates diverse signal transduction nodes that integrate numerous environmental cues to maintain cellular homeostasis. Hsp90 also is secreted from normal and transformed cells and regulates cell motility. Here, we have identified a conserved hydrophobic motif in a β-strand at the boundary between the N domain and charged linker of Hsp90, whose mutation not only abrogated Hsp90 secretion but also inhibited its function. These Hsp90 mutants lacked chaperone activity in vitro and failed to support yeast viability. Notably, truncation of the charged linker reduced solvent accessibility of this β-strand and restored chaperone activity to these mutants. These data underscore the importance of β-strand 8 for Hsp90 function and demonstrate that the functional consequences of weakened hydrophobic contacts in this region are reversed by charged-linker truncation.

Heat shock protein 90α (HSP90α), a substrate and chaperone of DNA-PK necessary for the apoptotic response.

The “apoptotic ring” is characterized by the phosphorylation of histone H2AX at serine 139 (γ-H2AX) by DNA-dependent protein kinase (DNA-PK). The γ-H2AX apoptotic ring differs from the nuclear foci patterns observed in response to DNA-damaging agents. It contains phosphorylated DNA damage response proteins including activated Chk2, activated ATM, and activated DNA-PK itself but lacks MDC1 and 53BP1, which are required to initiate DNA repair. Because DNA-PK can phosphorylate heat shock protein 90α (HSP90α) in biochemical assays, we investigated whether HSP90α is involved in the apoptotic ring. Here we show that HSP90α is phosphorylated by DNA-PK on threonines 5 and 7 early during apoptosis and that both phosphorylated HSP90α and DNA-PK colocalize in the apoptotic ring. We also show that DNA-PK is a client of HSP90α and that HSP90α is required for full DNA-PK activation, γ-H2AX formation, DNA fragmentation, and apoptotic body formation. In contrast, HSP90 inhibition by geldanamycin markedly enhances TRAIL-induced DNA-PK and H2AX activation. Together, our results reveal that HSP90α is a substrate and chaperone of DNA-PK in the apoptotic response. The response of phosphorylated HSP90α to TRAIL and its localization to the γ-H2AX ring represent epigenetic features of apoptosis that offer insights for studying and monitoring nuclear apoptosis.

Englerin A stimulates PKCθ to inhibit insulin signaling and to simultaneously activate HSF1: pharmacologically induced synthetic lethality.

The natural product englerin A (EA) binds to and activates protein kinase C-θ (PKCθ). EA-dependent activation of PKCθ induces an insulin-resistant phenotype, limiting the access of tumor cells to glucose. At the same time, EA causes PKCθ-mediated phosphorylation and activation of the transcription factor heat shock factor 1, an inducer of glucose dependence. By promoting glucose addiction, while simultaneously starving cells of glucose, EA proves to be synthetically lethal to highly glycolytic tumors.

Mesenchymal stem cells inhibit cutaneous radiation‐induced fibrosis by suppressing chronic inflammation

Exposure to ionizing radiation (IR) can result in the development of cutaneous fibrosis, for which few therapeutic options exist. We tested the hypothesis that bone marrow‐derived mesenchymal stem cells (BMSC) would favorably alter the progression of IR‐induced fibrosis. We found that a systemic infusion of BMSC from syngeneic or allogeneic donors reduced skin contracture, thickening, and collagen deposition in a murine model. Transcriptional profiling with a fibrosis‐targeted assay demonstrated increased expression of interleukin‐10 (IL‐10) and decreased expression of IL‐1β in the irradiated skin of mice 14 days after receiving BMSC. Similarly, immunoassay studies demonstrated durable alteration of these and several additional inflammatory mediators. Immunohistochemical studies revealed a reduction in infiltration of proinflammatory classically activated CD80+ macrophages and increased numbers of anti‐inflammatory regulatory CD163+ macrophages in irradiated skin of BMSC‐treated mice. In vitro coculture experiments confirmed that BMSC induce expression of IL‐10 by activated macrophages, suggesting polarization toward a regulatory phenotype. Furthermore, we demonstrated that tumor necrosis factor‐receptor 2 (TNF‐R2) mediates IL‐10 production and transition toward a regulatory phenotype during coculture with BMSC. Taken together, these data demonstrate that systemic infusion of BMSC can durably alter the progression of radiation‐induced fibrosis by altering macrophage phenotype and suppressing local inflammation in a TNF‐R2‐dependent fashion. Stem Cells 2013;31:2231–2241

Post-translational modification of heat-shock protein 90: impact on chaperone function

Heat-shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of many signaling proteins that are often activated, mutated or overexpressed in cancer cells and that underly cancer cell proliferation and survival. Hsp90 is a conformationally flexible protein that associates with a distinct set of cochaperones depending on ATP or ADP occupancy of an N-terminal binding pocket. Nucleotide exchange and ATP hydrolysis by Hsp90 itself, with the assistance of cochaperones, drive the Hsp90 chaperone machine to bind, chaperone and release client proteins. Cycling of the Hsp90 chaperone machine is critical to its function. Although ATP binding and hydrolysis have been convincingly implicated in regulating the Hsp90 cycle, growing evidence suggests that various post-translational modifications of Hsp90, including phosphorylation, acetylation and other modifications, provide an additional overlapping or parallel level of regulation. A more complete understanding of how these various protein modifications are regulated and interact with each other at the cellular level to modulate Hsp90 chaperone activity is critical to the design of novel approaches to inhibit this medically important molecular target.

Characterization of the interaction of Aha1 with components of the Hsp90 chaperone machine and client proteins

The activator of Hsp90 ATPase, Aha1, is an Hsp90 co-chaperone that has been suggested to act as a general stimulator of Hsp90 function. In this report, we have characterized the interaction of Aha1 with Hsp90 and its co-chaperones in rabbit reticulocyte lysate (RRL) and in HeLa cell extracts. Complexes formed by Aha1 with Hsp90 in RRL were stabilized by molybdate and contained the co-chaperones FKBP52 and p23/Sba1, but lacked HOP/Sti1 and Cdc37. Aha1 complexes isolated from HeLa cell extracts also contained Hsp70 and DNAJA1. Over-expression of Aha1 has been reported to stimulate the activity of v-Src and steroid hormone receptors ectopically expressed in yeast, however, no interaction between Aha1 and nascent v-Src or the progesterone receptor could be detected in RRL. Contrary to expectations, over-expression of Aha1 also inhibited the rate of Hsp90-dependent refolding of denatured luciferase. A number of potential client proteins that specifically associated with Aha1 were identified by liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and verified by Western blotting. The proteins identified suggest that Aha1 may play roles in modulating RNA splicing and DNA repair, in addition to other cellular processes.

Transforming Growth Factor Alpha is a Critical Mediator of Radiation Lung Injury

Radiation fibrosis of the lung is a late toxicity of thoracic irradiation. Epidermal growth factor (EGF) signaling has previously been implicated in radiation lung injury. We hypothesized that TGF-α, an EGF receptor ligand, plays a key role in radiation-induced fibrosis in lung. Mice deficient in transforming growth factor (TGF-α–/–) and control C57Bl/6J (C57-WT) mice were exposed to thoracic irradiation in 5 daily fractions of 6 Gy. Cohorts of mice were followed for survival (n ≥ 5 per group) and tissue collection (n = 3 per strain and time point). Collagen accumulation in irradiated lungs was assessed by Massons trichrome staining and analysis of hydroxyproline content. Cytokine levels in lung tissue were assessed with ELISA. The effects of TGF-α on pneumocyte and fibroblast proliferation and collagen production were analyzed in vitro. Lysyl oxidase (LOX) expression and activity were measured in vitro and in vivo. Irradiated C57-WT mice had a median survival of 24.4 weeks compared to 48.2 weeks for irradiated TGF-α–/– mice (P = 0.001). At 20 weeks after irradiation, hydroxyproline content was markedly increased in C57-WT mice exposed to radiation compared to TGF-α–/– mice exposed to radiation or unirradiated C57-WT mice (63.0, 30.5 and 37.6 μg/lung, respectively, P = 0.01). C57-WT mice exposed to radiation had dense foci of subpleural fibrosis at 20 weeks after exposure, whereas the lungs of irradiated TGF-α –/– mice were largely devoid of fibrotic foci. Lung tissue concentrations of IL-1β, IL-4, TNF-α, TGF-β and EGF at multiple time points after irradiation were similar in C57-WT and TGF-α–/– mice. TGF-α in lung tissue of C57-WT mice rose rapidly after irradiation and remained elevated through 20 weeks. TGF-α–/– mice had lower basal LOX expression than C57-WT mice. Both LOX expression and LOX activity were increased after irradiation in all mice but to a lesser degree in TGF-α–/– mice. Treatment of NIH-3T3 fibroblasts with TGF-α resulted in increases in proliferation, collagen production and LOX activity. These studies identify TGF-α as a critical mediator of radiation-induced lung injury and a novel therapeutic target in this setting. Further, these data implicate TGF-α as a mediator of collagen maturation through a TGF-β independent activation of lysyl oxidase.

Truncated Plasminogen Activator Inhibitor-1 Protein Protects From Pulmonary Fibrosis Mediated by Irradiation in a Murine Model.

PURPOSE To determine whether the delivery of recombinant truncated plasminogen activator inhibitor-1 (PAI-1) protein (rPAI-1(23)) would protect from the development of radiation-induced lung injury. METHODS AND MATERIALS C57Bl/6 mice received intraperitoneal injections of rPAI-1(23) (5.4 μg/kg/d) or vehicle for 18 weeks, beginning 2 days before irradiation (IR) (5 daily fractions of 6 Gy). Cohorts of mice were followed for survival (n=8 per treatment) and tissue collection (n=3 per treatment and time point). Fibrosis in lung was assessed with Masson-Trichrome staining and measurement of hydroxyproline content. Senescence was assessed with staining for β-galactosidase activity in lung and primary pneumocytes. RESULTS Hydroxyproline content in irradiated lung was significantly reduced in mice that received rPAI-1(23) compared with mice that received vehicle (IR+vehicle: 84.97 μg/lung; IR+rPAI-1(23): 56.2 μg/lung, P=.001). C57Bl/6 mice exposed to IR+vehicle had dense foci of subpleural fibrosis at 19 weeks, whereas the lungs of mice exposed to IR+rPAI-1(23) were largely devoid of fibrotic foci. Cellular senescence was significantly decreased by rPAI-1(23) treatment in primary pneumocyte cultures and in lung at multiple time points after IR. CONCLUSIONS These studies identify that rPAI-1(23) is capable of preventing radiation-induced fibrosis in murine lungs. These antifibrotic effects are associated with increased fibrin metabolism, enhanced matrix metalloproteinase-3 expression, and reduced senescence in type 2 pneumocytes. Thus, rPAI-1(23) is a novel therapeutic option for radiation-induced fibrosis.