Regulation of Molecular Chaperone Gene Transcription Involves the Serine Phosphorylation, 14-3-3ɛ Binding, and Cytoplasmic Sequestration of Heat Shock Factor 1

Heat shock factor 1 (HSF1) regulates the transcription of molecular chaperone hsp genes. However, the cellular control mechanisms that regulate HSF1 activity are not well understood. In this study, we have demonstrated for the first time that human HSF1 binds to the essential cell signaling protein 14-3-3ɛ. Binding of HSF1 to 14-3-3ɛ occurs in cells in which extracellular signal regulated kinase (ERK) is activated and blockade of the ERK pathway by treatment with the specific ERK pathway inhibitor PD98059 in vivo strongly suppresses the binding. We previously showed that ERK1 phosphorylates HSF1 on serine 307 and leads to secondary phosphorylation by glycogen synthase kinase 3 (GSK3) on serine 303 within the regulatory domain and that these phosphorylation events repress HSF1. We show here that HSF1 binding to 14-3-3ɛ requires HSF1 phosphorylation on serines 303 and 307. Furthermore, the serine phosphorylation-dependent binding of HSF1 to 14-3-3ɛ results in the transcriptional repression of HSF1 and its sequestration in the cytoplasm. Leptomycin B, a specific inhibitor of nuclear export receptor CRM1, was found to reverse the cytoplasmic sequestration of HSF1 mediated by 14-3-3ɛ, suggesting that CRM1/14-3-3ɛ directed nuclear export plays a major role in repression of HSF1 by the ERK/GSK3/14-3-3ɛ pathway. Our experiments indicate a novel pathway for HSF1 regulation and suggest a mechanism for suppression of its activity during cellular proliferation

Cell Surface Cdc37 Participates in Extracellular HSP90 Mediated Cancer Cell Invasion

<div><p>Cdc37 is a 50 kDa molecular chaperone which targets intrinsically unstable protein kinases to the molecular chaperone HSP90. It is also an over-expressed oncoprotein that mediates carcinogenesis and maintenance of the malignant phenotype by stabilizing the compromised structures of mutant and/or over-expressed oncogenic kinases. Here we report that Cdc37 is not restricted intracellularly but instead it is also present on the surface of MDA-MB-453 and MDA-MB-231 human breast cancer cells, where it is shown to participate in cancer cell motility processes. Furthermore, we demonstrate using an anti-Cdc37 cell impermeable antibody, that similarly to its intracellular counterpart, this surface pool of Cdc37 specifically interacts with HSP90 as well as the kinase receptors HER2 and EGFR on the cell surface, probably acting as a co-factor in HSP90's extracellular chaperoning activities. Finally, we show that functional inhibition of surface HSP90 using mAb 4C5, a cell impermeable monoclonal antibody against this protein, leads not only to disruption of the Cdc37/HSP90 complex but also to inhibition of the Cdc37/ErbB receptors complexes. These results support an essential role for surface Cdc37 in concert with HSP90 on the cell surface during cancer cell invasion processes and strengthen the therapeutic potential of mAb 4C5 for the treatment of cancer.</p> </div

Cdc37 is localized on the cell surface of MDA-MB 453 and MDA-MB 231 breast cancer cells.

<p><b>A</b>, Indirect immunofluorescence of live MDA-MB 453 cells using anti-Cdc37 antibody. The immunolabeling reveals the surface localization of Cdc37. Anti-HER2 and mAb 4C5 were used as positive controls. Anti –EGFR antibody was used as negative control. <b>B</b>, Indirect immunofluorescence of live MDA-MB 231 cells using anti-Cdc37 antibody. Anti-EGFR and mAb 4C5 were used as positive controls. Anti –HER2 antibodies was used as negative control. <b>C</b>, Cell fractionization of MDA-MB 453 cells followed by Western blot analysis confirmed the presence of Cdc37 in cell membrane fractions. Anti-HER2 antibodies were used as positive and negative controls in the membrane and cytosolic fractions respectively. <b>D</b>, Cell fractionization of MDA-MB 231 cells followed by western blot analysis confirmed the presence of Cdc37 on cell membrane fractions. Anti-EGFR antibodies were used as positive and negative controls in the membrane and cytosolic fractions respectively. In C and D antibody against the intracellular protein Cyclin D1 gave positive and negative immunostaining in the cytosolic and membrane fractions of both cell lines, respectively. CF, cytosolic fraction; MF, membrane fraction. Scale bar = 20 µm.</p

The anti-Cdc37 antibody is cell impermeable.

<p>Anti-Cdc37 antibody internalization was tested in both MDA-MB 453 and MDA-MB 231 cells. Live cells from the two cell lines were grown on coverslips and incubated at 37°C for 16 h with anti-Cdc37. MAb 4C5 and anti-HSP90 were used as positive and negative controls respectively. For detection of antibody internalization, cells were fixed and permeabilized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042722#s2" target="_blank">materials and methods</a> and antibodies were detected by confocal microscopy using an Alexa 488 secondary antibody. No internalization of the anti-Cdc37 antibody was observed even in both cell lines. Scale bar = 20 µm.</p