Human Fas-associated Factor 1 Interacts with Heat Shock Protein 70 and Negatively Regulates Chaperone Activity

Hee-Jung Kim,Eun Joo Song,Yun-Suk Lee,Eunhee Kim,Kong-Joo Lee

doi:10.1074/jbc.m406297200

Hee-Jung Kim, Eun Joo Song + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m406297200

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Abstract

We examined the cell death-inducing property of human Fas-associated factor 1 (hFAF1) in the heat shock signaling pathway. By employing co-immunoprecipitation and peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that hFAF1 binds to the 70-kDa heat shock protein family (Hsc70/Hsp70). Interaction mapping indicated that the 82-180 sequence of hFAF1 directly binds to the N-terminal region containing sequence 1-120 of Hsc70/Hsp70. This binding is very tight regardless of ATP and heat shock treatment. Hsc70/Hsp70 and hFAF1 co-localized in the cytosol and nucleus and concentrated to the perinuclear region by heat shock treatment. We examined how hFAF1 regulates Hsp70 function, and found that hFAF1 inhibited the Hsp70 chaperone activity of refolding denatured protein substrates, accelerated heat shock-induced SAPK/JNK activation, and raised heat shock-induced cell death in a binding dependent manner. These results suggest that hFAF1 prevents cells from recovery after stress by binding to and inhibiting the chaperone activity of Hsp70.

Highlights

We examined the cell death-inducing property of human Fas-associated factor 1 in the heat shock signaling pathway
FLAGtagged human Fas-associated factor 1 (hFAF1) was transiently overexpressed in HEK293T cells, and cellular proteins were metabolically labeled with 2 ␮Ci/ml [35S]methionine. hFAF1-interacting proteins were divided into two fractions and analyzed by two-dimensional gel electrophoresis
We identified spot numbers 1 and 2 as heat shock protein 70 cognate (Hsc70) and heat shock protein 70 inducible (Hsp70), respectively (Table I)

Summary

EXPERIMENTAL PROCEDURES

Plasmids—Full-length human hsp was subcloned into the pGEX4T-1 vector or 3ϫ FLAG-CMV-7.1 vector pGEX-4T-1/Hsp70⌬ABD(1–119, 429 – 640). Cells were disrupted with a buffer containing protease inhibitors (50 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 5 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 5 mM Na3VO4, 5 mM NaF) for 30 min on ice. The lysates were centrifuged at 12,000 rpm for 1 h, and the supernatant was incubated for 3 h at 4 °C with monoclonal anti-FLAG M2-affinity cross-linking agarose beads or incubated for 2 h with antiFLAG antibody and for an additional 1 h with protein A beads at 4 °C. The immunocomplexes were washed three times and incubated with a kinase buffer (50 mM Tris-Cl, pH 7.4, 10 mM MgCl2, 2 mM EGTA, 1 mM dithiothreitol, 0.1% Triton X-100) supplemented with GST-c-Jun and [␥-32P]ATP for 20 min at 37 °C.

RESULTS

Peptide coverage

DISCUSSION