Correlation of Fragile Histidine Triad (Fhit) Protein Structural Features with Effector Interactions and Biological Functions

Teresa Druck,Hiroshi Okumura,Kelly A Mccorkell,Sung-Suk Suh,Tatsuya Nakamura,Flavia Pichiorri,Carlo M Croce,Larry D Barnes,Pierluigi Gasparini,Kay Huebner,Preston N Garrison

doi:10.1074/jbc.m806638200

Abstract

We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G(2)/M or sub-G(1) fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.

Highlights

The earlier finding that Fhit interacts with the Hsp60/10 complex and is imported into mitochondria where it interacts with and stabilizes ferredoxin reductase (Fdxr) is of interest for a number of reasons
2) Fdxr accumulation under stress conditions increases the electron transport process, depleting the reduced NADPH pool (20), and in the absence of NADPH to detoxify reactive oxygen species (ROS), ROS-induced apoptosis is amplified (7). 3) In the absence of Fhit, a condition occurring in most cancers, there is less Fdxr in the mitochondria, allowing better detoxification of ROS and less apoptosis
4) This can allow survival of Fhit-deficient cells carrying a low level of oxidative damage that can contribute to neoplastic progression

Summary

Introduction

Tumor suppression studies showed that cancer cells stably transfected with wild type (WT) or H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested the hypothesis that the Fhit-substrate complex sends the tumor suppression signal (9, 10). To test this hypothesis, a series of FHIT alleles was designed to reduce substrate-binding and/or hydrolytic rates and was characterized by quantitative cell-death assays on cancer cells virally infected with each allele. It was possible that the alterations in Km and kcat values for phosphorylated forms of Fhit might favor formation and lifetime of the Fhit-Ap3A complex and enhance tumor suppressor activity (see Table 1 for characteristics of specific Fhit forms)

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