Correlation of Fragile Histidine Triad (Fhit) Protein Structural Features
with Effector Interactions and Biological
Functions |
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Authors: | Flavia Pichiorri Hiroshi Okumura Tatsuya Nakamura Preston N Garrison Pierluigi Gasparini Sung-Suk Suh Teresa Druck Kelly A McCorkell Larry D Barnes Carlo M Croce and Kay Huebner |
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Institution: | ‡Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University and Comprehensive Cancer Center, Columbus, Ohio 43210 and the §Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229 |
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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 G2/M or
sub-G1 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.Fhit protein is a powerful tumor suppressor that is frequently lost or
reduced in cancer cells because of rearrangement of the exquisitely DNA
damage-sensitive fragile FHIT gene. Restoration of Fhit expression
suppresses tumorigenicity of cancer cells of various types, and the ability to
induce apoptosis in cancer cells in vitro is reduced by specific Fhit
mutations (1,
2).Through studies of signal pathways affected by Fhit expression, by searches
for Fhit protein effectors, and by in vitro analyses of Fhit
activity, we and others have defined Fhit enzymatic activity in vitro
(3), apoptotic activity in
cells and tumors
(4–6),
and most recently identification of a Fhit protein complex that affects Fhit
stability, mitochondrial localization, and interaction with ferredoxin
reductase (Fdxr)5
(7). The complex includes Hsp60
and Hsp10 that mediate Fhit stability and may affect import into mitochondria,
where Fhit interacts with Fdxr, which is responsible for transferring
electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit
restoration in Fhit-deficient cancer cells increases production of
intracellular reactive oxygen species (ROS), followed by increased apoptosis
of cancer cells under oxidative stress conditions; conversely, Fhit-negative
cells escape apoptosis, likely carrying oxidative DNA damage that contributes
to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad
(HIT) family of proteins, suggested that the protein product would hydrolyze
diadenosine tetraphosphate or diadenosine triphosphate (Ap3A)
(8), and in vitro
studies showed that Ap3A was cleaved into ADP and AMP by Fhit. The
catalytic histidine triad within Fhit was essential for catalytic activity
(3), and a Fhit mutant that
substituted Asn for His at the central histidine (H96N mutant) was
catalytically inactive, although it bound substrate well
(3). Early 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. The allele series
covered defects as great as 100,000-fold in kcat and
increases as large as 30-fold in Km. Mutants with
2–7-fold increases in Km had significantly reduced
apoptotic indices and the mutant with a 30-fold increase in
Km retained little apoptotic function. Thus, the
proapoptotic function of Fhit, which is likely associated with tumor
suppressor function, is limited by substrate binding and is unrelated to
substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine
phosphorylation by the Src family protein kinases, which can phosphorylate
Tyr-114 of Fhit in vitro and in vivo
(12). After co-expression of
Fhit with the Elk tyrosine kinase in Escherichia coli to generate
phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated
Fhit were purified, and enzyme kinetics studies showed that monophosphorylated
Fhit exhibited monophasic kinetics with Km and
kcat values ∼2- and ∼7-fold lower, respectively,
than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic
kinetics; one site had Km and kcat
values ∼2- and ∼140-fold lower, respectively, than for
unphosphorylated Fhit; the second site had a Km
∼60-fold higher and a kcat ∼6-fold lower than for
unphosphorylated Fhit (13).
Thus, 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 | Open in a separate windowTo explore the in vivo importance of the Tyr-114 phosphorylation
site and define Fhit-mediated signaling events, Semba et al.
(14) compared the differential
biological effects of Ad-FHIT WT and Ad-FHIT Tyr-114 mutant
expression in human lung cancer cells. Caspase-dependent apoptosis was
effectively induced only by WT Fhit protein. However, the biological
significance of phosphorylation at Tyr-114 has been difficult to study because
the endogenous phosphorylated forms have very short half-lives; activation of
epidermal growth facto receptor family members induces Fhit phosphorylation by
Src and proteasome degradation of phosphorylated Fhit
(15).Although there are possible connections among the various pathways known to
be altered in Fhit-deficient cells, apoptosis, DNA damage-response checkpoint
activation, ROS production, and related biological effects of Fhit loss or
overexpression, details of the pathway(s) leading from Fhit overexpression to
cell death and tumor suppression have not been delineated. Now that a Fhit
signaling complex has been identified, we set out to examine which structural
features of Fhit protein might participate in individual steps of the pathway
leading from Fhit overexpression through complex formation, subcellular
localization, interaction with mitochondrial Fdxr, DNA damage induction, cell
cycle changes, apoptosis, and ultimately tumor suppression. The underlying
hypotheses were as follows: substrate-binding mutants would behave similarly
to WT; nonsubstrate-binding mutants would be defective in some step of the
pathway, perhaps complexing with heat shock proteins or Fdxr or perhaps
induction of DNA damage; and Tyr-114 mutants, which also affect formation or
stability of the enzyme-substrate complex, would also be defective in
executing some step of the Fhit overexpression pathway to cell death. One goal
was to identify specific mutants that exhibited deficiency in specific steps
of the pathway, so that such mutants could be used to dissect each step in
more detail. Using in vitro Fhit and Fhit-effector protein
interactions, we aimed to determine the following: 1) which proteins of the
complex interact directly with Fhit, and 2) the biological role of these
interactions in vivo. Using cancer cells expressing exogenous WT and
mutant Fhit proteins, we were able to examine the structural features of Fhit
that affect the direct interaction with its effectors, participate in ROS
production, and are necessary for tumor suppression activity. |