Fhit is a physiological target of the protein kinase Src

Abstract

The FHIT gene is a tumor suppressor that is frequently inactivated by genomic alterations at chromosomal region 3p14.2. In the last few years, a considerable amount of data describing inactivation of FHIT in a variety of human malignancies and demonstrating the tumor suppressor potential of Fhit have been reported. Despite the demonstration that FHIT functions as a tumor suppressor, the pathway through which Fhit induces apoptosis and inhibits growth of cancer cells is not known. Our data demonstrate that Fhit is a target of tyrosine phosphorylation by the Src protein kinase. We show that Src phosphorylates Y114 of Fhit in vitro and in vivo, providing insight into a biochemical pathway involved in Fhit signaling.

Fig. 1.

Src phosphorylates Fhit in vitro. One microgram of recombinant Fhit was incubated with 10 units of recombinant Src (lanes 1 and 2) or Lyn (lanes 3 and 4) at 30°C and immunoblotted with anti-phosphotyrosine (Upper) or anti-Fhit (Lower) antibodies. Incubations were carried out for 0 (lanes 1 and 3) or 30 (lanes 2 and 4) min.

Fig. 2.

Chromatographic separation of unphosphorylated and phosphorylated forms of Fhit. Dashed line, the MonoQ column elution profile of Fhit incubated in buffer with ATP and without Src kinase; solid line, the MonoQ column elution profile of Fhit incubated in the presence of ATP and Src kinase. (Inset) SDS gel electrophoresis pattern of Fhit samples corresponding to the indicated column peaks: U, unphosphorylated Fhit incubated without Src kinase; 1, residual, unphosphorylated Fhit after incubation with Src kinase; 2, Fhit phosphorylated on one subunit; and 3, Fhit phosphorylated on both subunits. Approximately 0.2 μg of each sample was subjected to electrophoresis, and the gel was stained with Sypro Ruby.

Fig. 3.

Mass spectral analysis of Fhit. (A-C) MALDI-TOF spectra of unphosphorylated, monophosphorylated, and diphosphorylated forms of Fhit, respectively, were acquired on an Applied Biosystems Voyager Elite as described in Materials and Methods. The m/z values shown in bold represent the average of three spectra obtained from the same sample spot on the MALDI target. The lower intensity peaks in each panel represent matrix adducts. (D and E) ESI tandem mass spectra were acquired on a Thermo Finnigan LCQ Classic spectrometer as described in Materials and Methods. (D) Tandem mass spectrum of the 2+ ion (m/z 619.8) of tryptic peptide 109-118 from unphosphorylated Fhit. (E) Tandem mass spectrum from the 2+ ion (m/z 659.6) of tryptic peptide 109-118 from phosphorylated Fhit. Spectra were obtained by targeted scans of the indicated ions. Peptide fragments are denoted on the spectra using the nomenclature of Roepstorff and Fohlman (23). Identification of the site of phosphorylation was obtained by comparison of the y-series ions in the two spectra.

Fig. 4.

Src phosphorylates Fhit in vivo. (A) 293 cells were cotransfected with WT FHIT and SRCDN (lane 1) or activated SRC (lane 2) and immunoblotted with anti-Fhit (Top), anti-Src (Middle), or anti-phosphotyrosine (Bottom) antibodies. Lysates were immunoprecipitated with anti-Fhit before Western blotting with anti-phosphotyrosine. Lane 3, an aliquot of in vitro phosphorylated Fhit was loaded for comparison. (B) 293 cells were cotransfected with SRCDN and WT FHIT (lane 2), FHITY114F (lane 3), FHITY145F (lane 4), and FHITY114F, Y145F (lane 5). Lysates were immunoblotted as in A. Lane 1, an aliquot of in vitro phosphorylated Fhit was loaded for comparison. (C) Western blot analysis of normal human tissues with anti-Fhit antibody; 100 μg of each lysate were loaded in each lane: lane 1, fetal liver; lane 2, fetal brain; lane 3, fetal lung; lane 4, lung; lane 5, fetal kidney; and lane 6, kidney. (D) One milligram of normal human kidney lysate was immunoprecipitated with anti-Fhit antibody (lane 1) or with rabbit IgG (lane 2) and immunoblotted with anti-Fhit (Upper) or with anti-phosphotyrosine (Lower).