Phosphorylation of the human Fhit tumor suppressor on tyrosine 114 in Escherichia coli and unexpected steady state kinetics of the phosphorylated forms

Preston N. Garrison, Angela K. Robinson, Yuri Pekarsky, Carlo M. Croce, Larry D. Barnes

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14 Scopus citations

Abstract

The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap3A hydrolase activity. We have recently discovered that Fhit is phosphorylated in vivo and is phosphorylated in vitro by Src kinase [Pekarsky, Y., Garrison, P. N., Palamarchuk, A., Zanesi, N., Aqeilan, R. I., Huebner, K., Barnes, L. D., and Croce, C. M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 3775-3779]. Now we have coexpressed Fhit with the elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit. Unphosphorylated Fhit, Fhit phosphorylated on one subunit, and Fhit phosphorylated on both subunits were purified to apparent homogeneity by column chromatography on anion-exchange and gel filtration resins. MALDI-TOF and HPLC-ESI tandem mass spectrometry of intact Fhit and proteolytic peptides of Fhit demonstrated that Fhit is phosphorylated on Y114 on either one or both subunits. Monophosphorylated Fhit exhibited monophasic kinetics with Km and kcat values ∼2- and ∼7-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics. One site had Km and kcat values ∼2- and ∼140-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. The second site had a K m ∼60-fold higher and a kcat ∼6-fold lower than the corresponding values for unphosphorylated Fhit. The unexpected kinetic patterns for the phosphorylated forms suggest the system may be enzymologically novel. The decreases in the values of Km and kcat for the phosphorylated forms in comparison to those of unphosphorylated Fhit favor the formation and lifetime of the Fhit - Ap3A complex, which may enhance the tumor suppressor activity of Fhit.

Original languageEnglish (US)
Pages (from-to)6286-6292
Number of pages7
JournalBiochemistry
Volume44
Issue number16
DOIs
StatePublished - Apr 26 2005

ASJC Scopus subject areas

  • Biochemistry

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