Phosphorylation of the human Fhit tumor suppressor on tyrosine 114 in Escherichia coli and unexpected steady state kinetics of the phosphorylated forms.
The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap(3)A 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 Y(114) on either one or both subunits. Monophosphorylated Fhit exhibited monophasic kinetics with K(m) and k(cat) values approximately 2- and approximately 7-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics. One site had K(m) and k(cat) values approximately 2- and approximately 140-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. The second site had a K(m) approximately 60-fold higher and a k(cat) approximately 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 K(m) and k(cat) for the phosphorylated forms in comparison to those of unphosphorylated Fhit favor the formation and lifetime of the Fhit-Ap(3)A complex, which may enhance the tumor suppressor activity of Fhit.
Garrison
Preston N
PN
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Robinson
Angela K
AK
Pekarsky
Yuri
Y
Croce
Carlo M
CM
Barnes
Larry D
LD
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D015226:Dinucleoside Phosphates; D009363:Neoplasm Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; D000099170:Bis(5'-adenosyl)triphosphatase; C011440:diadenosine triphosphate