Abstract
Chk2/CHEK2/hCds1 is a modular serine-threonine kinase involved in transducing DNA damage signals. Phosphorylation by ataxia telangiectasia-mutated kinase (ATM) promotes Chk2 self-association, autophosphorylation, and activation. Here we use expressed protein ligation to generate a Chk2 N-terminal regulatory region encompassing a fork-head-associated (FHA) domain, a stoichiometrically phosphorylated Thr-68 motif and intervening linker. Hydrodynamic analysis reveals that Thr-68 phosphorylation stabilizes weak FHA-FHA interactions that occur in the unphosphorylated species to form a high affinity dimer. Although clearly a prerequisite for Chk2 activation in vivo, we show that dimerization modulates potential phosphodependent interactions with effector proteins and substrates through either the pThr-68 site, or the canonical FHA phosphobinding surface with which it is tightly associated. We further show that the dimer-occluded pThr-68 motif is released by intra-dimer autophosphorylation of the FHA domain at the highly conserved Ser-140 position, a major pThr contact in all FHA-phosphopeptide complex structures, revealing a mechanism of Chk2 dimer dissociation following kinase domain activation.
Highlights
Chk2/CHEK2/hCds1 is a modular serine-threonine kinase involved in transducing DNA damage signals
We further show that the dimer-occluded pThr-68 motif is released by intra-dimer autophosphorylation of the FHA domain at the highly conserved Ser-140 position, a major pThr contact in all FHA-phosphopeptide complex structures, revealing a mechanism of Chk2 dimer dissociation following kinase domain activation
Chk2 was originally discovered as a Saccharomyces cerevisiae Rad53-related kinase that is primarily activated by the ataxia telangiectasia-mutated kinase (ATM)3 after DNA damage [1,2,3,4,5]
Summary
Strains and Reagents—Escherichia coli strain BL21 (DE3) RIL (Stratagene) was used for GST fusion protein expression and NovaBlue SinglesTM competent cells (Novagen) were used for plasmid construction and maintenance. The synthetic C-terminal thioester peptide at a concentration of 1 mM was incubated with 100 M purified Factor Xa-digested FHA proteins overnight at room temperature in 200 mM phosphate buffer, pH 8.0, 150 mM NaCl, 10 mM TCEP, 122 mM 2-mercaptoethanesulphonic acid (MESNA) to form the ligated pThr-68 FHA product (pT68 FHAlig). The steady state response for each concentration was obtained by subtracting responses from the ligand-immobilized flow cell with the correspondent blank flow cell These were plotted against analyte concentration and the data fitted with non-linear single-site binding model, BX Y ϭ KD ϩ X (Eq 1). The percentages of steady state signal remaining were calculated using the response from binding of 50 nM Plk PBD alone as 100% These were plotted against logarithm of concentration of the competitor. Daughter ion spectra were typically acquired at a collision energy of 30% and an isolation width of 4 Da
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