Chk2 oligomerization studied by phosphopeptide ligation: implications for regulation and phosphodependent interactions.

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.