The hCds1 (Chk2)-FHA Domain Is Essential for a Chain of Phosphorylation Events on hCds1 That Is Induced by Ionizing Radiation

Abstract

hCds1 (Chk2) (1-4) is an evolutionarily conserved kinase that functions in DNA damage response and cell cycle checkpoint. The Cds1 family of kinases are activated by a family of large phosphatidylinositol 3-kinase-like kinases. In humans, ataxia telangiectasia-mutated (ATM) (5) and ataxia-telangiectasia and Rad3-related (6) kinases activate hCds1 by phosphorylating Thr68 (7-9). hCds1 and Cds1-related kinases contain the FHA (fork-head-associated) domain (10), which appears to be important for integrating the DNA damage signal. It is not known how ATM phosphorylation activates hCds1 function and whether the phosphorylation is linked to the FHA. Here, we demonstrate that the hCds1-FHA domain is essential for Thr68 phosphorylation. Thr 68 phosphorylation, in turn, is required for ionizing radiation-induced autophosphorylation of two amino acid residues in hCds1, Thr383 and Thr387. These two amino acid residues, located in the activation loop of hCds1, are conserved in hCds1-related kinases and are essential for hCds1 activity. Thus, the hCds1-FHA domain mediates a chain of phosphorylation events on hCds1, which includes phosphorylation by ATM and hCds1 autophosphorylation, in response to DNA damage.