Conserved ATRMec1 phosphorylation-independent activation of Chk1 by single amino acid substitution in the GD domain.
Chk1 is a conserved kinase that comprises the first line of defense against DNA damage and replication blocks. Chk1 consists of two primary domains, the well conserved N-terminal kinase domain and the non-catalytic C-terminal domain that contains the two highly conserved TRF and GD sub-domains. Several studies suggested that the C-terminus of Chk1 acts as an inhibitory domain and that phosphorylation of the C-terminus by ATR serves to activate Chk1 by relieving the inhibitory effect of the C-terminus on the N-terminal catalytic domain. However, work carried out in many systems showed that phosphorylation on ATR sites was necessary but not sufficient to increase Chk1 kinase activity. In a recent manuscript we described a single amino acid substitution at an invariant Leucine in the conserved GD domain of the yeast Chk1 C-terminus (L506R) that led to a Chk1 protein that no longer required ATR(Mec1) phosphorylation at conserved sites for its function, and relieved the requirement of an upstream mediator, Rad9 (53BP1 homolog), for Chk1 activation. Here we show that this single amino acid substitution in the GD domain also led to constitutive phosphorylation of yeast and human Chk1 on ATR(Mec1) sites, suggesting that the protein was in a conformation in which it could be readily phosphorylated by ATR(Mec1). Unlike the phospho-mimetic mutants in earlier studies, the L505R and L449R modifications led to increased Chk1 activity both in vitro and in vivo. Therefore, we have uncovered a conserved mechanism for Chk1 regulation separate from the role of known ATR phosphorylation sites.
Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, NH, USA.
Elizabeth
E
Pereira
Yinhuai
Y
Chen
Yolanda
Y
Sanchez
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D064007:Ataxia Telangiectasia Mutated Proteins; D001665:Binding Sites; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018797:Cell Cycle Proteins; C069930:rad9 protein; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases