Abstract
The DNA damage-activated protein kinase Chk1 is known to undergo auto-phosphorylation, however the sites and functional significance of this modification remain poorly understood. We have identified two novel Chk1 auto-phosphorylation sites, threonines 378 and 382 (T378/382), located in a highly conserved motif within the C-terminal Kinase Associated 1 (KA1) domain. T378/382 occur within optimal consensus Chk1 phosphorylation motifs and substitution with phospho-mimetic aspartic acid residues results in a constitutively active mutant Chk1 kinase (Chk1-DD) that arrests cell cycle progression in G2 phase of the cell cycle in the absence of DNA damage. Remarkably, the mutant Chk1-DD protein is also subject to very rapid proteasomal degradation, with a half-life approximately one tenth that of wild-type Chk1. Consistent with this, T378/T382 auto-phosphorylation also accelerates the proteasomal degradation of constitutively active Chk1 KA1 domain structural mutants. T378/382 auto-phosphorylation and accelerated degradation of wild-type Chk1 occurs at low levels during unperturbed growth, but surprisingly, is not augmented in response to genotoxic stress. Taken together, these observations demonstrate that Chk1 T378/T382 auto-phosphorylation within the KA1 domain is linked to kinase activation and rapid proteasomal degradation, and suggest a non-canonical mechanism of regulation.
Highlights
The serine-threonine protein kinase Chk[1] is a key regulator of the DNA damage and replication checkpoints in vertebrate cells[1]
Activation of Chk[1] in response to genotoxic stress requires phosphorylation of multiple serine-glutamine (SQ) residues within the C-terminal regulatory region that is catalysed by the upstream regulatory kinase ATR1, most prominently serine 317 (S317) and serine 345 (S345)
Using a Chk[1] mutant bearing phospho-mimetic amino acid substitutions of T378/382, in combination with previously described structural mutants bearing point mutations within the Kinase Associated 1 (KA1) domain that confer enhanced kinase activity and constitutive biological activity on Chk[114], we show that auto-phosphorylation of these sites is linked both to Chk[1] activation and degradation
Summary
The serine-threonine protein kinase Chk[1] is a key regulator of the DNA damage and replication checkpoints in vertebrate cells[1]. During DNA synthesis inhibition, Chk[1] blocks the onset of mitosis in cells with incompletely replicated DNA whilst acting to stabilise stalled replication forks and inhibit late replication origin firing[1] These canonical interphase DNA structure checkpoint responses promote genomic stability and cell survival under conditions of genotoxic stress. Serine 296 (S296) has been identified as a Chk[1] auto-phosphorylation site[8,9] Modification of this residue is stimulated by DNA damage, contingent on prior modification of S317/S345 by ATR9, and plays an important role in dispersing Chk[1] through the nucleoplasm and targeting it to its substrate Cdc25A via interaction with 14-3-3 gamma[8]. Replacement of S296 with a non-phosphorylatable alanine impairs checkpoint proficiency[10] Taken together, these data suggest that auto-phosphorylation of S296 contributes to the conventional mechanism of Chk[1] activation by genotoxic stress in collaboration with ATR. Whether CRL4–DDB1–CDT2 and potentially other ubiquitin ligase complexes can interact with this putative degron remains unknown
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