Chk1-dependent constitutive phosphorylation of BLM helicase at serine 646 decreases after DNA damage.
BLM helicase, the protein mutated in Bloom syndrome, is involved in signal transduction cascades after DNA damage. BLM is phosphorylated on multiple residues by different kinases either after stress induction or during mitosis. Here, we have provided evidence that both Chk1 and Chk2 phosphorylated the NH(2)-terminal 660 amino acids of BLM. An internal region within the DExH motif of BLM negatively regulated the Chk1/Chk2-dependent NH(2)-terminal phosphorylation event. Using in silico analysis involving the Chk1 structure and its known substrate specificity, we predicted that Chk1 should preferentially phosphorylate BLM on serine 646 (Ser(646)). The prediction was validated in vitro by phosphopeptide analysis on BLM mutants and in vivo by usage of a newly generated phosphospecific polyclonal antibody. We showed that the phosphorylation at Ser(646) on BLM was constitutive and decreased rapidly after exposure to DNA damage. This resulted in the diminished interaction of BLM with nucleolin and PML isoforms, and consequently decreased BLM accumulation in the nucleolus and PML nuclear bodies. Instead, BLM relocalized to the sites of DNA damage and bound with the damage sensor protein, Nbs1. Mutant analysis confirmed that the binding to nucleolin and PML isoforms required Ser(646) phosphorylation. These results indicated that Chk1-mediated phosphorylation on BLM at Ser(646) might be a determinant for regulating subnuclear localization and could act as a marker for the activation status of BLM in response to DNA damage.
Kaur
Sarabpreet
S
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Modi
Priyanka
P
Srivastava
Vivek
V
Mudgal
Richa
R
Tikoo
Shweta
S
Arora
Prateek
P
Mohanty
Debasisa
D
Sengupta
Sagar
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001816:Bloom Syndrome / Q000201:enzymology / Q000473:pathology; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D053484:RecQ Helicases / Q000737:chemistry / Q000378:metabolism*
D010455:Peptides; D010769:Phosphothreonine; D010768:Phosphoserine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; C097151:Bloom syndrome protein; D053484:RecQ Helicases