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Chk1-dependent constitutive phosphorylation of BLM helicase at serine 646 decreases after DNA damage.

ABSTRACT: 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.

SUBMITTER: Kaur S

PROVIDER: S-EPMC2941563 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Chk1-dependent constitutive phosphorylation of BLM helicase at serine 646 decreases after DNA damage.

Kaur Sarabpreet S Modi Priyanka P Srivastava Vivek V Mudgal Richa R Tikoo Shweta S Arora Prateek P Mohanty Debasisa D Sengupta Sagar S

Molecular cancer research : MCR 20100818 9

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 in ...[more]

PMID: 20719863

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Project description:BK Ca(2+)-activated K(+) currents exhibit diverse properties across tissues. The functional variation in voltage- and Ca(2+)-dependent gating underlying this diversity arises from multiple mechanisms, including alternate splicing of Kcnma1, the gene encoding the pore-forming (α) subunit of the BK channel, phosphorylation of α subunits, and inclusion of β subunits in channel complexes. To address the interplay of these mechanisms in the regulation of BK currents, two native splice variants, BK0 and BKSRKR, were cloned from a tissue that exhibits dynamic daily expression of BK channel, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of mouse hypothalamus. The BK0 and BKSRKR variants differed by the inclusion of a four-amino acid alternate exon at splice site 1 (SRKR), which showed increased expression during the day. The functional properties of the variants were investigated in HEK293 cells using standard voltage-clamp protocols. Compared with BK0, BKSRKR currents had a significantly right-shifted conductance-voltage (G-V) relationship across a range of Ca(2+) concentrations, slower activation, and faster deactivation. These effects were dependent on the phosphorylation state of S642, a serine residue within the constitutive exon immediately preceding the SRKR insert. Coexpression of the neuronal β4 subunit slowed gating kinetics and shifted the G-V relationship in a Ca(2+)-dependent manner, enhancing the functional differences between the variants. Next, using native action potential (AP) command waveforms recorded from SCN to elicit BK currents, we found that these splice variant differences persist under dynamic activation conditions in physiological ionic concentrations. AP-induced currents from BKSRKR channels were significantly reduced compared with BK0, an effect that was maintained with coexpression of the β4 subunit but abolished by the mutation of S642. These results demonstrate a novel mechanism for reducing BK current activation under reconstituted physiological conditions, and further suggest that S642 is selectively phosphorylated in the presence of SRKR.

Dataset Information

Chk1-dependent constitutive phosphorylation of BLM helicase at serine 646 decreases after DNA damage.

Publications

Chk1-dependent constitutive phosphorylation of BLM helicase at serine 646 decreases after DNA damage.

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