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ATM phosphorylates ZBP-89 at Ser202 to potentiate p21waf1 induction by butyrate

Longchuan Bai1, Juanita L Merchant

  • 1Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

Biochemical and Biophysical Research Communications
|June 15, 2007
PubMed

Insights

Histone deacetylase inhibitors (HDACi) promote colon cancer cell growth arrest. ATM kinase-mediated phosphorylation of ZBP-89 is crucial for HDACi-induced p21(waf1) gene expression and therapeutic effects.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Biochemistry

Background:

  • Histone deacetylase inhibitors (HDACi) are investigated as chemotherapeutics, particularly for colon cancer.
  • HDACi induce cell growth arrest and differentiation, mediated partly by the cyclin-dependent kinase (CDK) inhibitor p21(waf1).
  • A complex involving ATM, ZBP-89, and p300 is known to regulate p21(waf1) expression following HDACi treatment.

Purpose of the Study:

  • To investigate the role of ATM kinase in the phosphorylation of ZBP-89.
  • To determine if ATM-mediated phosphorylation of ZBP-89 is essential for HDACi-induced p21(waf1) expression.
  • To elucidate the contribution of ZBP-89 phosphorylation to the mechanism of HDACi action in colonic cells.

Main Methods:

  • In vitro and in vivo phosphorylation assays of ZBP-89 by ATM kinase.
  • Site-directed mutagenesis to disrupt the ATM phosphorylation motif (202)SQ in ZBP-89.
  • Assays to evaluate the effect of ZBP-89 phosphorylation status on p21(waf1) activation by butyrate (an HDACi).
  • Measurement of endogenous p21(waf1) expression following butyrate treatment in cells expressing wild-type or mutated ZBP-89.

Main Results:

  • ZBP-89 was confirmed to be phosphorylated by ATM kinase both in vitro and in vivo.
  • Disruption of the ATM phosphorylation site (202)SQ in ZBP-89 significantly reduced its ability to enhance butyrate-induced p21(waf1) activation.
  • The mutated ZBP-89 lacking the functional ATM phosphorylation site failed to potentiate the induction of endogenous p21(waf1) expression by butyrate.

Conclusions:

  • ATM kinase phosphorylates ZBP-89, a critical step in the signaling pathway.
  • ATM-mediated phosphorylation of ZBP-89 is essential for the potentiation of p21(waf1) gene expression by HDAC inhibitors like butyrate.
  • This phosphorylation event is a key mechanism contributing to the therapeutic effects of HDAC inhibitors in colon cells.

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