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Research Description:
ATM, ATR, DNA-PK and SMG1 are phosphoinositide 3-kinase-related kinases (PIKKs) that safeguard genome integrity and determine cell survival following exposure to ionizing radiation (IR). ATM-deficient cells are radiosensitive and exhibit cell cycle checkpoint defects, increased chromosome breakage and delayed P53 stabilization following IR. While many substrates of the ATM kinase have been identified and implicated in cell-cycle checkpoint activation, ATM kinase-dependent signaling to P53 and mechanisms that control genome stability and cell survival are less well understood. We have previously shown that ATM activation requires the dissociation of inactive ATM homodimers into active ATM monomers. This activation process requires phosphorylation of serine-1981 in ATM and is detectable within seconds of cellular exposure to 0.1 Gy [137Cs]. Recently, it has been shown that ATM activation is also dependent on the MRE11 complex, the acetyltransferase TIP60 and the phosphatases PP2A and PP5. An aim of our laboratory is to identify additional phosphorylations, dephosphorylations and acetylations that regulate ATM activity in vivo. Direct cellular exposure to 1 mCi [32P]-orthophosphate (1.709 MeV) for 40 min is sufficient to activate ATM and stabilize P53. We therefore use direct cellular exposure to 1 mCi [32P]-orthophosphate to concurrently induce DNA-damage and label the cellular phosphoproteome. Small molecule inhibitors and siRNAs are used to identify ATM kinase-dependent signaling in the labeled nuclear phosphoproteome. ATM kinase-dependent radioprotective signaling is further isolated using siRNAs directed against the adaptor proteins 53BP1 and MDC1. Disruption of either of these ATM substrates results in radiosensitization. Dual labels are also being used to identify the DNA damage-dependent phosphoproteome in mammalian cells. Short exposures to [32P]-orthophosphate are used to label background “constitutive” phosphorylations while identical exposures to [33P]-orthophosphate are used to label both “constitutive” and ATM kinase-dependent signaling. An advantage of this approach is that the [32P]- and [33P]-labeled phosphoproteomes can be resolved in the same 2D gel and distinguished by shielding the low energy Β -particle emissions from [33P]. Also, since the activity of ATM associated TIP60 increases following IR, we are using [3H]-sodium acetate to identify ATM-dependent signaling in the acetylated proteome. Our overall hypothesis is that following IR, ATM is activated in a DNA damage recognition complex that is directly modulated by chromatin modifying activities, including that of the histone acetyltransferase TIP60, and that ATM kinase-dependent activity subsequently directly and indirectly mediates signal transduction pathways that involve kinase, phosphatase and acetyltransferase activities.
Education:
BSc (Hons), Biochemistry/Chemistry, University of Liverpool, UK, 1989.
PhD Imperial College, University of London, UK, 1994
Postdoctoral Fellow, St. Jude Children's Research Hospital, Memphis, TN, 1999-2005.
Important Publications:
- Bakkenist CJ, R Drissi, J Wu, MB Kastan and JS Dome. ATM activation is a marker of cellular aging and a precursor to replicative senescence. Cancer Res 64:3748-3752, 2004
- Horejsi Z, J Falck, CJ Bakkenist, M Kastan, J Lukas and J Bartek. Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation. Oncogene 23:3122-3127, 2004
- Kitagawa R, CJ Bakkenist, PJ McKinnon and MB Kastan. Phosphorylation of SMC1 is the critical downstream event in the ATM-NBS1-BRCA1 pathway. Genes and Development 18:1423-1438, 2004
- Bakkenist CJ and MB Kastan. Initiating cellular stress responses. Cell 118:9-17, 2004
- Bakkenist CJ and MB Kastan. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421:499-506, 2003
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