Abstract
De novo expression of human T-cell leukemia virus type 1 Tax results in cellular genomic instability. We demonstrated previously that Tax associates with the cell cycle check point regulator Chk2 and proposed that the inappropriate activation of Chk2 provides a model for Tax-induced loss of genetic integrity (Haoudi, A., Daniels, R. C., Wong, E., Kupfer, G., and Semmes, O. J. (2003) J. Biol. Chem. 278, 37736-37744). Here we provide an explanation for how Tax induces some Chk2 activities but represses others. We show that Tax interaction with Chk2 generates two activation signals in Chk2, oligomerization and autophosphorylation. However, egress of Chk2 from chromatin, normally observed in response to ionizing radiation, was repressed in Tax-expressing cells. Analysis of chromatin-bound Chk2 from Tax-expressing cells revealed phosphorylation at Thr(378), Ser(379), Thr(383), Thr(387), and Thr(389). In contrast, chromatin-bound Chk2 in the absence of Tax was phosphorylated at Thr(383) and Thr(387) in response to ionizing radiation. We further establish that Tax binds to the kinase domain of Chk2. Confocal microscopy revealed a redistribution of Chk2 to colocalize with Tax in Tax speckled structures, which we have shown previously to coincide with interchromatin granules. We propose that Tax binding via the Chk2 kinase domain sequesters phosphorylated Chk2 within chromatin, thus hindering chromatin egress and appropriate response to DNA damage.
Highlights
Tax regulates a wide range of cellular functions, there is no evidence that Tax directly interacts with DNA in a manner that would result in DNA damage (11)
Tax Expression Results in Binding to and Activation of Endogenous Chk2—We demonstrated previously that Tax interacts with Chk2 by co-immunoprecipitation (26)
We examined the impact of Tax on phosphorylation of chromatin-bound Chk2 using direct mass spectrometry analysis
Summary
It appears that Tax expression results in Chk2 that is able to generate activation of some of its cognate downstream targets, as is reflected in cellular G2/M accumulation, but that is impaired in its overall response to DNA damage. The affinity isolation of Tax protein from cells resulted in a multiprotein complex containing endogenous Chk2 as a component (Fig. 1A). Tax or control cell nuclear lysate was added at increasing amounts to the purified GST-Chk2 protein, and kinase assays were performed to assess autophosphorylation of Chk2.
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