Abstract
The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.
Highlights
The cellular DNA damage response comprises mechanisms to repair DNA, as well as pathways that lead to senescence or apoptosis, depending on the level of damage and internal and external cellular conditions
Lats2 inhibited c-Abl phosphorylation of Homeodomain-interacting protein kinase 2 (HIPK2), and HIPK2 accumulation and phosphorylation of p53 at Ser46 were inhibited at high cell density. These findings demonstrate a new link between the tyrosine kinase c-Abl and the serine/threonine kinase HIPK2 in promoting DNA damage-induced apoptosis and help to explain how apoptosis is inhibited at high cell density
HIPK2 Accumulation and Activity Are Inhibited at High Cell Density—DNA damage-induced apoptosis is inhibited at high cell density, and we have recently demonstrated that the Hippo pathway, activated at high cell density, is involved in mediating this effect
Summary
Cells and Cell Culture—The cell lines used were human embryonic kidney cells HEK 293 and HEK 293T, HepG2, and Hep3B cells. Human pCDNA3 FLAG-HIPK2 full-length, K221A (kinase-dead), truncation, and GFP-fused constructs and HA-Siah-1 constructs have been previously described [16]. FLAG-HIPK2 constructs were transferred to the pTriEx expression vector (Novagen, Merck Millipore), for bacterial expression of N-terminally His-tagged proteins. HEK293 cells were transfected with c-Abl constructs. Bacterially expressed and purified recombinant proteins or control were added to the tubes containing the immunoprecipitated c-Abl (not eluted from the beads). Other antibodies used were: anti-HA, monoclonal anti--tubulin, anti--actin, and anti-FLAG M5 (Sigma); anti-c-Abl K12, anti-c-Abl 8E9, and anti-general phosphotyrosine (phospho-Tyr (PY20), Santa Cruz Biotechnology, Santa Cruz, CA); anti-cleaved caspase-3, anti-phospho-Ser p53 (Cell Signaling, Beverly, MA). Imaging Flow Cytometry—HEK293T cells were transfected with GFP-HIPK2 and H2B-RFP or mRFP-PML IV. C-Abl led to a dramatic increase in the accumulation of wild-type HIPK2, HIPK2 1-1019 551-1191
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