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Updated: Jun 27, 2026
Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Published on: March 21, 2022
c-Abl phosphorylates Hdmx and regulates its interaction with p53
Valentina Zuckerman1, Kristiaan Lenos, Grzegorz M Popowicz
1Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel.
Abstract:
Upon exposure to DNA damage the p53 tumor suppressor is accumulated and activated to stall cellular growth. For this to occur, p53 must be relieved from its major inhibitors, Mdm2 (Hdm2 in humans) and Mdmx (Mdm4; Hdmx in humans). A key mechanism controlling this relief is the post-translational modifications of p53 and its inhibitors. We have previously demonstrated that the stress-activated tyrosine kinase, c-Abl, contributes to the relief of p53 from Hdm2. Because Hdmx is the major inhibitor of p53 activity, the additional possibility that c-Abl protects p53 through targeting Hdmx was explored in this study. c-Abl was found to interact with and to phosphorylate Hdmx. This phosphorylation was enhanced in response to DNA damage. Importantly, we mapped the sites of phosphorylation to the p53 binding domain of Hdmx. One of these phosphorylations, on tyrosine 99, inhibited Hdmx interaction with p53. This inhibition is consistent with the predicted role of this residue in the interaction with p53 based on the crystal structure of the interaction site. Our results show that c-Abl not only targets Hdm2, but also Hdmx, which together contribute to p53 activation in response to DNA damage.
Insights
The stress-activated tyrosine kinase c-Abl targets both Hdm2 and Hdmx, major inhibitors of the p53 tumor suppressor. This dual targeting by c-Abl promotes p53 activation following DNA damage, crucial for halting cellular growth.
Area of Science:
- Molecular Biology
- Cellular Biology
- Oncology
Background:
- The p53 tumor suppressor is activated upon DNA damage to prevent uncontrolled cell proliferation.
- p53 activity is tightly regulated by its inhibitors, primarily Mdm2 and Mdmx (also known as Hdm2 and Hdm4 in humans).
- Post-translational modifications of p53 and its inhibitors are critical for modulating p53 activity.
Purpose of the Study:
- To investigate the role of the stress-activated tyrosine kinase c-Abl in regulating p53 activity through its interaction with Hdmx.
- To determine if c-Abl targets Hdmx, similar to its previously established role with Hdm2, in the context of DNA damage response.
Main Methods:
- Co-immunoprecipitation assays to assess the interaction between c-Abl and Hdmx.
- In vitro kinase assays to confirm Hdmx phosphorylation by c-Abl.
- Site-directed mutagenesis and structural analysis to map phosphorylation sites and their functional impact on Hdmx-p53 binding.
Main Results:
- c-Abl directly interacts with and phosphorylates Hdmx, with increased phosphorylation observed upon DNA damage.
- Phosphorylation sites on Hdmx were mapped to its p53 binding domain.
- Specific phosphorylation, notably at tyrosine 99, was shown to inhibit the interaction between Hdmx and p53.
Conclusions:
- c-Abl plays a dual role in p53 regulation by targeting both Hdm2 and Hdmx.
- Phosphorylation of Hdmx by c-Abl, particularly at tyrosine 99, is a key mechanism for relieving p53 inhibition.
- These findings elucidate a novel pathway for c-Abl-mediated p53 activation in response to DNA damage.
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