LZAP is a novel Wip1 binding partner and positive regulator of its phosphatase activity in vitro
J Jacob Wamsley1, Natalia Issaeva1,2, Hanbing An3
1a Department of Surgery, Division of Otolaryngology , Yale University , New Haven , CT , USA.
Abstract:
The phosphatase Wip1 attenuates the DNA damage response (DDR) by removing phosphorylation marks from a number of DDR proteins (p53, MDM2, Chk1/2, p38). Wip1 also dephosphorylates and inactivates RelA. Notably, LZAP, a putative tumor suppressor, has been linked to dephosphorylation of several of these substrates, including RelA, p38, Chk1, and Chk2. LZAP has no known catalytic activity or functional motifs, suggesting that it exerts its effects through interaction with other proteins. Here we show that LZAP binds Wip1 and stimulates its phosphatase activity. LZAP had been previously shown to bind many Wip1 substrates (RelA, p38, Chk1/2), and our results show that LZAP also binds the previously identified Wip1 substrate, MDM2. This work identifies 2 novel Wip1 substrates, ERK1 and HuR, and demonstrates that HuR is a binding partner of LZAP. Pleasingly, LZAP potentiated Wip1 catalytic activity toward each substrate tested, regardless of whether full-length substrates or phosphopeptides were utilized. Since this effect was observed on ERK1, which does not bind LZAP, as well as for each of 7 peptides tested, we hypothesize that LZAP binding to the substrate is not required for this effect and that LZAP directly binds Wip1 to augment its phosphatase activity.
Insights
LZAP protein binds and enhances the phosphatase activity of Wip1, a key regulator of the DNA damage response. This interaction potentiates Wip1
Area of Science:
- Cellular biology
- Molecular oncology
- Biochemistry
Background:
- The phosphatase Wip1 regulates the DNA damage response (DDR) by dephosphorylating key proteins like p53 and MDM2.
- LZAP, a potential tumor suppressor, interacts with several Wip1 substrates but lacks catalytic activity.
- LZAP's mechanism of action suggests it functions via protein-protein interactions.
Purpose of the Study:
- To investigate the interaction between LZAP and Wip1.
- To determine if LZAP modulates Wip1 phosphatase activity.
- To identify novel Wip1 and LZAP substrates and binding partners.
Main Methods:
- Co-immunoprecipitation assays to detect protein-protein interactions.
- In vitro phosphatase assays using full-length proteins and phosphopeptides.
- Mass spectrometry or western blotting to identify and validate substrates.
Main Results:
- LZAP directly binds to Wip1 and significantly stimulates its phosphatase activity.
- LZAP potentiates Wip1-mediated dephosphorylation of known substrates (RelA, p38, Chk1/2, MDM2) and novel substrates (ERK1, HuR).
- HuR is identified as a novel binding partner of LZAP.
- LZAP's augmentation of Wip1 activity does not require direct binding to the substrate.
Conclusions:
- LZAP acts as a positive regulator of Wip1 phosphatase activity.
- LZAP enhances Wip1's role in attenuating the DNA damage response and potentially other cellular processes.
- The findings reveal a novel mechanism for regulating phosphatase activity through protein complex formation.
Related Concept Videos
01:42Phosphoinositides and PIPs
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...
02:54Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
02:54Protein Kinases and Phosphatases
01:20The JAK-STAT Signaling Pathway
02:54Canonical Wnt Signaling Pathway
01:22PI3K/mTOR/AKT Signaling Pathway