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Proteolytic processing of delta-like 1 by ADAM proteases
Emilia Dyczynska1, Danqiong Sun, Haiqing Yi
1Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
The Journal of Biological Chemistry
|November 17, 2006
Summary
Delta-like 1 (Dll1) is processed by ADAM proteases, including ADAM12, ADAM9, and ADAM17. This cleavage by ADAM family members regulates Notch signaling pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Delta-like 1 (Dll1) is a key ligand in Notch pathway signaling.
- Dll1's interaction with Notch receptors can either activate or inhibit signaling depending on cis or trans orientation.
- Proteolytic processing of Dll1 by ADAM10 is known, but its regulation by other ADAM family members is less understood.
Purpose of the Study:
- To investigate the role of various ADAM family members in the proteolytic processing of Dll1.
- To determine how ADAM-mediated Dll1 cleavage affects Notch pathway activation.
- To identify specific ADAM proteases involved in Dll1 processing and their functional consequences.
Main Methods:
- Co-transfection assays with Dll1 and different ADAM family members (ADAM9, ADAM12, ADAM15, ADAM17).
- Analysis of Dll1 cleavage products and release of N-terminal fragments.
- Co-immunoprecipitation to assess protein interactions.
- Notch reporter assays to measure pathway activation.
- Experiments using ADAM-deficient mouse embryonic fibroblasts (MEFs).
- Studies on endogenous Dll1 cleavage in primary mouse myoblasts using small interfering RNAs (siRNAs).
Main Results:
- Dll1 is constitutively cleaved by ADAM12, releasing its N-terminal fragment in a cell density-dependent manner.
- ADAM12-mediated cleavage of Dll1 occurs in cis and enhances Notch signaling cell-autonomously.
- ADAM9 and ADAM17 also process Dll1, while ADAM15 does not cleave Dll1 but still interacts with it.
- A specific asparagine residue (Asn-353) in the catalytic motif is crucial for Dll1 cleavage by ADAM12, ADAM9, and ADAM17.
- Dll1 cleavage is reduced in ADAM9/12/15(-/-) MEFs, indicating a role for endogenous ADAMs.
- Endogenous Dll1 cleavage occurs during myoblast differentiation and is reduced by ADAM12 knockdown.
Conclusions:
- ADAM12, ADAM9, and ADAM17 are novel proteases that cleave Dll1, expanding the known regulators of this ligand.
- ADAM-mediated Dll1 processing plays a significant role in modulating Notch signaling.
- These findings highlight the intricate regulation of Notch signaling through proteolytic processing of its ligands by the ADAM family.
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