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Published on: November 30, 2018
Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation
Paul Mellor1, Jeremy D S Marshall1,2, Xuan Ruan1
1Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada.
Abstract:
The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.
Insights
Cancer-associated p85α mutations disrupt PTEN and Rab5 interactions, altering signaling pathways crucial for oncogenesis. These mutations impact protein binding and enzymatic activity, potentially driving cancer development.
Area of Science:
- Molecular Biology
- Cell Signaling
- Cancer Research
Background:
- The p85α regulatory subunit is central to the PI3K/PTEN pathway and Rab GTPase regulation.
- Dysregulation of these pathways is implicated in cancer development.
- Specific p85α domains bind and modulate PTEN and Rab5 functions.
Purpose of the Study:
- To investigate the functional impact of cancer patient-derived p85α mutations.
- To determine how these mutations affect PTEN and Rab5 binding and activity.
- To elucidate the structural basis for altered p85α function in cancer.
Main Methods:
- Analysis of patient-derived p85α mutations.
- Biochemical assays measuring PTEN lipid phosphatase and Rab5 GAP activity.
- Protein binding assays.
- Crystal structure determination of p85α BH domain mutants.
Main Results:
- One mutation (L30F) reduced PTEN binding but enhanced PTEN activity.
- Other mutations (E137K, K288Q, E297K) altered PTEN regulation.
- Many mutations decreased Rab5 binding (e.g., L30F, I69L).
- Several mutations impaired p85α's GTPase-activating protein (GAP) activity towards Rab5.
- Crystal structures revealed no significant changes in overall BH domain structure.
Conclusions:
- Cancer-derived p85α mutations can deregulate PTEN and Rab5 pathways.
- These alterations contribute to oncogenesis by disrupting critical cellular signaling.
- Specific residues (L191, V263) were identified as critical for Rab5 interaction and regulation.
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