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Published on: September 30, 2016
Mass spectroscopic phosphoprotein mapping of Ral binding protein 1 (RalBP1/Rip1/RLIP76)
Mikael C Herlevsen1, Dan Theodorescu1
1Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Abstract:
RalBP1, a multifunctional protein implicated in cancer cell proliferation, radiation and chemoresistance, and ligand dependent receptor internalization, is upregulated in bladder cancer and is a downstream effector of RalB, a GTPase associated with metastasis. RalBP1 can be regulated by phosphorylation by protein kinase C (PKC). No studies have comprehensively mapped RalBP1 phosphorylation sites or whether RalB affects these. We identified 14 phosphorylation sites of RalBP1 in human bladder carcinoma UMUC-3 and embryonic kidney derived 293T cells. The phosphorylated residues are concentrated at the N-terminus. Ten of the first 100 amino acids of the primary structure were phosphorylated. Nine were serine residues, and one a threonine. We evaluated the effect of RalB overexpression on RalBP1 phosphorylation and found the largest change in phosphorylation status at S463 and S645. Further characterization of these sites will provide novel insights on RalBP1 biology, its functional relationship to RalB and possible avenues for therapeutic intervention.
Insights
Researchers mapped 14 phosphorylation sites on RalBP1, a protein linked to bladder cancer progression. RalB overexpression significantly altered phosphorylation at specific sites, offering potential therapeutic targets.
Area of Science:
- Molecular biology
- Cancer research
- Biochemistry
Background:
- Ral Binding Protein 1 (RalBP1) is a key protein in cancer cell proliferation, chemoresistance, and metastasis.
- RalBP1 is upregulated in bladder cancer and regulated by protein kinase C (PKC) phosphorylation.
- The precise phosphorylation sites of RalBP1 and the influence of RalB on these sites remain largely uncharacterized.
Purpose of the Study:
- To comprehensively map RalBP1 phosphorylation sites in human bladder carcinoma cells.
- To investigate the impact of RalB overexpression on RalBP1 phosphorylation.
- To identify potential therapeutic targets for bladder cancer intervention.
Main Methods:
- Phosphoproteomic analysis of RalBP1 in human bladder carcinoma UMUC-3 and HEK 293T cells.
- Overexpression of RalB to assess its effect on RalBP1 phosphorylation patterns.
- Identification and characterization of specific phosphorylated residues.
Main Results:
- Identified 14 novel phosphorylation sites on RalBP1.
- Concentration of phosphorylated residues within the N-terminal region, with 10 sites in the first 100 amino acids.
- Significant alterations in phosphorylation status at Serine 463 (S463) and Serine 645 (S645) upon RalB overexpression.
Conclusions:
- This study provides the first comprehensive map of RalBP1 phosphorylation sites.
- RalB significantly influences RalBP1 phosphorylation at specific sites (S463, S645).
- Further investigation of these sites may reveal novel insights into RalBP1-RalB interactions and potential therapeutic strategies for bladder cancer.
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