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Updated: Jul 16, 2025
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Phosphorylation at tyrosine 317 and 508 are crucial for PIK3CA/p110α to promote CRC tumorigenesis
Ting Wang1, Longci Sun2, Chengkun Chen1
1State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200032, China.
Background:
PI3K/AKT signaling pathway plays important role in tumorigenesis of human cancer. Protein phosphorylation is crucial for signaling transduction of this pathway. PIK3CA, encoding the catalytic subunit p110α of PI3K complex, is one of the most frequently mutated oncogenes in human cancers. However, phosphorylation sites of PIK3CA/p110α and their underlying mechanism in tumorigenesis are largely unknown.
Methods:
Tyrosine phosphorylation sites of PIK3CA/p110α are identified with Mass-Spectrum. Crispr/CAS9 strategy is applied to generate Y317F and Y508F mutant knock-in cell clones. The growth and metastasis abilities of cells are evaluated in vitro and in vivo. Phospho-proteomics analysis and Western blots are used to demonstrate downstream signaling pathways of PIK3CA/p110α tyrosine phosphorylation. In vitro kinase assay is applied to identify the kinase of PIK3CA/p110α tyrosine phosphorylation.
Results:
Tyrosine phosphorylation of PIK3CA/p110α is stimulated by growth factors such as EGF, HGF and PDGF. Two tyrosine residues, Y317 and Y508, are identified on PIK3CA/p110α. Either Y317 or Y508 phosphorylation is essential for tumorigenesis of CRC. Mutation at Y317 of p110α reduces the proliferation, migration, and invasion of cancer cells through Src-MLC2 pathway, while mutation at Y508 of p110α impairs AKT signaling. Moreover, Src interacts with and phosphorylates p110α.
Conclusions:
PIK3CA/p110α phosphorylation at Y317 and Y508 play important role in tumorigenesis of colorectal cancer through two independent pathways.
Insights
Phosphorylation of PIK3CA/p110α at Y317 and Y508 is crucial for colorectal cancer (CRC) tumorigenesis. These sites regulate cancer cell growth and metastasis through distinct signaling pathways, offering potential therapeutic targets.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- The PI3K/AKT pathway is vital in cancer development, with PIK3CA mutations frequently observed in human cancers.
- Understanding PIK3CA/p110α phosphorylation is key to elucidating its role in tumorigenesis.
Purpose of the Study:
- To identify and characterize tyrosine phosphorylation sites on PIK3CA/p110α.
- To investigate the functional impact of these phosphorylation events on cancer progression.
Main Methods:
- Mass spectrometry to identify tyrosine phosphorylation sites.
- CRISPR/Cas9 gene editing to create PIK3CA Y317F and Y508F mutants.
- In vitro and in vivo assays for cell growth and metastasis.
- Phospho-proteomics and Western blot analyses for downstream signaling.
- In vitro kinase assays to identify responsible kinases.
Main Results:
- Two key tyrosine phosphorylation sites, Y317 and Y508, were identified on PIK3CA/p110α.
- Phosphorylation at Y317 impacts proliferation, migration, and invasion via the Src-MLC2 pathway.
- Phosphorylation at Y508 affects AKT signaling, crucial for tumorigenesis.
- Src kinase was identified as interacting with and phosphorylating PIK3CA/p110α.
Conclusions:
- PIK3CA/p110α phosphorylation at Y317 and Y508 are critical for colorectal cancer (CRC) tumorigenesis.
- These phosphorylation events regulate cancer progression through two independent signaling pathways.
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