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Enhanced phosphatidylinositol 3-kinase activity and high phosphorylation state of its downstream signalling molecules
H Murakami1, T Iwashita, N Asai
1Department of Pathology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
Abstract:
We compared the intracellular signalling pathways through Ret tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF), multiple endocrine neoplasia (MEN) 2A, or MEN 2B mutation. Tyrosine phosphorylation of Grb2-associated binder-1 (Gab1) and activation of phosphatidylinositol 3-kinase (PI 3-kinase) were induced at higher levels by GDNF stimulation or the MEN 2B mutation than by the MEN 2A mutation. Tyrosine-phosphorylated Gab1 was a major component that interacted with the active PI 3-kinase in vivo. In addition, we found that p62Dok and PKB/Akt were phosphorylated in a PI 3-kinase-dependent manner and the levels of their phosphorylation were significantly higher in the MEN 2B transfectant than in the MEN 2A transfectant. Tyrosine phosphorylation of p62Dok resulted in its complex formation with the Ras GTPase-activating protein (RasGAP) and the Nck adaptor protein. These findings thus suggested that high levels of activation of PI 3-kinase and of phosphorylation of its downstream signalling molecules may be associated with the clinical phenotype of MEN 2B.
Insights
Glial cell line-derived neurotrophic factor (GDNF) and mutations in multiple endocrine neoplasia (MEN) 2B activate intracellular signaling pathways more strongly than MEN 2A. This suggests higher PI 3-kinase activation may link to MEN 2B clinical features.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncology
Background:
- The Ret tyrosine kinase pathway is crucial in cellular signaling and is implicated in various cancers.
- Multiple Endocrine Neoplasia (MEN) types 2A and 2B are genetic disorders associated with Ret mutations, presenting distinct clinical phenotypes.
Purpose of the Study:
- To compare intracellular signaling pathway activation by glial cell line-derived neurotrophic factor (GDNF) and distinct Multiple Endocrine Neoplasia (MEN) mutations (2A vs. 2B).
- To elucidate the role of phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream effectors in the differential signaling observed.
Main Methods:
- Investigated Ret tyrosine kinase signaling pathways.
- Utilized cell transfection models expressing MEN 2A or MEN 2B mutations.
- Measured tyrosine phosphorylation of key signaling proteins including Gab1, p62Dok, and PKB/Akt.
- Assessed the activation of PI 3-kinase and its interactions with downstream molecules.
Main Results:
- GDNF stimulation and MEN 2B mutations induced higher levels of Gab1 tyrosine phosphorylation and PI 3-kinase activation compared to MEN 2A mutations.
- Phosphorylated Gab1 was identified as a key interactor with active PI 3-kinase.
- PKB/Akt and p62Dok phosphorylation occurred in a PI 3-kinase-dependent manner, with significantly higher levels in MEN 2B transfectants.
- MEN 2B-associated p62Dok phosphorylation led to complex formation with RasGAP and Nck.
Conclusions:
- The study demonstrates differential activation of intracellular signaling pathways by GDNF and MEN mutations.
- Higher activation of PI 3-kinase and its downstream signaling molecules in MEN 2B is associated with the observed clinical phenotype.
- These findings provide insights into the molecular mechanisms underlying MEN 2B pathogenesis.