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Intracellular calcium regulates the tyrosine kinase receptor encoded by the MET oncogene
L Gandino1, L Munaron, L Naldini
1Department of Biomedical Sciences and Oncology, University of Torino Medical School, Italy.
Abstract:
Previous work (Gandino, L., Di Renzo, M. F., Giordano, S., Bussolino, F., and Comoglio, P.M. (1990) Oncogene 5, 721-725) has shown that the tyrosine kinase activity of the receptor encoded by the MET protooncogene is negatively modulated by protein kinase C (PKC). We now show that an increase of intracellular Ca2+ has a similar inhibitory effect in vivo, via a PKC-independent mechanism. In GTL-16 cells the p145MET kinase is overexpressed and constitutively phosphorylated on tyrosine. A rapid and reversible decrease of p145MET tyrosine phosphorylation was induced by treatment with the calcium ionophores A23187 or ionomycin. Experiments performed with the ionophores in absence of extracellular calcium showed that a rise in cytoplasmic Ca2+ concentration to 450 nM (due to release from intracellular stores) resulted in a similar effect. These Ca2+ concentrations had no effect on p145MET autophosphorylation in an in vitro kinase assay. This suggests that the effect of Ca2+ on p145MET tyrosine phosphorylation is not direct but may be mediated by Ca(2+)-activated proteins(s). Involvement of Ca(2+)-dependent tyrosine phosphatases was ruled out by experiments carried out in presence of Na2VO4. In vivo labeling with [32P]orthophosphate showed that the rise of intracellular Ca2+ induces serine phosphorylation of p145MET on a specific phosphopeptide. This suggests that Ca2+ negatively modulates p145MET kinase through the phosphorylation of a critical serine residue by a Ca(2+)-activated serine kinase distinct from PKC.
Insights
Increased intracellular calcium (Ca2+) inhibits MET protooncogene tyrosine kinase activity independently of protein kinase C (PKC). This calcium-mediated inhibition occurs via serine phosphorylation of the p145MET kinase.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- The MET protooncogene encodes a receptor tyrosine kinase.
- Protein kinase C (PKC) negatively modulates MET tyrosine kinase activity.
- Dysregulation of MET signaling is implicated in cancer development.
Purpose of the Study:
- To investigate the effect of intracellular calcium (Ca2+) on MET protooncogene tyrosine kinase activity.
- To elucidate the mechanism of Ca2+-mediated inhibition of p145MET.
- To determine if Ca2+ acts via a PKC-independent pathway.
Main Methods:
- Utilized GTL-16 cells overexpressing p145MET.
- Administered calcium ionophores (A23187, ionomycin) to alter intracellular Ca2+ levels.
- Performed in vitro kinase assays and in vivo [32P]orthophosphate labeling.
- Investigated the role of Ca2+-dependent tyrosine phosphatases using Na2VO4.
Main Results:
- Increased intracellular Ca2+ rapidly and reversibly decreased p145MET tyrosine phosphorylation in vivo.
- The effect was observed with Ca2+ release from intracellular stores, independent of extracellular calcium.
- Ca2+ did not directly affect p145MET autophosphorylation in vitro.
- Intracellular Ca2+ induced serine phosphorylation of p145MET on a specific phosphopeptide.
- Ca2+-mediated inhibition was independent of PKC and Ca2+-dependent tyrosine phosphatases.
Conclusions:
- Intracellular Ca2+ negatively modulates p145MET kinase activity through a PKC-independent mechanism.
- This modulation involves the phosphorylation of a critical serine residue on p145MET by a Ca2+-activated serine kinase.
- Calcium signaling represents a novel regulatory pathway for MET tyrosine kinase activity.