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08:05
Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Sphingosine kinase type 2 activation by ERK-mediated phosphorylation
Nitai C Hait1, Andrea Bellamy, Sheldon Milstien
1Department of Biochemistry and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, USA.
The Journal of Biological Chemistry
|February 22, 2007
Summary
Epidermal growth factor (EGF) activates sphingosine kinase 2 (SphK2) through ERK1-mediated phosphorylation. This phosphorylation is crucial for EGF-stimulated migration in human breast cancer cells.
Area of Science:
- Biochemistry
- Cell Biology
- Cancer Research
Background:
- Sphingosine 1-phosphate (S1P) is a lipid mediator regulating biological responses in cancer.
- Sphingosine kinases (SphK1 and SphK2) tightly control cellular S1P levels.
- SphK1 activation is understood, but SphK2 activation mechanisms are largely unknown.
Purpose of the Study:
- To elucidate the mechanism of SphK2 activation by agonists.
- To investigate the role of phosphorylation in SphK2 regulation.
- To understand SphK2's contribution to human breast cancer cell migration.
Main Methods:
- Investigated epidermal growth factor (EGF) and phorbol ester effects on SphK2 phosphorylation.
- Utilized MEK1/ERK pathway inhibition and ERK1 down-regulation.
- Performed in vitro phosphorylation assays with recombinant ERK1 and SphK2.
- Conducted site-directed mutagenesis and analyzed cell migration.
Main Results:
- EGF and phorbol ester induced rapid SphK2 phosphorylation, dependent on the MEK1/ERK pathway.
- ERK1 directly phosphorylated SphK2 in vitro, increasing its activity.
- ERK1 and SphK2 formed a complex in vivo.
- Phosphorylation of SphK2 at Ser-351 and Thr-578 by ERK1 was essential for EGF-stimulated MDA-MB-453 cell migration.
Conclusions:
- Established the first insights into agonist-mediated SphK2 activation.
- Demonstrated ERK1-dependent phosphorylation regulates SphK2 activity.
- Highlighted the role of SphK2 phosphorylation in EGF-induced breast cancer cell motility.
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