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Phosphorylation of basic fibroblast growth factor by a protein kinase associated with the outer surface of a target
1Department of Molecular and Cellular Growth Biology, Whittier Institute for Diabetes and Endocrinology, La Jolla, California 92037.
Abstract:
A protein kinase capable of phosphorylating basic fibroblast growth factor (FGF) can be localized on the outer cell surface of human hepatoma cells (SK-Hep cells). The addition of [gamma-32P]ATP, but not H3(32)PO4, results in a rapid (less than 10 min) incorporation of 32P into exogenously added basic FGF. The reaction is time and concentration dependent (apparent Km, 170 nM) and is stimulated by the addition of cAMP (EC50, 0.5 microM), but not the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate. There is also no tyrosine protein kinase detected on the cell surface. The inhibition of basic FGF binding to its low and/or high affinity sites decreases the phosphorylation of basic FGF by the ecto-protein kinase. Accordingly, pretreatment of cells with heparinase for 30 min or coincubation with heparin (0.1-10 micrograms/ml) decreases phosphorylation in a dose-dependent manner. Furthermore, the addition of a nonphosphorylatable peptide analog of basic FGF ([Val112] basic FGF-(106-146)NH2) that can compete with basic FGF binding to cells prevents the phosphorylation of basic FGF. Together, these observations suggest that 1) exogenous basic FGF must associate with its low and/or high affinity binding sites to be phosphorylated, and 2) the kinase is cAMP dependent and associated with the outer cell surface, and support the hypothesis that phosphorylation may regulate the activity and/or bioavailability of the growth factor.
Insights
A cell surface protein kinase phosphorylates basic fibroblast growth factor (FGF) on human hepatoma cells. This cAMP-dependent process requires FGF binding to cell surface receptors, suggesting phosphorylation regulates FGF activity.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Basic fibroblast growth factor (FGF) plays a crucial role in cell growth and development.
- The extracellular activity and regulation of FGF are not fully understood.
- Investigating cell surface modifications of FGF can reveal new regulatory mechanisms.
Purpose of the Study:
- To identify and characterize a protein kinase on human hepatoma cells that phosphorylates basic FGF.
- To elucidate the conditions and requirements for this extracellular phosphorylation event.
- To explore the potential implications of FGF phosphorylation for its biological activity.
Main Methods:
- Incubation of human hepatoma cells (SK-Hep) with radiolabeled ATP and exogenous basic FGF.
- Assessing 32P incorporation into basic FGF under various conditions (e.g., presence of cAMP, heparin, competing peptides).
- Enzyme kinetic analysis to determine the apparent Km and EC50 values.
Main Results:
- A protein kinase localized on the outer cell surface of SK-Hep cells was found to rapidly phosphorylate exogenous basic FGF.
- Phosphorylation was dependent on time, FGF concentration, and stimulation by cAMP, but not by a phorbol ester.
- FGF binding to its cell surface receptors, as well as heparin, was necessary for efficient phosphorylation, and a nonphosphorylatable FGF analog inhibited the reaction.
Conclusions:
- Exogenous basic FGF must associate with its cell surface binding sites to be phosphorylated by a cell-surface, cAMP-dependent kinase.
- These findings support the hypothesis that extracellular phosphorylation of basic FGF may regulate its activity and/or bioavailability.