Abstract
A phosphorylated basic fibroblast growth factor (FGF) can be detected in extracts of bovine capillary endothelial cells and human hepatoma cells. Accordingly, human basic FGF contains consensus sequences that account for its phosphorylation on Thr-112 by the catalytic subunit of the cAMP-dependent protein kinase A (PK-A) and on Ser-64 by the calcium- and phospholipid-dependent protein kinase C (PK-C). A kinetic analysis of both of these reactions revealed that basic FGF is among the better substrates for these enzymes. Although the kinase responsible for the phosphorylation in vivo has not yet been identified, we examined the effects of phosphorylation on the biological activity, heparin-binding capacity, and receptor-binding capacity of phosphorylated basic FGF. No effects of phosphorylation were observed when the mitogen was phosphorylated by PK-C. In contrast, when basic FGF was phosphorylated in the receptor-binding domain with PK-A, the growth factor was 3-8 times better at displacing radiolabeled basic FGF in the radioreceptor assay. No effects were seen on the binding of this FGF to immobilized heparin or cell-associated glycosaminoglycans, suggesting that this phosphorylation modifies the affinity of basic FGF for its receptor. Biological assays for basic FGF failed to identify differences between the phosphorylated and unphosphorylated forms of recombinant basic FGFs presumably because of the presence of ectophosphatases and the experimental conditions of proliferation and mitogenic assays (37 degrees C, 24-96 hr). Because the relative affinity of basic FGF for its receptor and cell-associated glycosaminoglycans may regulate its activity, the identification of a modified form of basic FGF may be of particular importance in understanding the mechanisms that regulate its biological activity, bioavailability, and processing to and from the extracellular matrix.