By means of successive gel filtration on a Superdex 30 pg column and Mono S column chromatography, a 5-kDa polypeptide (p5) was highly purified from the low molecular weight (LMW) fraction separated from the partially purified lactoferrin (bLF) fraction of bovine milk, and biochemically characterized as a phosphate acceptor for two protein kinases [cAMP-dependent protein kinase (PKA) and casein kinase 18 (CK1 delta)] in vitro. Purified p5 was identified as a fragment (N-terminal positions 24-51, 28 amino acid residues) cleaved from fibroblast growth factor-binding protein (FGF-BP, p37). Both purified p5 and synthetic p5 (sp5) were effectively phosphorylated by PKA, and also phosphorylated by CK1 delta in the presence of two sulfated lipids [sulfatide or cholesterol-3-sulfate (CH-3S), SCS] in vitro. A novel phosphorylation site (RNRRGS) for CK1 delta and a potent SCS-binding site (RNRR) on p5 were identified. The PKA-mediated phosphorylation of p5 was highly stimulated when incubated with either acidic FGF (aFGF) or bLF in vitro, but this phosphorylation was more sensitive to SCS than H-89 (a specific PKA inhibitor). Immunoprecipitate experiments revealed p5, but not the phosphorylated p5, to be directly bound to aFGF in vitro. These results show that (i) p5 has a high binding affinity with aFGF as well as bLF; (ii) the binding of SCS to p5 results in the selective inhibition of its phosphorylation by PKA; and (iii) SCS functions as an effective stimulator for the phosphorylation of p5 by CK1 delta in vitro. In addition, p5 may play an important physiological role as a trafficking factor for the physiological interaction between aFGF group including endothelial cell growth factors and their binding proteins in vivo. (C) 2007 Published by Elsevier B.V.
