Biochemical characterization of a N-terminal fragment (p5) cleaved from fibroblast growth factor-binding protein (FGF-BP) in bovine milk in vitro.
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 1delta (CK1delta)] 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 CK1delta in the presence of two sulfated lipids [sulfatide or cholesterol-3-sulfate (CH-3S), SCS] in vitro. A novel phosphorylation site (RNRRGS) for CK1delta 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 CK1delta 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.
Ohtsuki
Kenzo
K
Laboratory of Genetical Biochemistry and Signal Biology, Graduate School of Medical Sciences, Kitasato University, Sagamihara 228-8555, Japan. ken@kitasato-u.ac.jp
Hirayama
Kyoko
K
Kawakami
Fumitaka
F
Kato
Tomoki
T
Kawakami
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D047389:Casein Kinase I / Q000378:metabolism; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D066298:In Vitro Techniques; D007781:Lactoferrin / Q000737:chemistry / Q000378:metabolism; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D013433:Sulfoglycosphingolipids / Q000378:metabolism
D010455:Peptides; D013433:Sulfoglycosphingolipids; D005346:Fibroblast Growth Factors; D000242:Cyclic AMP; D047389:Casein Kinase I; D007781:Lactoferrin