Identification of the major sites of phosphorylation in IGF binding protein-3.
Insulin-like growth factor binding protein-3 (IGFBP-3) is the major carrier of insulin-like growth factor I and II in the circulation. IGFBP-3 is secreted by various tissues and cell lines as a glycosylated phosphoprotein. We have identified two major serine phosphorylation sites located at amino acids 111 and 113 of the human protein. These serine residues and neighboring amino acids potentially involved in defining a protein kinase recognition sequence were mutated to alanine using PCR. Single and double point mutants were stably transfected into CHO-cells and analyzed for their level of phosphorylation. Mutation of both serines reduced phosphorylation by > 80% in the full-length protein and completely abolished phosphorylation in a 17 kDa IGFBP-3 fragment, derived from digestion with EndoProteinase Lys-C. The 17 kDa fragment contained serines 111 and 113. S111A/S113A, a double serine-to-alanine mutant at positions 111 and 113, showed a strongly reduced glycosylation pattern that appears to be the result of amino acid substitutions rather than lack of phosphorylation. Mutant S111A/S113A, despite being non-phosphorylated and non-glycosylated, is functionally similar to the wild-type IGFBP-3 in terms of IGF-I binding. These results enhance our understanding on the functional role of glycosylation and phosphorylation of IGFBP-3.
Hoeck
W G
WG
Department of BioAnalytical Technology, Genentech, Inc., South San Francisco, California 94080.
Mukku
V R
VR
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006224:Cricetinae; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011384:Proinsulin / Q000096:biosynthesis; D011494:Protein Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014162:Transfection
D002352:Carrier Proteins; D017931:DNA Primers; D018969:Insulin-Like Growth Factor Binding Proteins; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D012694:Serine; D011384:Proinsulin; D011494:Protein Kinases; D000409:Alanine