Identification of the major sites of phosphorylation in IGF binding protein‐3

Abstract

Insulin‐like growth factor binding protein‐3 (IGFBP‐3) is the major carrier of insulin‐like growth dactor 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 ngighboring amino acids potentially involved in defining a protrein 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 17kDa fragment containd 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 anino acid substitutions rather tha n lack of phosphrylation. Mutant S111A/S113A, despite being non‐phosphorylated and non‐glycosylated, is functionally similar to the wild‐type IGFBP‐3 in terms of IGF‐1 binding. These results enhance our 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.