The effect of phosphorylation by casein kinase 2 on the activity of insulin-like growth factor-binding protein-3

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is known to be secreted as a phosphoprotein, constitutively phosphorylated at casein kina se 2 (CK2) sites. To examine the effect of phosphorylation by CK2 on the pr opel-ties of glycosylated human IGFBP-3, we phosphorylated plasma-derived I GFBP-3, containing less than 1 mol/mol phosphoserine, in vitro. As judged b y incorporated P-32, enzymatic deglycosylation did not decrease the phospha te content of phospho-IGFBP-3. Phosphorylation had no effect on IGF-T or IG F-II binding, but was inhibitory to acid-labile subunit binding in the pres ence of either IGF. Determined in simian virus 40-transformed human fibrobl asts, cell association by phospho-IGFBP-3 was inhibited approximately 50% c ompared with that of the nonphosphorylated preparation. Phospho-IGFBP-3 sho wed significant resistance to proteolysis by plasmin and a cysteine proteas e secreted by MCF-7 cells. However, no difference was seen between the two preparations in their inhibition of IGF-I-stimulated DNA synthesis when coi ncubated with IGF-I in neonatal skin fibroblasts or MCF-7 breast cancer cel ls, and little difference was found in their ability to potentiate IGF-I-st imulated DNA synthesis when preincubated with fibroblasts. These results in dicate that IGFBP-3 interaction with acid-labile subunit and with the cell surface, both of which involve basic carboxyl-terminal residues, may be mod ulated by phosphorylation. Relative resistance to proteolysis and poor bind ing to cells suggest that CK2-phospho-IGFBP-3 may be a significant inhibito r of ICF activity in the extracellular environment.