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 kinase 2 (CK2) sites. To examine the effect of phosphorylation by CK2 on the properties of glycosylated human IGFBP-3, we phosphorylated plasma-derived IGFBP-3, containing less than 1 mol/mol phosphoserine, in vitro. As judged by incorporated 32P, enzymatic deglycosylation did not decrease the phosphate content of phospho-IGFBP-3. Phosphorylation had no effect on IGF-I or IGF-II binding, but was inhibitory to acid-labile subunit binding in the presence of either IGF. Determined in simian virus 40-transformed human fibroblasts, cell association by phospho-IGFBP-3 was inhibited approximately 50% compared with that of the nonphosphorylated preparation. Phospho-IGFBP-3 showed significant resistance to proteolysis by plasmin and a cysteine protease secreted by MCF-7 cells. However, no difference was seen between the two preparations in their inhibition of IGF-I-stimulated DNA synthesis when coincubated with IGF-I in neonatal skin fibroblasts or MCF-7 breast cancer cells, and little difference was found in their ability to potentiate IGF-I-stimulated DNA synthesis when preincubated with fibroblasts. These results indicate that IGFBP-3 interaction with acid-labile subunit and with the cell surface, both of which involve basic carboxyl-terminal residues, may be modulated by phosphorylation. Relative resistance to proteolysis and poor binding to cells suggest that CK2-phospho-IGFBP-3 may be a significant inhibitor of IGF activity in the extracellular environment.

Phosphorylation of insulin-like growth factor binding proteins.

Insulin-like growth factor (IGF) binding proteins (IGFBPs) play a key role in regulating the availability of IGFs in the circulation and the extracellular environment. Three of these proteins-IGFBP-1, IGFBP-3 and IGFBP-5-are known to be serine-phosphorylated in their central domains, and the others have possible target sites for serine/threonine kinases. Whereas nonphosphorylated IGFBP-1 may potentiate IGF action in certain cells, phosphorylation increases its affinity for IGFs, and converts the protein to an inhibitory form. The highly phosphorylated protein predominates in the circulation, where it may acutely regulate IGF bioavailability. IGFBP-3 is also secreted as a phosphoprotein, and can be phosphorylated in vitro by protein kinases A and C, and casein kinase II. De-phosphorylation has no effect on IGF-binding, but may increase its ability to bind to the acid-labile subunit and to associate with cell surfaces. Although no specific functions have yet been ascribed to phosphorylated forms of the other IGFBPs, current evidence supports the proposal that IGFBP phosphorylation plays an important role in the regulation of IGFBP function.

Regulation of insulin-like growth factor (IGF) binding protein-3 phosphorylation by IGF-I.

Insulin-like growth factor (IGF) action is modulated by six IGF-binding proteins (IGFBP-1 to -6). IGFBP-3 is the main IGFBP in serum and is produced by many cell types, which can modify it post-translationally to yield glycosylation, proteolysis and phosphorylation products. This study investigates the regulation of IGFBP-3 phosphorylation by IGF-I in human neonatal skin fibroblasts. Fibroblasts were incubated with IGF peptides and 32P-orthophosphate for 4 h, and phosphorylated IGFBP-3 (P-IGFBP-3) was immunoprecipitated from the medium and analysed by SDS-PAGE. Media collected from parallel experiments without radioactivity were assayed for immunoreactive IGFBP-3 (I-IGFBP-3). IGF-I (50 ng/ml) increased levels of P-IGFBP-3 and I-IGFBP-3 in conditioned medium to 205 +/- 9% and 198 +/- 10% of control, respectively (n = 5). Stimulation of I-IGFBP-3 was consistent with IGF-mediated release of cell-associated IGFBP-3, since treatment with an IGF-I analogue with reduced affinity for IGFBPs did not increase I-IGFBP-3 levels, whereas treatment with an analogue with reduced affinity for receptor but normal affinity for binding proteins did. In contrast, stimulation of P-IGFBP-3 occurred independently of IGF binding to IGFBP, instead requiring interaction of IGF-I with its receptor. While the functional significance of IGFBP-3 phosphorylation is unclear, we propose that it plays a regulatory role in IGFBP-3 action.

Insulin-like growth factor-binding protein-3 production by MCF-7 breast cancer cells: stimulation by retinoic acid and cyclic adenosine monophosphate and differential effects of estradiol.

The insulin-like growth factors (IGFs) stimulate the proliferation of human breast cancer cells, including the estrogen-dependent cell line MCF-7. These cells secrete regulatory IGF-binding proteins (IGFBPs) which may enhance or attenuate IGF-stimulated cell proliferation. In this study, we have used RIA to quantify the production and regulation of IGFBP-3 and IGFBP-6 by MCF-7 cells in vitro. Under basal (serum- and phenol red-free) conditions, IGFBP-3 and IGFBP-6 accumulated in 72 h-conditioned MCF-7 medium to concentrations of approximately 0.18 nM and 0.02 nM, respectively. Treatment with retinoic acid (RA, 100 nM) increased medium concentrations of IGFBP-3 to 175 +/- 8% (mean +/- SE, n = 4), and IGFBP-6 to 217 +/- 20% of control values. Forskolin (0.5 microM) or dibutyryl cAMP (db-cAMP, 1 mM) increased both proteins 2- to 3-fold. In the presence of 100 nM RA, the stimulation elicited by these agents was enhanced, with IGFBP-3 levels increasing to 6-fold above that seen with RA alone. IGFBP-6 increased 12-fold with RA + forskolin and 20-fold with RA + dbcAMP. Estrogen (10 nM estradiol) reduced basal IGFBP-3 levels by 25% but increased IGFBP-6 1.5- to 2-fold. The stimulatory effect of RA + forskolin on IGFBP-3 was partially reversed by estrogen, whereas RA + forskolin-stimulated IGFBP-6 levels were further increased by estrogen. Increased IGFBP-3 and -6 production in response to RA + forskolin was accompanied by a decrease in IGF-stimulated thymidine incorporation into DNA; by contrast, the bioactivity of an IGF analog that does not bind with IGFBPs, [Gln3, Ala4, Tyr15, Leu16]IGF-I, was unchanged under these conditions. These data demonstrate that modulating the production of IGFBPs can lead to changes in the sensitivity of breast cancer cells to IGFs, and as a result change the cell proliferative effects of these growth factors. Further, IGFBP-3 and IGFBP-6 are differentially regulated by estrogen. Dissecting the roles of the individual IGFBPs is essential to understanding how such differential regulation will ultimately affect IGF-stimulated cell proliferation in breast cancer.

A cell-surface opsonic receptor on leucocytes from the phylogenetically primitive vertebrate, Eptatretus stouti.

A humoral recognition molecule that is homologous to the mammalian complement components C3, C4 and C5 has recently been identified in the Pacific hagfish, Eptatretus stouti. One function of this complement-like protein (CLP) is to opsonize foreign material for phagocytosis by hagfish leucocytes. Here, we demonstrate that CLP's opsonic activity can be abrogated by pre-incubating phagocytes with an anti-hagfish leucocyte mAb (1B1). Moreover, antigen-activated CLP can block the binding of the 1B1 antibody to hagfish leucocytes. Flow cytometry and immunoprecipitation indicate that 1B1 recognizes a 105 kDa cell-surface, monomeric protein that is expressed exclusively on phagocytic hagfish leucocytes. It is concluded that this 105 kDa protein represents the cell surface receptor by which CLP mediates the phagocytosis of opsonized targets.

Regulation of immunoreactive insulin-like growth factor binding protein-6 in normal and transformed human fibroblasts.

Insulin-like growth factor-binding proteins (IGFBPs) have been shown to both potentiate and inhibit IGF bioactivity in vitro; thus, changes to the type or amount of IGFBPs present in the cellular environment will ultimately affect insulin-like growth factor action. In this study, we have investigated the production of immunoreactive IGFBP-6 by normal human fibroblasts (NHF) and an SV-40-transformed human fibroblast line (AG2804). When analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting, IGFBP-6 appeared as a doublet of 32-34-kDa in conditioned medium of both cell lines, with the lower molecular mass band predominating in the NHF cell line. Measured by a specific radioimmunoassay, serum-free NHF, and AG2804 cultures secreted IGFBP-6 at 1.44 +/- 0.09 and 1.23 +/- 0.08 ng/10(4) cells (mean +/- S.E.), respectively. Despite a relatively weak IGFBP-6 signal by ligand blot compared with IGFBP-3, the two proteins were secreted in similar molar concentrations by NHF. Retinoic acid increased IGFBP-6 by 3-fold in NHF and AG2804-conditioned media, maximal at approximately 100 nM retinoic acid. In contrast, IGFBP-6 production was inhibited by transforming growth factor-beta 1 and agents that increase intracellular cAMP concentrations, including dibutyryl cAMP, forskolin, isobutylmethylxanthine, and cholera toxin. This study indicates that IGFBP-6 has a pattern of regulation unique among the IGFBPs, supporting the concept of specific roles for each binding protein in regulating cell growth and metabolism.