Increased IGFBP-1 phosphorylation in response to leucine deprivation is mediated by CK2 and PKC

Abstract

Insulin-like growth factor binding protein-1 (IGFBP-1), secreted by fetal liver, is a key regulator of IGF-I bioavailability and fetal growth. IGFBP-1 phosphorylation decreases IGF-I bioavailability and diminishes its growth-promoting effects. Growth-restricted fetuses have decreased levels of circulating essential amino acids. We recently showed that IGFBP-1 hyperphosphorylation (pSer101/119/169) in response to leucine deprivation is regulated via activation of the amino acid response (AAR) in HepG2 cells. Here we investigated nutrient-sensitive protein kinases CK2/PKC/PKA in mediating IGFBP-1 phosphorylation in leucine deprivation. We demonstrated that leucine deprivation stimulated CK2 activity (enzymatic assay) and induced IGFBP-1 phosphorylation (immunoblotting/MRM-MS). Inhibition (pharmacological/siRNA) of CK2/PKC, but not PKA, prevented IGFBP-1 hyperphosphorylation in leucine deprivation. PKC inhibition also prevented leucine deprivation-stimulated CK2 activity. Functionally, leucine deprivation decreased IGF-I-induced-IGF-1R autophosphorylation when CK2/PKC were not inhibited. Our data strongly support that PKC promotes leucine deprivation-induced IGFBP-1 hyperphosphorylation via CK2 activation, mechanistically linking decreased amino acid availability and reduced fetal growth.

Exposure of decidualized HIESC to low oxygen tension and leucine deprivation results in increased IGFBP-1 phosphorylation and reduced IGF-I bioactivity

“…To identify/quantify the relative changes in sites of phosphorylation of decidual IGFBP-1 in response to hypoxia or leucine deprivation, we first investigated the changes at three known serine sites (Ser101, Ser119 and Ser169) (Damerill et al, 2016; Malkani et al, 2016). The HIESC conditioned media samples were analyzed by MRM-MS using an internal IGFBP-1 peptide to normalize data as described previously (Damerill et al, 2016).…”

“…Leucine has several times greater impact on protein synthesis than any other amino acid. The 450 μM Leu for HIESC culture is equivalent to normal DMEM/F-12 routinely used in vitro (Seferovic et al, 2009; Malkani et al, 2016, 2015). …”

“…Subsequently, prior to harvesting, the cells were washed in PBS and cultured in DMEM F12 lacking the essential amino acid leucine (Leu 0 μM) as previously (Seferovic et al, 2009; Malkani et al, 2016, 2015). In the control treatment the cells were washed and cultured in DMEM/F12 media containing the standard amino acid concentrations including leucine (450 μM).…”

“…We performed in-solution digestions using the IP samples of IGFBP-1 from cell media isolated from decidualized HIESC cultured in low-oxygen tension or leucine deprivation as previously described (Damerill et al, 2016; Malkani et al, 2016). In brief, the IGFBP-1 sample was first digested using endoproteinase Asp-N incubated overnight at 37 °C (Roche Diagnostics, Laval, QC, Canada) and followed by a subsequent digestion using trypsin (Roche Diagnostics) overnight at 37 °C following the manufacturers instruction.…”

“…In brief, the IGFBP-1 sample was first digested using endoproteinase Asp-N incubated overnight at 37 °C (Roche Diagnostics, Laval, QC, Canada) and followed by a subsequent digestion using trypsin (Roche Diagnostics) overnight at 37 °C following the manufacturers instruction. IGFBP-1 digests were subsequently analyzed by LC-MS/MS with a triple quadruple mass spectrometer (4000 QTRAP AB Sciex, Concord, ON, Canada) using methods that we previously optimised for the detection of site-specific IGFBP-1 phosphorylation (Damerill et al, 2016; Malkani et al, 2016). A NanoAcquity UPLC system (Waters, Milford, MA, USA) equipped with a C18 analytical column (1.7 μm, 75 μm × 200 mm) was used to separate the peptides at a flow rate of 300 nl/min and operating pressure of 8000 psi.…”