Functional and Complementary Phosphorylation State Attributes of Human Insulin-like Growth Factor-Binding Protein-1 (IGFBP-1) Isoforms Resolved by Free Flow Electrophoresis

Nissum, Mikkel; Shehab, Majida Abu; Sukop, Ute; Khosravi, Javad; Wildgruber, Robert; Eckerskorn, Christoph; Han, V. K. M.; Gupta, Madhulika B.

doi:10.1074/mcp.m800571-mcp200

Cited by 22 publications

(48 citation statements)

References 42 publications

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“…It is possible that IGFBP-1 phosphorylation at these new sites may elicit functional effects on IGF-I affinity directly or through synergistic interactions with Ser101, 119 or 169 or other alternate sites. For example, phosphorylation of IGFBP-1 at Ser95 and Ser98 has been previously identified by us (Abu Shehab et al 2010, Nissum et al 2009) as well as others (Dolcini et al 2009) and might be functionally relevant in modulating IGFBP-1 phosphorylation via CK2/PKC in FGR.…”

Section: Discussionmentioning

confidence: 58%

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

Malkani

Biggar

Shehab

et al. 2016

Molecular and Cellular Endocrinology

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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.

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Section: Discussionmentioning

confidence: 58%

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

Malkani

Biggar

Shehab

et al. 2016

Molecular and Cellular Endocrinology

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“…Our MRM-MS data show that the doubly phosphorylated peptide (Ser169/Ser174) elutes first suggesting it to be most negatively charged while the single phosphorylated peptides (Ser98 and Ser101) eluted at the end indicating they were least negatively charged (Table 1). Given that we previously have demonstrated, using BIACORE analysis, that the most negatively charged phosphopeptides contributed to highest binding affinity for IGF-I (Nissum et al, 2009), the current data are intriguing as phosphorylation at most negatively charged Ser169/Ser174 was highly induced in both low oxygen tension and leucine privation. These data suggest the biological relevance of phosphorylation at Ser169/Ser174 potentially to be key in reducing IGF-I bioavailability under cellular stress conditions.…”

Section: Discussionmentioning

confidence: 73%

“…Further, we provide additional novel information showing increases in all three decidual cytoplasmic IGFBP-1 phosphoisoforms in response to low oxygen tension/leucine deprivation. The three known residues play a major functional role in IGF-I binding (Nissum et al, 2009), suggests a strongly controlled action of IGFBP-1 phosphorylation in reducing IGF-I bioavailability in decidual cells in response to hypoxia or/and decreased nutrient availability consistent with placental insufficiency.…”

Section: Discussionmentioning

confidence: 99%

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

Shehab

Biggar

Singal

et al. 2017

Molecular and Cellular Endocrinology

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Phosphorylation of decidual IGFBP-1 enhances binding of IGF-I, limiting the bioavailability of this growth factor which may contribute to reduced placental and fetal growth. The mechanisms regulating decidual IGFBP-1 phosphorylation are incompletely understood. Using decidualized human immortalized endometrial stromal cells we tested the hypothesis that low oxygen tension or reduced leucine availability, believed to be common in placental insufficiency, increase the phosphorylation of decidual IGFBP-1. Multiple reaction monitoring-MS (MRM-MS) was used to quantify IGFBP-1 phosphorylation. MRM-MS validated the novel phosphorylation of IGFBP-1 at Ser58, however this site was unaffected by low oxygen tension/leucine deprivation. In contrast, significantly elevated phosphorylation was detected for pSer119, pSer98/pSer101 and pSer169/pSer174 sites. Immunoblotting and dual-immunofluorescence using phosphosite-specific IGFBP-1 antibodies further demonstrated increased IGFBP-1 phosphorylation in HIESC under both treatments which concomitantly reduced IGF-I bioactivity. These data support the hypothesis that down regulation of IGF-I signaling links decidual IGFBP-1 hyperphosphorylation to restricted fetal growth in placental insufficiency.

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“…As a traditional separation mode, continuous free‐flow zone electrophoresis (FFZE) has been well used in sample pretreatment of complex protein sample in proteomics . One of the most important pretreatments of protein sample is to exclude or decrease high‐content proteins, e.g.…”

Section: Introductionmentioning

confidence: 99%

Quantitative investigation of resolution increase of free‐flow electrophoresis via simple interval sample injection and separation

et al. 2012

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Interval free-flow zone electrophoresis (FFZE) has been used to suppress sample band broadening greatly hindering the development of free-flow electrophoresis (FFE). However, there has been still no quantitative study on the resolution increase of interval FFZE. Herein, we tried to make a comparison between bandwidths in interval FFZE and continuous one. A commercial dye with methyl green and crystal violet was well chosen to show the bandwidth. The comparative experiments were conducted under the same sample loading of the model dye (viz. 3.49, 1.75, 1.17, and 0.88 mg/h), the same running time (viz. 5, 10, 15, and 20 min), and the same flux ratio between sample and background buffer (= 10.64 × 10⁻³). Under the given conditions, the experiments demonstrated that (i) the band broadening was evidently caused by hydrodynamic factor in continuous mode, and (ii) the interval mode could clearly eliminate the hydrodynamic broadening existing in continuous mode, greatly increasing the resolution of dye separation. Finally, the interval FFZE was successfully used for the complete separation of two-model antibiotics (herein pyoluteorin and phenazine-1-carboxylic acid coexisting in fermentation broth of a new strain Pseudomonas aeruginosa M18), demonstrating the feasibility of interval FFZE mode for separation of biomolecules.

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Functional and Complementary Phosphorylation State Attributes of Human Insulin-like Growth Factor-Binding Protein-1 (IGFBP-1) Isoforms Resolved by Free Flow Electrophoresis

Cited by 22 publications

References 42 publications

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

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

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

Quantitative investigation of resolution increase of free‐flow electrophoresis via simple interval sample injection and separation

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