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PLoS One
2016 Apr 21;114:e0154294. doi: 10.1371/journal.pone.0154294.
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SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR.
Thomas JT
,
Chhuy-Hy L
,
Andrykovich KR
,
Moos M
.
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In an attempt to identify the cell-associated protein(s) through which SMOC (Secreted Modular Calcium binding protein) induces mitogen-activated protein kinase (MAPK) signaling, the epidermal growth factor receptor (EGFR) became a candidate. However, although in 32D/EGFR cells, the EGFR was phosphorylated in the presence of a commercially available human SMOC-1 (hSMOC-1), only minimal phosphorylation was observed in the presence of Xenopus SMOC-1 (XSMOC-1) or human SMOC-2. Analysis of the commercial hSMOC-1 product demonstrated the presence of pro-EGF as an impurity. When the pro-EGF was removed, only minimal EGFR activation was observed, indicating that SMOC does not signal primarily through EGFR and its receptor remains unidentified. Investigation of SMOC/pro-EGF binding affinity revealed a strong interaction that does not require the C-terminal extracellular calcium-binding (EC) domain of SMOC or the EGF domain of pro-EGF. SMOC does not appear to potentiate or inhibit MAPK signaling in response to pro-EGF, but the interaction could provide a mechanism for retaining soluble pro-EGF at the cell surface.
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27101391
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Fig 3. XSMOC-1 binds to pro-EGF and co-localizes with pro-EGF in vivo.(A) Immunoblot of pro-EGF following co-immunoprecipitation of pro-EGF with XSMOC-1, XSMOC-1 δEC, or XSMOC-1 EC in the presence of TBST/0.1% SDS; pro-EGF binds to XSMOC-1 and XSMOC-1 δEC, but not XSMOC-1EC. (B) RT-PCR analysis of HEK293 cells showing positive signal for pro-EGF(C) Representative confocal image showing co-localization of XSMOC-1 and pro-EGF (red fluorophore) on HEK293 cells using the PLA method. Nuclei are stained blue with DAPI. (D, E) Representative whole mount hybridization in situ images of Xenopus neurula embryos (stage 26) stained for XSMOC-1 (C) or pro-EGF (D). The locations of the eye (e) and pronephros (pn) are indicated.
Fig 4. XSMOC-1 does not potentiate MAPK signaling by pro-EGF, but can bind to pro-EGF when bound to heparin sepharose.(A) Immunoblot of 32D/EGFR cell lysates showing Erk phosphorylation (dpErk) following a six minute exposure to a dilution series (0â500ng/ml) of pro-EGF. Total Erk is shown as loading control. (B) Graph showing relative dpErk fluorescence obtained on immunoblots from triplicate experiments of 32D/EGFR cells following a six minute exposure to submaximal concentrations of pro-EGF (2ng or 5ng/ml) in the presence or absence of XSMOC-1 (100μg/ml).The level of dpErk by pro-EGF was not significantly affected by XSMOC-1. (C) Coomassie-stained SDS-PAGE showing the heparin sepharose (HS) elution profile of pro-EGF in the presence of increasing concentrations of NaCl (D) Coomasie-stained SDS-PAGE showing HS elution profiles (±) following incubation of 5μg XSMOC-1 with 5μg of either pro-EGF or EGF in PBS/500mM NaCl, compared to each protein alone. A standard (std) lane is provided for pro-EGF and EGF to demonstrate their expected migration position.
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