Epidermal growth factor and/or growth hormone induce differential, side-specific signal transduction protein phosphorylation in enterocytes.
BACKGROUND
Epidermal growth factor (EGF) plus growth hormone (GH) enhances luminal glutamine transport into rabbit and human intestinal cells. Our objective was to screen for activation status of signal proteins in C2(BBe)1 cells (enterocyte-like cell line) in response to side-specific EGF or GH treatment and to investigate the dependence of EGF receptor (EGFR) phosphorylation status on its tyrosine kinase.


METHODS
C2(BBe)1 cells on Transwells were treated for 15 minutes on either the basolateral or apical-side with EGF or GH. Lysates underwent Kinetworks phospho site-screen-2.1 analysis (duplicate experiments). In addition, lysates from cells treated as above with or without tyrphostin AG1478 (a specific EGFR tyrosine kinase inhibitor) underwent Western blot analysis for total EGFR and EGFR phosphorylated on tyrosine 1173, 1086 or 1068 (4-7 experiments).


RESULTS
Kinetworks phospho-screening demonstrated a broad range of interactions dependent on both side of exposure and protein studied. From this screen, it appears that ErbB2, Met, and insulin receptor (R)/insulin-like growth factor 1 R are not involved in the growth factors signals. For EGFR phosphorylation, basolateral, but not apical, EGF was a strong activator. Synergism was seen, but only with apical EGF plus basolateral GH. All EGFR phosphorylations were EGFR tyrosine kinase dependent. In contradistinction, apical EGF phosphorylated FAK and MAPKs.


CONCLUSIONS
Kinetworks phosphoprotein screens can suggest pathways involved in side-specific and synergistic interaction between EGF and GH. For EGFR, synergism by EGF + GH was noticed only with Ap EGF plus Bl GH and was EGFR tyrosine kinase dependent. Adaptive intestinal responses due to enterally administrated EGF might be accelerated by the availability of parenteral GH.
Avissar
Nelly E
NE
University of Rochester School of Medicine and Dentistry, Department of Surgery, Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA. nelly_avissar@urmc.rochester.edu
Toia
Liana
L
Sax
Harry C
HC
D015153:Blotting, Western; D002478:Cells, Cultured; D004357:Drug Synergism; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D019382:Human Growth Hormone / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014443:Tyrosine / Q000378:metabolism
D019382:Human Growth Hormone; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases