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Role of GSK3 beta and ERK in the human neutrophil response to fungal beta‐glucan (1046.5)
Author(s) -
Johnson Courtney,
Byrd Angel,
Reichner Jonathan
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1046.5
Subject(s) - phosphorylation , mapk/erk pathway , blot , chemistry , tyrosine phosphorylation , microbiology and biotechnology , extracellular , signal transduction , fibronectin , western blot , biology , biochemistry , extracellular matrix , gene
Complement Receptor 3 (CR3), an integrin found on neutrophils, plays an important role in the neutrophil recognition and response to fungal components in the context of extracellular matrix proteins. Co‐ligation of CR3 with fibronectin (Fn) at the I‐domain and ß‐glucan (B) at the lectin‐like domain is possible due to spatially distinct locations and creates unique cellular activity not seen with ligation of either domain separately. Dual ligation of CR3 with Fn+B induces homotypic aggregation of primed neutrophils, a response representative of the immune cells encounter with fungi within tissues. To understand the signaling pathways involved in these CR3‐mediated responses, we employed various molecular techniques to determine the key proteins. Mass spectroscopy revealed a significant increase in tyrosine phosphorylation of nineteen proteins in human neutrophil samples exposed to FN+B but not in samples exposed to FN alone. Western blotting and immunopreciptation techniques validate these findings by showing an increase in two of the proteins, p‐ERK and p‐GSK. Further work using inhibitors UO126 (ERK inhibitor) and TZDZ‐8 (GSK3Beta inhibitor), show differing effects on cluster formation. Use of UO126 caused a complete abrogation of cluster formation and a significant loss of ERK phosphorylation. However, use of TZDZ‐8 caused a partial inhibition of cluster formation with little effect on GSK3Beta phosphorylation. Blotting for protein levels, we were able to show the phosphorylation of ERK increases over time while the phophorylation of GSK decreases. Use of TZDZ‐8 decreased the phosphorylation of ERK, while inhibition with UO126 had little effect on GSK3 phosphoryaltion. This result indicates a upstream role for GSK3 Beta in the phosphorylation of ERK. In addition, monoclonal antibody blocking of CR3 led to an increase in GSK3 Beta phosphorylation indicating CR3 signaling of p‐ERK may be regulated through GSK3 Beta phosphylation. Grant Funding Source : Supported by NIH GM‐066194