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Evidence of the Regulation of JNK2 through Oligomerization
Author(s) -
Kaoud Tamer S,
Riggs Austin F.,
Dalby Kevin N.
Publication year - 2013
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.27.1_supplement.789.22
Subject(s) - tetramer , phosphorylation , chemistry , biophysics , monomer , kinase , mutant , recombinant dna , hek 293 cells , microbiology and biotechnology , transfection , biochemistry , biology , enzyme , receptor , gene , organic chemistry , polymer
c‐Jun N‐terminal kinases (JNKs) are mediators of cellular signals associated with stress and inflammation. Activation of the JNKs is mediated cooperatively by MKK4 & MKK7. Uniquely, JNK2 isoforms can self‐activate, which underlies the constitutive activation of JNK2 in primary glial tumors. Herein, we investigate the regulation of JNK2 self‐activation in both cell‐free and cell‐based systems. Size exclusion chromatography & light scattering analysis (SEC‐LS) suggests that unphosphorylated, recombinant JNK2α2 exists in solution as a mixture of monomers, dimers and tetramers. JNK2α2 self‐phosphorylation occurs rapidly at low concentration leading to its activation. At higher concentrations of JNK2, where the formation of a JNK2 tetramer is favored, self‐phosphorylation is suppressed in vitro. Transfection of pcDNA‐JNK2α2 into HEK293 cells suppresses constitutive activation of JNK2 at higher levels of expression in a dose‐dependent manner. The F170R mutant of JNK2, which is predicted to favor an inactive conformation, stabilizes the tetramer as determined by SEC‐LS. When HEK293 cells are incubated with BIRB796 (which binds an inactive conformation of JNK2) and the cell‐permeable crosslinker disuccinimidyl suberate, a crosslinked JNK2 tetramer is detectable by SDS PAGE. We conclude that oligomerization of JNK2 has potential to contribute to the regulation of its self‐activation in mammalian cells.