Novel regulatory mechanisms and biological functions of Hog1/p38s and Mpk1/ERKs revealed by the use of constitutively active variants.

David Engelberg, The Hebrew University of Jerusalem, Jerusalem, Israel

David Engelberg, Jonah Beenstock, Tal Goshen-Lago, Karin Smorodinsky, Masha Tesker, Navit Mooshayf, Dafna Mordechai, Anat Carp, Galina Otonin, Oded Livnah.

As MAP kinases are commonly activated in parallel with other signaling cascades, it is difficult to reveal the bona fide functions of each given MAPK molecule in the cell. To address this matter in an accurate manner we developed an arsenal of intrinsically active variants of Hog1, p38alpha-delta, Mpk1 and ERK1+2. Expression of each of these variants allows identification of the specific substrates, target genes and biological effects of the expressed molecule per se. We report that the active MAPK molecules disclosed novel mechanisms of MAPK regulation including autophosphorylation at sites outside the TXY motif, and novel regulatory elements, including the alpha-G-helix and MAPK insert. We further report the use of active variants of Hog1 to reveal the entire Hog1-dependent transcriptome and proteomoe and the use of active variants of ERKs to test which ERK isoform is the mediator of the Ras/Raf/MEK oncogenic pathway. As active variants of ERK1, but not of ERK2, manifest oncogenic properties, it seems that the mediator is ERK1.