Characterizing the role of the actin-interacting MAPKKK Ssk2p in the recovery of the actin cytoskeleton after osmotic stress.
Blaine T. Bettinger, Tatiana Yuzyuk, David C Amberg
Biochemistry & Molecular Bio, SUNY Upstate Medical Univ., 750 E. Adams St., Syracuse, NY, 13207, USA
Osmotic stress causes actin cytoskeleton disassembly, cell cycle arrest, and activation of the High Osmolarity Growth (HOG) MAP Kinase Pathway. Ssk2p, a MAPKKK of the HOG pathway, promotes actin cytoskeleton recovery of osmotically stressed cells (Yuzyuk et al, 2002). Previous experiments have shown that Ssk2's role in promoting recovery of the actin cytoskeleton is separate from its role as a signal transducer in the HOG pathway. In particular, Ssk2p can promote actin recovery in the absence of Ssk1p, the upstream activator of Ssk2p in the HOG pathway. Recent biochemical data from the lab supports the theory of Ssk1p-independent activation of Ssk2p. We are therefore attempting to identify the pertinent upstream activators of Ssk2p upon osmotic shock using a number of techniques including mass spectrometry. Using a modified TAP tag system, we are trying to identify proteins that co-purify with Ssk2p following osmotic stress. One of the most prominent co-purifying proteins is actin, which we have previously shown to bind to Ssk2p. Interestingly, the removal of actin from the purified complex abrogates the Ssk1-independent kinase activity of Ssk2p, suggesting that actin binding plays an important role in activating or maintaining activation of Ssk2p in the actin recovery pathway.
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