Thiol-based direct threat sensing by the stress-activated protein kinase Hog1.

The yeast stress-activated protein kinase Hog1 is best known for its role in mediating the response to osmotic stress, but it is also activated by various mechanistically distinct environmental stressors, including heat shock, endoplasmic reticulum stress, and arsenic. In the osmotic stress response, the signal is sensed upstream and relayed to Hog1 through a kinase cascade. Here, we identified a mode of Hog1 function whereby Hog1 senses arsenic through a direct physical interaction that requires three conserved cysteine residues located adjacent to the catalytic loop. These residues were essential for Hog1-mediated protection against arsenic, were dispensable for the response to osmotic stress, and promoted the nuclear localization of Hog1 upon exposure of cells to arsenic. Hog1 promoted arsenic detoxification by stimulating phosphorylation of the transcription factor Yap8, promoting Yap8 nuclear localization, and stimulating the transcription of the only known Yap8 targets, ARR2 and ARR3, both of which encode proteins that promote arsenic efflux. The related human kinases ERK1 and ERK2 also bound to arsenic in vitro, suggesting that this may be a conserved feature of some members of the mitogen-activated protein kinase (MAPK) family. These data provide a mechanistic basis for understanding how stress-activated kinases can sense distinct threats and perform highly specific adaptive responses.

Rapid induction of p62 and GABARAPL1 upon proteasome inhibition promotes survival before autophagy activation.

Proteasome inhibitors are used as research tools and to treat multiple myeloma, and proteasome activity is diminished in several neurodegenerative diseases. We therefore studied how cells compensate for proteasome inhibition. In 4 h, proteasome inhibitor treatment caused dramatic and selective induction of GABARAPL1 (but not other autophagy genes) and p62, which binds ubiquitinated proteins and GABARAPL1 on autophagosomes. Knockdown of p62 or GABARAPL1 reduced cell survival upon proteasome inhibition. p62 induction requires the transcription factor nuclear factor (erythroid-derived 2)-like 1 (Nrf1), which simultaneously induces proteasome genes. After 20-h exposure to proteasome inhibitors, cells activated autophagy and expression of most autophagy genes by an Nrf1-independent mechanism. Although p62 facilitates the association of ubiquitinated proteins with autophagosomes, its knockdown in neuroblastoma cells blocked the buildup of ubiquitin conjugates in perinuclear aggresomes and of sumoylated proteins in nuclear inclusions but did not reduce the degradation of ubiquitinated proteins. Thus, upon proteasome inhibition, cells rapidly induce p62 expression, which enhances survival primarily by sequestering ubiquitinated proteins in inclusions.