SAPKs are key elements for intracellular signalling networks that respond and adapt to extracellular changes. Upon osmostress, the p38-related SAPK Hog1 has a deep impact on different aspects of cellular physiology, such as cell cycle (Clotet and Posas, 2007
; Yaakov et al, 2009
), cytoskeleton reorganization, ion homoeostasis and metabolic adjustments (Hohmann, 2002
; Hohmann et al, 2007
), as well as major effects on gene expression (de Nadal and Posas, 2010
; Martinez-Montanes et al, 2010
; Weake and Workman, 2010
). Here, we provide evidence that the ubiquitin protease Ubp3 is a novel target of the Hog1 SAPK for modulating gene expression in response to extracellular input. First, Ubp3 copurifies with Hog1 in an osmotic stress-dependent manner. Second, Hog1 phosphorylates Ubp3 at serine 695 upon osmotic stress. Third, this phosphorylation is essential for the transcriptional response to osmostress because the non-phosphorylated mutant shows impaired osmoresponsive gene expression and reduced binding of RNA Pol II.
The balance of ubiquitylation is crucial to the modulation of many activities. There are several examples of control of E3 ubiquitin ligases; however, little is known about the regulation of ubiquitin proteases in response to external stimuli. Here, we show that Ubp3 phosphorylated by the Hog1 SAPK upon stress alters its activity. Ubp3p is also phosphorylated under basal conditions in alternative phosphorylation sites than Hog1, suggesting that Ubp3 can be targeted by alternative kinases in different growth conditions. Actually, analysis by mass spectrometry of purified Ubp3 from yeast cells identified several Ubp3 phosphorylation sites that were not osmo-regulated. How Ubp3 de-ubiquitylase activity is regulated is a major question. Ubp3 function is modulated by binding cofactor Bre5 (Li et al, 2005
). Because the phosphorylatable Ubp3 serine 695 is far away from its binding domain with Bre5 in the tertiary structure, it is unlikely that the interaction of Ubp3 with Bre5 is altered by phosphorylation. An alternative scenario is that binding between Ubp3 and its substrates is altered upon phosphorylation of Ubp3. Again, this is an unlikely scenario because the affinity between RNA Pol II, a well-known Ubp3 substrate, and the de-ubiquitylase is not affected by osmostress or by the presence of the SAPK (Supplementary Figure S7
). Interestingly, our data suggest that Ubp3 activity might be up-regulated by phosphorylation that occurs on the catalytic domain of Ubp3 and the enzyme, which is able to de-ubiquitylate ubiquitylated RNA Pol II in vitro
, does it more efficiently when phosphorylated by the Hog1 SAPK. Correspondingly, the activity of the non-phosphorylated Ubp3S695A
mutant does not change in response to osmostress. In addition, the complementation of the 6-AU sensitivity of the ubp3
mutant by Ubp3S695A
shows that this allele is able to perform some of the functions of Ubp3 under normal growth conditions. Thus, these results suggest that Ubp3 activity is increased by Hog1 phosphorylation in response to stress.
Ubiquitin proteases are implicated in multiple processes, including the reversal of ubiquitylation, which functions to prevent degradation or to modify substrate activity. In this study, we found that Ubp3 is implied in regulating both initiation and elongation of transcription in response to stress. Ubp3 reverses the ubiquitylation of Tbp1/Spt15 during transcriptional activation (Chew et al, 2010
), which opens the possibility that Ubp3 recruited by the Hog1 SAPK protects promoter-bound Tbp1/Spt15. Indeed, Tbp1/Spt15 occupancy at stress-responsive promoters is reduced in the absence of UBP3
. Alternatively, the transcription factor Msn2, which acts downstream of Hog1, is known to be ubiquitylated (Chi et al, 2001
). It is noteworthy that binding of Msn2 in osmoresponsive promoters is reduced in ubp3
cells. Thus, it seems likely that the basic machinery involved in transcription initiation upon osmostress is altered in UBP3
-deficient cells. Correspondingly, there is less expression of a reporter construct that contains only a stress-responsive promoter fused to LacZ and there is a strong reduction of the amount of RNA Pol II recruited at stress-responsive genes upon osmostress. Binding of Ubp3 occurs in both promoters and ORFs of stress-activated genes, indicating a role of this ubiquitin protease in transcriptional initiation and in elongation in response to osmostress. Ubp3 has a role in rescuing RNA Pol II from degradation by reversing the ubiquitylation of RNA Pol II (Kvint et al, 2008
). We show that Ubp3 is important for induction of stress-activated genes at coding regions, suggesting that Ubp3 also targets elongating RNA Pol II upon osmostress. Thus, although we cannot exclude other targets of Ubp3 on osmostress-mediated transcriptional activation, Ubp3 clearly has a prominent role in this process. The Hog1 SAPK interacts and travels with elongating RNA Pol II through the coding regions of osmoresponsive genes (Proft et al, 2001
). Because Hog1 associates specifically with ORF of osmoresponsive genes in response to stress, it seems likely that the SAPK itself dictates the specificity of targeting RNA Pol II and, presumably also Ubp3, to osmoresponsive ORFs. Together, our data suggest that the control of Ubp3 activity by the HOG pathway in response to extracellular stimuli controls the balance of ubiquitylated proteins and this is a key factor in determining the outcome of gene expression in response to external stimuli.