Yeast cells respond to increases in osmolarity in the extracellular environment by activating the stress-activated MAPK Hog1. A major outcome of the activation of the Hog1 MAPK is the regulation of gene expression. One of the most common mechanisms by which SAPKs regulate gene expression is by modification of specific transcription factors (9
), and several transcription regulators have been proposed to be controlled by the Hog1 MAPK. However, due to their DNA binding specificities and the profile of gene induction upon stress shown by DNA microarrays, they cannot account for the regulation of all of the genes under the control of Hog1. In addition, a mutant strain carrying deletions in hot1
, and msn4
is not osmosensitive (19
From these considerations, we suspected that additional transcription factors were required for the osmostress-induced regulation of gene expression by the MAPK Hog1. In this report, we describe a genetic screen in which we identified Smp1, a MEF2-like protein, as one such factor. The results of this study demonstrate that both overexpression and deletion of SMP1
result in altered expression of osmoresponsive genes such as STL1
but not of others, such as ALD3
. It was reported that Smp1 has MEF2-related DNA-binding specificities (4
), and a conserved sequence similar to the predicted DNA binding site for Smp1 was consistently present on the promoters of the osmoresponsive STL1
but not HXT1
genes (Fig. and data not shown).
Several lines of evidence suggest that Smp1 is actually a direct substrate for the MAPK. In vivo studies suggested that Hog1 was able to interact with Smp1 and that Smp1 was phosphorylated upon stress in a HOG1
-dependent manner. The relationship of Smp1 and Hog1 in vivo are further supported by the in vitro evidence that Hog1 phosphorylates Smp1 directly. Four independent phosphorylaton sites were mapped within 28 amino acids. Similarly, phosphorylation of Sko1 by Hog1 was restricted to three phosphorylation sites clustered within 19 amino acids (15
). The introduction of several phosphates in a small pocket of amino acids might result in conformational changes required for switching the function of those transcriptional regulators. In vitro phosphorylation studies also showed that full-length Smp1 is phosphorylated less efficiently by Hog1 than by the C-terminal domain alone (Fig. , lanes 1 and 3). This observation raised the possibility that a region of Smp1 could be limiting the access of the MAPK to the phosphorylation sites. Consistent with this observation was the fact that simultaneous incubation of the C-terminal domain with an N-terminal domain (which is not phosphorylated by Hog1) resulted in a dramatic decrease of C-terminal phosphorylation (Fig. , lane 4). This might suggest that Smp1 must be in a preactivated state (i.e., bound to DNA or interacting with other factors) to be accessible for phosphorylation and activation by the MAPK.
Phosphorylation of Smp1 by Hog1 is important for Smp1 function. Upon osmotic stress, Smp1 is strongly phosphorylated, and mutation of the phosphorylation sites to Ala results in an Smp1 that is unable to regulate gene expression (Fig. ). In yeast, there exists a second MEF2-related protein, Rlm1, that is under the control of the Slt2/Mpk1 MAPK. Interestingly, Mpk1 phosphorylates Rlm1 in a region similar to that found for Smp1, and this phosphorylation results in an increase of its transcriptional activity (4
). Thus, two independent MAPK signaling pathways could be controlling the two MEF2-related factors by a similar mechanism. In mammals, regulation of MEF2A and MEF2C factors has been shown to be under the control of the p38 MAPKs, among other kinases (reviewed in references 9
). Phosphorylation of the transcription activator domain of these factors by p38 stimulates MEF2 activity, which is analogous to the mechanism proposed for the yeast Smp1.
Apart from the role of Smp1 in the regulation of a subset of osmoresponsive genes under the control of the Hog1 MAPK (and thus a role in osmoadaptation), we found that smp1Δ
cells lose viability in the stationary phase, as occurs with hog1Δ
cells (Fig. ). This phenotype is reminiscent of that observed upon deletion of the Spc1/Sty
I MAPK in Schizosaccharomyces pombe
), and although the molecular mechanism(s) of this deficiency remains uncharacterized, the formal possibility exists that this could be caused by the lack of some specific transcript required for this phase of growth. As shown in Fig. , Smp1 concentrates into the nucleus when cells enter into the stationary phase, and this could be of relevance for the induction of specific genes. In hog1Δ
cells, Smp1 nuclear accumulation is clearly diminished, and thus this could impede the normal function of Smp1. Because smp1Δ
and smp1Δ hog1Δ
cells display similar viability, it is likely that the deficiency observed in hog1Δ
cells could be caused by the improper function of Smp1 rather than other mechanisms.
Taken together, our results show that Smp1 is a direct target for the Hog1 MAPK and that two different levels of regulation can be controlling the activity of this transcription factor. Under stress, Hog1 phosphorylates the putative transcription activation domain stimulating Smp1 activity, and, when entering into the stationary phase, Hog1 is required for proper localization of the transcription factor.