Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase

Abstract

SummaryThe Mpk1 MAP kinase of the Saccharomyces cerevisiae cell wall integrity signalling pathway phosphorylates and activates the Rlm1 transcription factor in response to cell wall stress. Rlm1 is related to mammalian MEF2 isoforms, and shares a similar DNAbinding specificity. Signalling through Rlm1 regulates the expression of at least 25 genes, most of which have been implicated in cell wall biogenesis. We report here the transcriptional induction by agents of cell wall stress of a set of lacZ reporter plasmids derived from several Rlm1-responsive genes. Analysis of substitution mutations at putative Mpk1 phosphorylation sites within Rlm1 revealed that Ser427 and Thr439 are important for its stress-induced transcriptional activation of these reporter plasmids. Assessment of Rlm1 activation potency when fused to a heterologous DNA-binding domain showed that the identified seryl and threonyl residues are necessary for the Rlm1 transcriptional activation function independently of its DNA binding. We also demonstrate that a MAP kinase docking site, shown recently to mediate activation of MEF2A and MEF2C, is conserved in Rlm1 and is required for its ability to mediate transcriptional activation in response to agents that induce cell wall stress. Finally, intracellular localization analyses show that Rlm1 resides in the nucleus regardless of its activation and phosphorylation status. Together these observations support the inference that Mpk1 regulates the Rlm1 transcriptional activation function by phosphorylation of Ser427 and Thr439.

The Hsp110 protein chaperone Sse1 is required for yeast cell wall integrity and morphogenesis

“…It is possible that the Hsp90 chaperone complex may differentially promote productive interaction between kinase and substrates, potentially as a scaffold. Because the strongest transcriptional defects in sse1Δ cells were observed with the Rlm1-dependent reporter, more precise investigation of these later stages in the pathway await development of an in vivo assay for Rlm1 phosphorylation by Slt2 (Jung et al 2002).…”

“…PST1 induction in response to heat shock requires Slt2, and thus directly reports on cell integrity pathway signaling (Garcia et al 2004). Although Slt2 phosphorylation attenuates rapidly, Rlm1-dependent transcription as assayed by reporter fusion increases over time up to 15 h (Jung et al 2002). In wild type cells, expression of PST1-lacZ was low at 30°C but induced nearly eightfold by a 1.5-h heat shock at 37°C.…”

“…Slt2 is known to phosphorylate and activate at least two transcription factors, Rlm1 and the Swi4/Swi6 heterodimer SBF (Watanabe et al 1995;Dodou and Treisman 1997;Madden et al 1997;Jung et al 2002)). To determine if Rlm1 function is impaired in sse1Δ cells, we constructed a transcriptional reporter using promoter sequences amplified from the known Rlm1 target gene PST1 and β-galactosidase (lacZ).…”

“…Cell wall damage is transmitted by protein kinase C to a MAP kinase module terminating in the MAPK Slt2 (Torres et al 1991;Levin and Bartlett-Heubusch 1992;Paravicini et al 1992). Slt2 (also known as Mpk1) phosphorylates and activates two transcription factors, Rlm1 and the Swi4/6 cell-cycle box binding factor (SBF), the latter a heterodimer composed of Swi4 and Swi6 proteins (Watanabe et al 1995;Dodou and Treisman 1997;Madden et al 1997;Jung et al 2002). Rlm1 and SBF activate expression of distinct sets of genes with predicted roles in cell wall synthesis and repair (Lagorce et al 2003;Garcia et al 2004).…”

Dissecting the transcriptional activation function of the cell wall integrity MAP kinase

“…Finally, agents that interfere with cell wall biogenesis, such as the chitin antagonist Calcofluor white (Ketela et al, 1999), Congo red, or zymolyase (de Nobel et al, 2000), also stimulate signalling. Activation of CWI signalling can be detected by measurement of Slt2p protein kinase activity , immunological detection of phosphorylated Slt2p (Martín et al, 2000;de Nobel et al, 2000) or by induction of transcriptional reporters (Zhao et al, 1998;Jung et al, 2002;Kuranda et al, 2006).…”