Characterization of a Serum Response Factor-Like Protein in Saccharomyces cerevisiae, Rlm1, Which Has Transcriptional Activity Regulated by the Mpk1 (Slt2) Mitogen-Activated Protein Kinase Pathway

Abstract

The Mpk1 (Slt2) mitogen-activated protein (MAP) kinase has been implicated in several biological processes in Saccharomyces cerevisiae. The Rlm1 protein, a member of the MADS box family of transcription factors, functions downstream of Mpk1 in the pathway. To characterize the role of Rlm1 in mediating the transcriptional activation by the Mpk1 pathway, we constructed a LexA-Rlm1⌬N chimera in which sequences, including the MADS box domain of the Rlm1 protein, were replaced by the LexA DNA binding domain and tested the ability of this chimera to activate a LexA operator-controlled reporter gene. In this assay, the Rlm1 protein was found to activate transcription in a manner regulated by the Mpk1 pathway. The Mpk1 protein kinase phosphorylated Rlm1⌬N in vitro and the LexA-Rlm1⌬N chimera protein was phosphorylated in vivo in a Mpk1-dependent manner. These results suggest that Mpk1 regulates the transcriptional activity of Rlm1 by directly phosphorylating it. We identified a Mpk1-like protein kinase, Mlp1, as an Rlm1-associated protein by using the yeast two-hybrid system. Overexpression of MLP1 suppresses the caffeine-sensitive phenotype of the bck1⌬ mutation. The additivity of the mlp1⌬ defect with the mpk1⌬ defect with regard to the caffeine sensitivity, combined with the results of genetic epistasis experiments, suggested that the activity of Rlm1 is regulated independently by Mpk1 MAP kinase and the Mlp1 MAP kinase-like kinase.Extracellular molecules that regulate cell proliferation and differentiation in eukaryotes exert their effects through pathways that detect signals at the cell surface and transmit them through the cytoplasm to the nucleus. Transcription factors in the nucleus are typically the ultimate targets of these signaling pathways, and they function to elicit alterations in gene expression that in turn regulate cellular events. One mechanism for transmitting these signals involves a protein phosphorylation cascade leading to activation of mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated kinases (ERKs) (1,19,20). Thus, these enzymes are thought to function as intermediaries between membrane-associated signaling molecules and the nucleus. MAPKs/ERKs are activated through protein kinase cascades that are comprised of three highly conserved core components. The MAPKs are activated in response to dual phosphorylation on tyrosine and threonine residues catalyzed by a family of dual-specificity protein kinases, termed MEK (MAPK or ERK kinase) or MAPKK (MAPK kinase). MAPKK/MEK itself is phosphorylated and activated by upstream regulators, MAPKK kinases (MAPKKKs), that include Raf, MEK kinase (MEKK), and Mos (19). Each of these upstream components also functions in multiple cell signaling processes.Defining the targets of MAPKs is of fundamental importance for understanding at a molecular level how intracellular processes are dramatically altered in response to environmental signals. The targets of MAPKs include a number of transcription factors, and it is presumed that it is by p...

Oxidative Stress ActivatesFUS1andRLM1Transcription in the YeastSaccharomyces cerevisiaein an Oxidant-dependent Manner

“…Genetic analysis performed in an mpk1⌬ strain surprisingly revealed that cells lacking Mpk1 are still able to activate RLM1 reporter, although the induction was attenuated. Two-hybrid analysis has shown that RLM1 interacts with two MAPKs: Mpk1 and its homologue Mlp1 (Watanabe et al, 1997). Interestingly, cells lacking mitochondrial glutaredoxin exhibit a high induction of Mlp1 as revealed by transcriptome analysis (Belli et al, 2004).…”

The effect of tea tree oil and antifungal agents on a reporter for yeast cell integrity signalling

“…A hybrid transcription factor containing the Nterminal DNA-binding domain of the bacterial lexA protein and the C-terminal two-thirds of the yeast Rlm1 sequence (encoding amino acids 222-676) has been shown to activate transcription of a lexA-operator-lacZ fusion in yeast, strictly depending on the specific phosphorylation of the Rlm1 part at Ser427 and Thr439 by Mpk1/Slt2 (Watanabe et al, 1997;Jung et al, 2002). As reported previously, all necessary genes for this reporter system have been placed onto a yeast CEN-ARS plasmid (pHPS100; Kirchrath et al, 2000).…”

“…On the other hand, it has been observed that substitution of the DNA-binding domain of the MADSbox transcription factor Rlm1 (i.e. a direct target of Mpk1/Slt2) for the bacterial lexA sequence can be used to drive phosphorylation-dependent expression of a lacZ reporter construct (Watanabe et al, 1997). This then serves as a less laborious, albeit indirect, measure of pathway activity.…”

Differential regulation of cell wall biogenesis during growth and development in yeast

“…A transcription factor downstream of the Slt2-MAP kinase module is Rlm1p (Dodou & Treisman, 1997 ;Jung & Levin, 1999 ;Watanabe et al, 1997), but it is not the only one involved in the transcriptional response to cell wall stress. Deletion of RLM1 does not lead to defects in cell wall integrity (Watanabe et al, 1997), and, more importantly, binding sites for Rlm1p in the promoter of FKS2, the best-defined target of the PKC1-SLT2 pathway, are present and involved in transcription activation (Zhao et al, 1998), but the region of the FKS2 promoter responsible for heat shock activation of the gene through the PKC1-SLT2 pathway is separate from that containing the Rlm1p-binding sites. This suggests that another, currently unknown, transcription factor might be involved (Zhao et al, 1998).…”