scholarly journals The Extracellular Domain of the Saccharomyces cerevisiae Sln1p Membrane Osmolarity Sensor Is Necessary for Kinase Activity

1999 ◽  
Vol 181 (8) ◽  
pp. 2527-2534 ◽  
Author(s):  
Darin B. Ostrander ◽  
Jessica A. Gorman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Leucine Zipper ◽  
Transmembrane Domain ◽  
Active Form ◽  
Extracellular Domain ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
High Osmolarity ◽  
Mitogen Activated Protein ◽  
Regulation Of Activity

ABSTRACT The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeastSaccharomyces cerevisiae. Truncations of SLN1that retain an intact kinase domain are capable of complementing the lethality of an sln1Δ strain. By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various SLN1 constructions were determined. In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity. Removal of the first TMD (ΔTMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated. When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated. Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality. When the ECD of the ΔTMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated. In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive. These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD.

scholarly journals Phosphorylated Ssk1 Prevents Unphosphorylated Ssk1 from Activating the Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase in the Yeast High-Osmolarity Glycerol Osmoregulatory Pathway

2008 ◽  
Vol 28 (17) ◽  
pp. 5172-5183 ◽  
Author(s):  
Tetsuro Horie ◽  
Kazuo Tatebayashi ◽  
Rika Yamada ◽  
Haruo Saito
Keyword(s):  
Protein Kinase ◽  
Active Form ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Function ◽  
Response Regulators ◽  
High Osmolarity ◽  
Inactive Form ◽  
Mitogen Activated Protein ◽  
Mutant Phenotypes

ABSTRACT In Saccharomyces cerevisiae, external high osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which controls various aspects of osmoadaptation. Ssk1 is a homolog of bacterial two-component response regulators and activates the Ssk2 MAPK kinase kinase upstream of Hog1. It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) is the active form and that Ssk1 phosphorylated (Ssk1∼P) at Asp554 by the Sln1-Ypd1-Ssk1 multistep phosphorelay mechanism is the inactive form. In this study, we show that constitutive activation of Ssk2 occurs when Ssk1 phosphorylation is blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits its interaction with Ypd1, the donor of phosphate to Ssk1. Thus, Ssk1-OH is indeed necessary for Ssk2 activation. However, overexpression of wild-type Ssk1 or of an Ssk1 mutant that cannot bind Ssk2 prevents constitutively active Ssk1 mutants from activating Ssk2. Therefore, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself. We also found that Ssk1 exists mostly as a dimer within cells. From mutant phenotypes, we deduce that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. Hence, because Ssk1∼P binds to and inhibits Ssk1-OH, moderate fluctuation of the level of Ssk1-OH does not lead to nonphysiological and detrimental activation of Hog1.

scholarly journals Osmotic Stress-Induced Gene Expression in Saccharomyces cerevisiae Requires Msn1p and the Novel Nuclear Factor Hot1p

1999 ◽  
Vol 19 (8) ◽  
pp. 5474-5485 ◽  
Author(s):  
Martijn Rep ◽  
Vladimír Reiser ◽  
Ulrike Gartner ◽  
Johan M. Thevelein ◽  
Stefan Hohmann ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Osmotic Stress ◽  
Transcriptional Response ◽  
Mitogen Activated Protein Kinase ◽  
The Novel ◽  
Hog Pathway ◽  
High Osmolarity ◽  
Nuclear Factors ◽  
Mitogen Activated Protein ◽  
Protective Genes

ABSTRACT After a sudden shift to high osmolarity, Saccharomyces cerevisiae cells respond by transiently inducing the expression of stress-protective genes. Msn2p and Msn4p have been described as two transcription factors that determine the extent of this response. Inmsn2 msn4 mutants, however, many promoters still show a distinct rise in transcriptional activity upon osmotic stress. Here we describe two structurally related nuclear factors, Msn1p and a newly identified protein, Hot1p (for high-osmolarity-induced transcription), which are also involved in osmotic stress-induced transcription.hot1 single mutants are specifically compromised in the transient induction of GPD1 and GPP2, which encode enzymes involved in glycerol biosynthesis, and exhibit delayed glycerol accumulation after stress exposure. Similar to agpd1 mutation, a hot1 defect can rescue cells from inappropriately high HOG pathway activity. In contrast, Hot1p has little influence on the osmotic stress induction of CTT1, where Msn1p appears to play a more prominent role. Cells lacking Msn1p, Msn2p, Msn4p, and Hot1p are almost devoid of the short-term transcriptional response of the genes GPD1,GPP2, CTT1, and HSP12 to osmotic stress. Such cells also show a distinct reduction in the nuclear residence of the mitogen-activated protein kinase Hog1p upon osmotic stress. Thus, Hot1p and Msn1p may define an additional tier of transcriptional regulators that control responses to high-osmolarity stress.

scholarly journals Cooperative Regulation of DOG2, Encoding 2-Deoxyglucose-6-Phosphate Phosphatase, by Snf1 Kinase and the High-Osmolarity Glycerol–Mitogen-Activated Protein Kinase Cascade in Stress Responses of Saccharomyces cerevisiae

2000 ◽  
Vol 182 (18) ◽  
pp. 5121-5126 ◽  
Author(s):  
Yoshiyuki Tsujimoto ◽  
Shingo Izawa ◽  
Yoshiharu Inoue
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Osmotic Stress ◽  
Mitogen Activated Protein Kinase ◽  
High Osmolarity ◽  
High Osmolarity Glycerol ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade

ABSTRACT We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZtransposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression ofDOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels ofDOG2 gene expression were increased in amig1Δ mutant, while the derepression of DOG2was not observed in a snf1Δ mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptantspbs2Δ, hog1Δ, and snf1Δ. However, the osmotic induction was completely abolished in both thesnf1Δ pbs2Δ mutant and the snf1Δ hog1Δ mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol–mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.

Induced, selective proteolysis of MLK3 negatively regulates MLK3/JNK signalling

2010 ◽  
Vol 427 (3) ◽  
pp. 435-443 ◽  
Author(s):  
Geou-Yarh Liou ◽  
Hua Zhang ◽  
Eva M. Miller ◽  
Steve A. Seibold ◽  
Weiqin Chen ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Active Form ◽  
Homology Model ◽  
Kinase Domain ◽  
Small Gtpase ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Mixed Lineage Kinase 3

MLK3 (mixed lineage kinase 3) is a MAP3K [MAPK (mitogen-activated protein kinase) kinase kinase] that activates multiple MAPK pathways, including the JNK (c-Jun N-terminal kinase) pathway. Immunoblotting of lysates from cells ectopically expressing active MLK3 revealed an additional immunoreactive band corresponding to a CTF (C-terminal fragment) of MLK3. In the present paper we provide evidence that MLK3 undergoes proteolysis to generate a stable CTF in response to different stimuli, including PMA and TNFα (tumour necrosis factor α). The cleavage site was deduced by Edman sequencing as between Gln251 and Pro252, which is within the kinase domain of MLK3. Based on our homology model of the kinase domain of MLK3, the region containing the cleavage site is predicted to reside on a flexible solvent-accessible loop. Site-directed mutagenesis studies revealed that Leu250 and Gln251 are required for recognition by the ‘MLK3 protease’, reminiscent of the substrate specificity of the coronavirus 3C and 3CL proteases. Whereas numerous mammalian protease inhibitors have no effect on MLK3 proteolysis, blockade of the proteasome through epoxomicin or MG132 abolishes PMA-induced production of the CTF of MLK3. This CTF is able to heterodimerize with full-length MLK3, and interact with the active form of the small GTPase Cdc42, resulting in diminished activation loop phosphorylation of MLK3 and reduced signalling to JNK. Thus this novel proteolytic processing of MLK3 may negatively control MLK3 signalling to JNK.

scholarly journals Response of the Saccharomyces cerevisiae Mpk1 Mitogen-Activated Protein Kinase Pathway to Increases in Internal Turgor Pressure Caused by Loss of Ppz Protein Phosphatases

2004 ◽  
Vol 3 (1) ◽  
pp. 100-107 ◽  
Author(s):  
Stephanie Merchan ◽  
Dolores Bernal ◽  
Ramón Serrano ◽  
Lynne Yenush
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Posttranslational Modifications ◽  
Active Form ◽  
Turgor Pressure ◽  
Mitogen Activated Protein Kinase ◽  
Ph Homeostasis ◽  
Expression Of Genes ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

ABSTRACT The Mpk1 pathway of Saccharomyces cerevisiae is a key determinant of cell wall integrity. A genetic link between the Mpk1 kinase and the Ppz phosphatases has been reported, but the nature of this connection was unclear. Recently, the Ppz phosphatases were shown to be regulators of K+ and pH homeostasis. Here, we demonstrate that Ppz-deficient strains display increased steady-state K+ levels and sensitivity to increased KCl concentrations. Given these observations and the fact that K+ is the major determinant of intracellular turgor pressure, we reasoned that the connection between PPZ1 and -2 and MPK1 was due to the combination of increased internal turgor pressure in Ppz-deficient strains and cell wall instability observed in strains lacking MPK1. Accordingly, the MPK1 gene was up-regulated, the Mpk1 protein was overexpressed, and the phosphorylated active form was more abundant in Ppz-deficient strains. Moreover, the expression of genes previously identified as targets of the Mpk1 pathway are also up-regulated in strains lacking PPZ1 and -2. The transcriptional and posttranslational modifications of Mpk1 were not observed when the internal K+ concentration (and thus turgor pressure) was lowered by disrupting the TRK1 and -2 K+ transporter genes or when the cell wall was stabilized by the addition of sorbitol. Moreover, we present genetic evidence showing that both the Wsc1 and Mid2 branches of the Mpk1 pathway contribute to this response. Finally, despite its role in G1/S transition, increased levels of activated Mpk1 do not appear to be responsible for the cell cycle phenotype observed in Ppz-deficient strains.

scholarly journals Regulation of the Saccharomyces cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine phosphatases.

1997 ◽  
Vol 17 (3) ◽  
pp. 1289-1297 ◽  
Author(s):  
S M Wurgler-Murphy ◽  
T Maeda ◽  
E A Witten ◽  
H Saito
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Mitogen Activated Protein Kinase ◽  
Yeast Cells ◽  
High Osmolarity ◽  
Mapk Kinase ◽  
Protein Tyrosine ◽  
Mitogen Activated Protein

In response to increases in extracellular osmolarity, Saccharomyces cerevisiae activates the HOG1 mitogen-activated protein kinase (MAPK) cascade, which is composed of a pair of redundant MAPK kinase kinases, namely, Ssk2p and Ssk22p, the MAPK kinase Pbs2p, and the MAPK Hog1p. Hog1p is activated by Pbs2p through phosphorylation of specific threonine and tyrosine residues. Activated Hog1p is essential for survival of yeast cells at high osmolarity. However, expression of constitutively active mutant kinases, such as those encoded by SSK2deltaN and PBS2(DD), is toxic and results in a lethal level of Hog1p activation. Overexpression of the protein tyrosine phosphatase Ptp2p suppresses the lethality of these mutations by dephosphorylating Hog1p. A catalytically inactive Cys-to-Ser Ptp2p mutant (Ptp2(C/S)p) is tightly bound to tyrosine-phosphorylated Hog1p in vivo. Disruption of PTP2 leads to elevated levels of tyrosine-phosphorylated Hog1p following exposure of cells to high osmolarity. Disruption of both PTP2 and another protein tyrosine phosphatase gene, PTP3, results in constitutive Hog1p tyrosine phosphorylation even in the absence of increased osmolarity. Thus, Ptp2p and Ptp3p are the major phosphatases responsible for the tyrosine dephosphorylation of Hog1p. When catalytically inactive Hog1(K/N)p is expressed in hog1delta cells, it is constitutively tyrosine phosphorylated. In contrast, Hog1(K/N)p, expressed together with wild-type Hog1p, is tyrosine phosphorylated only when cells are exposed to high osmolarity. Thus, the kinase activity of Hog1p is required for its own tyrosine dephosphorylation. Northern blot analyses suggest that Hog1p regulates Ptp2p and/or Ptp3p activity at the posttranscriptional level.

scholarly journals Characterization of an activated human ros gene.

1986 ◽  
Vol 6 (9) ◽  
pp. 3109-3116 ◽  
Author(s):  
C Birchmeier ◽  
D Birnbaum ◽  
G Waitches ◽  
O Fasano ◽  
M Wigler
Keyword(s):  
Gene Transfer ◽  
Protein Kinases ◽  
Transmembrane Domain ◽  
Extracellular Domain ◽  
Kinase Domain ◽  
Specific Protein ◽  
Normal Human

A human oncogene, mcf3, previously detected by a combination of DNA-mediated gene transfer and a tumorigenicity assay, derives from a human homology of the avian v-ros oncogene. Both v-ros and mcf3 can encode a protein with homology to tyrosine-specific protein kinases, and both mcf3 and v-ros encode a potential transmembrane domain N terminal to the kinase domain. mcf3 probably arose during gene transfer from a normal human ros gene by the loss of a putative extracellular domain. There do not appear to be any other gross rearrangements in the structure of mcf3.

scholarly journals A single amino acid substitution (N297A) in the conserved NPXXY sequence of the human N-formyl peptide receptor results in inhibition of desensitization and endocytosis, and a dose-dependent shift in p42/44 mitogen-activated protein kinase activation and chemotaxis

2000 ◽  
Vol 352 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Jeannie M. GRIPENTROG ◽  
Algirdas J. JESAITIS ◽  
Heini M. MIETTINEN
Keyword(s):  
Protein Kinase ◽  
Transmembrane Domain ◽  
Mitogen Activated Protein Kinase ◽  
Formyl Peptide Receptor ◽  
Wild Type ◽  
Peptide Receptor ◽  
Formyl Peptide ◽  
Mitogen Activated Protein ◽  
Dose Dependent Increase ◽  
Dose Dependent

The formyl peptide receptor (FPR) is a G-protein-coupled receptor (GPCR) that mediates chemotaxis and stimulates the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase pathway. We have examined the functional effects of substitutions of a conserved aspartic acid residue in the second transmembrane domain (D71A) and of residues in the conserved NPXXY motif in the seventh transmembrane domain (N297A and Y301A). These mutated receptors, expressed in Chinese hamster ovary (CHO) cells, bind ligand with affinities similar to wild-type FPR, but the D71A mutant is uncoupled from G-protein [Miettinen, Mills, Gripentrog, Dratz, Granger and Jesaitis (1997) J. Immunol 159, 4045–4054]. In the present study, we show that both the D71A and N297A mutations resulted in defective endocytosis. The N297A substitution also prevented desensitization, as determined by intracellular calcium mobilization by sequential stimulation with ligand. In chemotaxis assays, the N297A mutation resulted in cell migration towards gradients of up to 100nM N-formyl-methionyl-leucyl-phenylalanine (fMLF), whereas cells expressing the wild-type FPR and the Y301A mutant were no longer chemotactically responsive at 10–100nM fMLF. Maximal activation of p42/44 MAPK occurred in CHO cells expressing wild-type FPR at 10nM–100nM fMLF, whereas cells expressing the N297A mutant showed a dose-dependent increase in the amount of phosphorylated p42/44 MAPK up to 1–10µM fMLF. Since the MAPK kinase inhibitor PD98059 blocked fMLF-induced chemotaxis, our results suggest that the dose-dependent increase in p42/44 MAPK activation may correlate with the increased chemotactic migration of N297A transfectants at 10nM–100nM fMLF.

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