scholarly journals Identification of Ste4 as a potential regulator of Byr2 in the sexual response pathway of Schizosaccharomyces pombe.

1996 ◽  
Vol 16 (10) ◽  
pp. 5597-5603 ◽  
Author(s):  
M M Barr ◽  
H Tu ◽  
L Van Aelst ◽  
M Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Binding Site ◽  
Sexual Differentiation ◽  
Leucine Zipper ◽  
Regulatory Region ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
Two Hybrid ◽  
Erk Kinase

A conserved MAP kinase cascade is central to signal transduction in both simple and complex eukaryotes. In the yeast Schizosaccharomyces pombe, Byr2, a homolog of mammalian MAPK/ERK kinase kinase and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation. A screen for S. pombe proteins that interact with Byr2 in a two-hybrid system led to the isolation of Ste4, a protein that is known to be required for sexual function. Ste4 binds to the regulatory region of Byr2. This binding site is separable from the binding site for Ras1. Both Ste4 and Ras1 act upstream of Byr2 and act at least partially independently. Ste4 contains a leucine zipper and is capable of homotypic interaction. Ste4 has regions of homology with STE50, an S. cerevisiae protein required for sexual differentiation that we show can bind to STE11.

scholarly journals Skh1, the MEK component of the mkh1 signaling pathway in Schizosaccharomyces pombe

2000 ◽  
Vol 113 (1) ◽  
pp. 153-160 ◽  
Author(s):  
R. Loewith ◽  
A. Hubberstey ◽  
D. Young
Keyword(s):  
Map Kinase ◽  
Schizosaccharomyces Pombe ◽  
Genetic Evidence ◽  
Yeast Two Hybrid ◽  
Beta Glucanase ◽  
Map Kinase Cascade ◽  
Mutant Strains ◽  
Kinase Cascade ◽  
Delta Cells ◽  
Two Hybrid

We previously reported the identification of Mkh1, a MEK kinase in Schizosaccharomyces pombe that is required for cell wall integrity, and we presented genetic evidence that Pmk1/Spm1, a MAP kinase, functions downstream from Mkh1 in the same pathway. Here, we report the identification of Skh1, a MEK (MAP kinase kinase) in S. pombe. The sequence of Skh1 is nearly identical to that of the recently reported Pek1 sequence. We present biochemical and genetic evidence that Skh1 is the MEK component of the Mkh1-Spm1 MAP kinase cascade. Our yeast two-hybrid results indicate that Mkh1, Skh1, and Spm1 physically interact to form a ternary complex. Deletion of mkh1, skh1 or spm1 results in identical phenotypes, including sensitivity to (beta)-glucanase treatment, growth inhibition on media containing KCl, and filamentous growth on medium containing caffeine. Double mutant strains exhibit phenotypes that are identical to the single mutant strains. Furthermore, expression of an activated HA-Skh1(DD)protein suppressed these defects in mkh1(delta) cells, and overexpression of Spm1 suppressed these defects in skh1(delta) cells. We also show that HA-Spm1 is hyper-phosphorylated on tyrosine residues in cells co-expressing the activated HA-Skh1(DD) protein. Furthermore, we found the phosphorylated/activated form of GFP-HA-Spm1 at detectable levels in wild-type cells, but not at appreciable levels in mkh1(delta) or skh1(delta) cells expressing this fusion protein. Together, our results indicate that Mkh1, Skh1 and Spm1 constitute a MAPK cascade in fission yeast.

scholarly journals The Saccharomyces cerevisiae GATA Factors Dal80p and Deh1p Can Form Homo- and Heterodimeric Complexes

1998 ◽  
Vol 180 (21) ◽  
pp. 5682-5688 ◽  
Author(s):  
Vladimir V. Svetlov ◽  
Terrance G. Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Leucine Zipper ◽  
Nitrogen Catabolite Repression ◽  
Leucine Zipper Motif ◽  
Gata Factors ◽  
Two Factors ◽  
The Difference ◽  
Two Hybrid ◽  
Specific Orientation

ABSTRACT GATA family proteins Gln3p, Gat1p, Dal80p, and Deh1p mediate the regulation of nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces cerevisiae. Thus far, Gln3p, Dal80p, and Deh1p have been shown to bind to GATA sequences in NCR-sensitive promoters, in some cases to exactly the same GATA sequences. A minimal Gln3p binding site consists of a single GATA sequence, whereas a Dal80p binding site consists of two GATA sequences in specific orientation, 15 to 35 bp apart, suggesting that Dal80p may bind to DNA as a dimer. Additionally, both Dal80p and Deh1p are predicted to contain a leucine zipper motif near their C termini. Therefore, we tested whether they could form homo- and/or heterodimers in two-hybrid assays. We show that Dal80p-Dal80p, Dal80p-Dal80pLZ (leucine zipper), Dal80pLZ-Dal80pLZ, Dal80p-Deh1pLZ, Dal80pLZ-Deh1pLZ, and Deh1pLZ-Deh1pLZ complexes can form. Dal80p-Dal80p and Dal80pLZ-Dal80pLZ complexes yield 5- to 10-fold stronger signals than the other possible dimers. If Dal80p and Deh1p bind to DNA only after dimerization, then the difference in ability to form complexes could significantly affect their affinity for binding DNA and thus the degree of regulation exerted by each of the two factors.

scholarly journals Interaction of a Swi3 homolog with Sth1 provides evidence for a Swi/Snf-related complex with an essential function in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (4) ◽  
pp. 1768-1775 ◽  
Author(s):  
I Treich ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Leucine Zipper Motif ◽  
Cell Extracts ◽  
Essential Function ◽  
Self Association ◽  
Two Hybrid

The Saccharomyces cerevisiae Swi/Snf complex has a role in remodeling chromatin structure to facilitate transcriptional activation. The complex has 11 components, including Swi1/Adr6, Swi2/Snf2, Swi3, Snf5, Snf6, Snf11, Swp73/Snf12, and Tfg3. Mammalian homologs of these proteins have been shown to form multiple Swi/Snf-related complexes. Here we characterize an S. cerevisiae Swi3 homolog (Swh3) and present evidence that it associates in a complex with a Snf2 homolog, Sthl. We identified Swh3 as a protein that interacts with the N terminus of Snf2 in the two-hybrid system. Swh3 and Swi3 are functionally distinct, and overexpression of one does not compensate for loss of the other. Swh3 is essential for viability and does not activate transcription of reporters. The Snf2 sequence that interacts with Swh3 was mapped to a region conserved in Sth1. We show that Swh3 and Sth1 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. We also map interactions between Swh3 and Sth1 and examine the role of a leucine zipper motif in self-association of Swh3. These findings, together with previous analysis of Sth1, indicate that Swh3 and Sth1 are associated in a complex that is functionally distinct from the Swi/Snf complex and essential for viability.

scholarly journals Cyclic AMP-Dependent Protein Kinase Regulates Pseudohyphal Differentiation in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (7) ◽  
pp. 4874-4887 ◽  
Author(s):  
Xuewen Pan ◽  
Joseph Heitman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Map Kinase ◽  
Filamentous Growth ◽  
Dependent Protein Kinase ◽  
Catalytic Subunits ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
Dependent Protein ◽  
Pseudohyphal Differentiation

ABSTRACT In response to nitrogen starvation, diploid cells of the yeastSaccharomyces cerevisiae differentiate to a filamentous growth form known as pseudohyphal differentiation. Filamentous growth is regulated by elements of the pheromone mitogen-activated protein (MAP) kinase cascade and a second signaling cascade involving the receptor Gpr1, the Gα protein Gpa2, Ras2, and cyclic AMP (cAMP). We show here that the Gpr1-Gpa2-cAMP pathway signals via the cAMP-dependent protein kinase, protein kinase A (PKA), to regulate pseudohyphal differentiation. Activation of PKA by mutation of the regulatory subunit Bcy1 enhances filamentous growth. Mutation and overexpression of the PKA catalytic subunits reveal that the Tpk2 catalytic subunit activates filamentous growth, whereas the Tpk1 and Tpk3 catalytic subunits inhibit filamentous growth. The PKA pathway regulates unipolar budding and agar invasion, whereas the MAP kinase cascade regulates cell elongation and invasion. Epistasis analysis supports a model in which PKA functions downstream of the Gpr1 receptor and the Gpa2 and Ras2 G proteins. Activation of filamentous growth by PKA does not require the transcription factors Ste12 and Tec1 of the MAP kinase cascade, Phd1, or the PKA targets Msn2 and Msn4. PKA signals pseudohyphal growth, in part, by regulating Flo8-dependent expression of the cell surface flocculin Flo11. In summary, the cAMP-dependent protein kinase plays an intimate positive and negative role in regulating filamentous growth, and these findings may provide insight into the roles of PKA in mating, morphogenesis, and virulence in other yeasts and pathogenic fungi.

scholarly journals Methylglyoxal Activates the Target of Rapamycin Complex 2-Protein Kinase C Signaling Pathway in Saccharomyces cerevisiae

2015 ◽  
Vol 35 (7) ◽  
pp. 1269-1280 ◽  
Author(s):  
Wataru Nomura ◽  
Yoshiharu Inoue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Map Kinase ◽  
Target Of Rapamycin ◽  
Dependent Manner ◽  
Content Type ◽  
Protein Levels ◽  
Map Kinase Cascade ◽  
Phosphorylated Akt ◽  
Mitogen Activated Protein ◽  
Kinase Cascade

Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeastSaccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr1125and Ser1143. Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser1143, which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1T1125affected the phosphorylation of Pkc1 at Ser1143, in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser473. Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.

scholarly journals A Third Osmosensing Branch in Saccharomyces cerevisiae Requires the Msb2 Protein and Functions in Parallel with the Sho1 Branch

2002 ◽  
Vol 22 (13) ◽  
pp. 4739-4749 ◽  
Author(s):  
Sean M. O'Rourke ◽  
Ira Herskowitz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Map Kinase ◽  
Cross Talk ◽  
Dna Microarrays ◽  
Hog Pathway ◽  
Gene Sets ◽  
Map Kinase Cascade ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Membrane Spanning

ABSTRACT Two Saccharomyces cerevisiae plasma membrane-spanning proteins, Sho1 and Sln1, function during increased osmolarity to activate a mitogen-activated protein (MAP) kinase cascade. One of these proteins, Sho1, utilizes the MAP kinase kinase kinase Ste11 to activate Pbs2. We previously used the FUS1 gene of the pheromone response pathway as a reporter to monitor cross talk in hog1 mutants. Cross talk requires the Sho1-Ste11 branch of the HOG pathway, but some residual signaling, which is STE11 dependent, still occurs in the absence of Sho1. These observations led us to propose the existence of another osmosensor upstream of Ste11. To identify such an osmosensor, we screened for mutants in which the residual signaling in a hog1 sho1 mutant was further reduced. We identified the MSB2 gene, which encodes a protein with a single membrane-spanning domain and a large presumptive extracellular domain. Assay of the FUS1-lacZ reporter (in a hog1 mutant background) showed that sho1 and msb2 mutations both reduced the expression of the reporter partially and that the hog1 sho1 msb2 mutant was severely defective in the expression of the reporter. The use of DNA microarrays to monitor gene expression revealed that Sho1 and Msb2 regulate identical gene sets in hog1 mutants. A role for MSB2 in HOG1 strains was also seen in strains defective in the two known branches that activate Pbs2: an ssk1 sho1 msb2 strain was more osmosensitive than an ssk1 sho1 MSB2 strain. These observations indicate that Msb2 is partially redundant with the Sho1 osmosensing branch for the activation of Ste11.

scholarly journals The CLIP-170 Homologue Bik1p Promotes the Phosphorylation and Asymmetric Localization of Kar9p

2006 ◽  
Vol 17 (1) ◽  
pp. 178-191 ◽  
Author(s):  
Jeffrey K. Moore ◽  
Sonia D'Silva ◽  
Rita K. Miller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Mitotic Spindle ◽  
Phosphorylation Site ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Basic Domain ◽  
Pole Body ◽  
Two Hybrid ◽  
Insight Into

Accurate positioning of the mitotic spindle in Saccharomyces cerevisiae is coordinated with the asymmetry of the two poles and requires the microtubule-to-actin linker Kar9p. The asymmetric localization of Kar9p to one spindle pole body (SPB) and microtubule (MT) plus ends requires Cdc28p. Here, we show that the CLIP-170 homologue Bik1p binds directly to Kar9p. In the absence of Bik1p, Kar9p localization is not restricted to the daughter-bound SPB, but it is instead found on both SPBs. Kar9p is hypophosphorylated in bik1Δ mutants, and Bik1p binds to both phosphorylated and unphosphorylated isoforms of Kar9p. Furthermore, the two-hybrid interaction between full-length KAR9 and the cyclin CLB5 requires BIK1. The binding site of Clb5p on Kar9p maps to a short region within the basic domain of Kar9p that contains a conserved phosphorylation site, serine 496. Consistent with this, Kar9p is found on both SPBs in clb5Δ mutants at a frequency comparable with that seen in kar9-S496A strains. Together, these data suggest that Bik1p promotes the phosphorylation of Kar9p on serine 496, which affects its asymmetric localization to one SPB and associated cytoplasmic MTs. These findings provide further insight into a mechanism for directing centrosomal inheritance.

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