Page 127 Workshop E: Cell regulation Talk 101
Osmostress signaling by the Wis4-Win1 MAPKKK heteromer stabilized by the Mcs4 response regulator
Kaz Shiozaki
a,b
, Susumu Morigasaki
a
, Aminah Ikner
b
, Hisashi Tatebe
a
a
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan
b
Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616, USA
Presented by: Shiozaki, Kaz
The Spc1 MAP kinase MAPK cascade in fission yeast is activated by two MAPK kinase kinases MAPKKKs, Wis4 and Win1, in response to multiple
forms of environmental stress. Interestingly, these MAPKKK paralogs do not appear to be redundant, and it was proposed that Win1, but not Wis4, is
responsible for transmitting osmostress signals to the Wis1 MAPKK-Spc1 MAPK cascade [1,2]. However, we have found that both Wis4 and Win1 are
required for osmostress signaling and that these MAPKKKs function as a heteromer. Intriguingly, only one of the MAPKKKs in the heteromer complex
needs to be catalytically active, but disruption of the complex results in reduced MAPKKK-MAPKK interaction and consequently, compromised MAPK
activation. Although some of mammalian MAPKKKs, such as B-Raf and C-Raf, are known to form a heteromer, the Wis4-Win1 association is the first example
of a functional MAPKKK heteromer in fungal species.
Peroxide stress signals are sensed and transmitted to the Spc1 MAPK cascade by the phosphorelay module composed of the Mak sensor kinases, the Mpr1
histidine phosphotransferase and the Mcs4 response regulator [3,4]. It has been a conundrum that the mcs4 null mutant is defective in transmitting not only
peroxide stress but also osmostress signals to Spc1 MAPK. We have discovered that Mcs4 is required for stable heteromer formation between Wis4 and Win1
MAPKKKs and that the destabilized MAPKKK heteromer in the mcs4 null mutant is responsible for compromised activation of Spc1 in response to
osmostress. Such phosphorelay-independent function of Mcs4 is quite unexpected, because response regulator proteins normally act as terminal
effectors of phosphorelay signaling in both bacteria and eukaryotes.
[1] Samejima et al., EMBO J. 16, 6162 1997 [2] Samejima et al., Mol. Biol. Cell 9, 2325 1998
[3] Nguyen et al., Mol. Biol. Cell 11, 1169 2000 [4] Buck et al., Mol. Biol. Cell 12, 407 2001
Page 128 Workshop E: Cell regulation Talk 102
Growth regulation via opposite effects of TORC1 and TORC2 on amino acid homeostasis
Dana Laor
a
, Adi Cohen
b
, Martin Kupiec
a
, Ronit Weisman
b
a
Tel Aviv University, Israel
b
Open University of Israel
Presented by: Weisman, Ronit
Cell growth and proliferation are conserved and highly regulated processes. The target of rapamycin TOR is a key regulator of cellular growth in response to
nutrition status. Fission yeast contains two conserved TOR complexes, TORC1 and TORC2. TORC1 is an essential complex, while TORC2 is required under
stress conditions. Previously, we observed that deletion mutations in the TSC complex
tsc1 or tsc2 render cells highly sensitive to rapamycin on proline medium. Here we show that this rapamycin sensitive phenotype is mediated via
TOR complex 1 TORC1 and is suppressed by over expression of isp7+, a putative 2-oxoglutarate-FeII dependent oxygenase, which acts to induce
TORC1 activity. Functional and transcriptional analysis of isp7 mutant cells revealed that Isp7 is a master regulator of amino acid permease expression and
amino acid uptake. Isp7, similar to TORC1 and opposite to TORC2, suppresses transcription of nitrogen-starvation induced permeases. Overexpression of Isp7
induced TORC1-dependnet phosphorylation towards the ribosomal protein S6 even under nitrogen starvation, conditions under which Rps6 is normally de-
phsophorylated. Thus, high activity of Isp7 may mimic nitrogen or amino acid sufficiency. Remarkably, the transcription of isp7+ is downregulated by TORC1
and upregulated by TORC2, revealing an isp7+-dependent regulatory loops that involves both TORC1 and TORC2 and an elaborate mechanism to control amino
acid homoeostasis.
Page 129 Workshop E: Cell regulation Talk 103
The TOR kinases and the SAGA transcriptional co-activator coordinately control gene expression in response to nutrient
availability
Thomas Laboucarié, Gwenda Lledo, Yves Romeo, Ghislaine Yagoubi, Dom Helmlinger
Macromolecular Biochemistry Research Center, CNRS UMR 5237, Montpellier, France
Presented by: Helmlinger, Dom
The regulation of gene expression plays a fundamental role in the ability of cells to respond to external changes. Although there are multiple levels of regulation,
including transcription and translation, little is known about how these processes are synchronized. Using
S. pombe
as a model system, we are addressing this issue in the context of cell fate control by nutrient availability. We and others
have established that the TOR kinases and the SAGA transcriptional co-activator complex are critical for
S. pombe
to respond to a change in nutrient levels and to decide whether to proliferate or differentiate. We have started to determine
whether SAGA is directly regulated by the TOR pathway. Our preliminary genetic analyses show that SAGA functions downstream of the TOR pathway to
control the expression of genes induced in response to nutrient starvation. In addition, we and others have recently discovered that one SAGA subunit, Tra1,
directly associates with an HSP90 co-chaperone, the ASTRA complex. Work in
S. pombe
and in mammals has recently established that ASTRA is a novel, important regulator of TOR kinase activity. Therefore, we hypothesize that Tra1
plays a central role in nutrient sensing and, through its function in ASTRA, synchronizes the activity of the TOR pathway with the regulatory roles of SAGA
at specific promoters. Mass spectrometry analysis of ASTRA purified from either wild-type or tra1 deletion mutant cells showed that Tra1 controls the
association of the TOR kinases with ASTRA. This observation suggests that Tra1 plays a direct role within ASTRA to regulate the TOR kinases. We will
discuss how these results contribute to our understanding of the control of transcription by signal transduction pathways.
Page 130 Workshop E: Cell regulation Talk 104
Full TORC1 inhibition, reduce Wee1 levels and advance mitotic commitment in fission yeast and mammalian cells
Jane Atkin, Jennifer Ferguson, Claudia Wellbrock, Janni Petersen
University of Manchester, Michael Smith Building, Manchester M13 9PT, UK
Presented by: Petersen, Janni
The highly conserved protein kinase, Target Of Rapamycin TOR is a key regulator of cell growth and cell division.
S. pombe
contains two TOR proteins Tor1 and Tor2, which exist as part of at least two distinct protein complexes:
TORC1 mainly containing Tor2 and TORC2 predominantly containing Tor1. It is widely established that rapamycin inhibits a subset of TOR activities by
binding to the FRB domain adjacent to the kinase domain of TORC1 only. We previously demonstrated that rapamycin treated cells advance mitosis to reduce
cell size however cells continue to proliferate. We now characterise an ATP- competitive Tor1Tor2 inhibitor. In contrast to the mild impact of rapamycin on
cell division, blocking the catalytic site of TOR kinases with this competitive inhibitor completely arrests growth. This growth arrest occurs without death or a
specific arrest in the G1 phase of the cell cycle. Specific read-outs of both TORC1 and TORC2 activities establish that the activities of both complexes are
inhibited. A screen for inhibitor-resistant mutants identified mutations in Tor2 TORC1
– the essential complex, confirming the specificity of the ATP- competitor for TOR. Furthermore this shows that, unlike rapamycin, the
competitive inhibitor fully inhibits TORC1. We have exploited this mutation to show that Wee1 levels are reduced and cell advance mitotic commitment as soon
as TORC1 is completely inhibited. Growth arrest follows this acceleration of the cell cycle. Experiments in mammalian cells mirror these results from fission
yeast as mTORC1 inhibition advances mitosis, as Wee1 is lost.
Page 131
Identification of novel upstream regulators of fission yeast transcription factors by Ssynthetic dosage lethality
Kate Chatfield-Reed, Eun-Joo Kwon, Amy Laderoute, Gordon Chua
University of Calgary, Canada
Presented by: Chatfield-Reed, Kate
Mapping the interactions of transcription factors with upstream regulatory pathways is critical to enhancing our comprehension of transcriptional control
and gene regulatory networks. These pathways involve regulatory proteins that affect the expression and activity of the transcription factor by altering its
structural conformation, localization or rate of degradation. Approximately half of fission yeast transcription factors have fitness defects when overexpressed,
likely due to their hyperactivation and the aberrant expression of their target genes. In contrast, the remaining transcription factors exhibit minor or no
changes in colony size as assessed by synthetic genetic array SGA technology. These transcription factors are not toxic to the cell because their overexpression
may not be sufficient for activation. If this is the case, then deletion backgrounds that sensitize the overexpression of these transcription factors may represent
negative regulators. We are developing an SGA-based synthetic dosage lethality method to identify novel upstream regulators of fission yeast transcription
factors. An
nmt1
-driven transcription factor gene is systematically overexpressed in every mutant of a regulator deletion miniarray by SGA, and the double
mutants are assayed for decreased colony size. This miniarray contains over 250 Bioneer gene deletions of regulatory molecules including kinases, phosphatases,
acetylases and ubiquitin ligases. We will present preliminary data on synthetic dosage lethality screens of the transcription factors Cbf11, Scr1, Toe1, Tos4 and
Yox1. These screens can be used to unravel another level of control in the
S. pombe
gene regulatory network.
Page 132
Quantitative analysis of fission yeast genome expression at population and single-cell levels
Samuel Marguerat
a,b
