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Laboratory of Molecular Genetics (Frontier Research Unit)
遺伝子解析施設（フロンティア研究領域）
Professor
Makoto Nakanishi, M.D., Ph.D.
Associate Professor Kazuo Tatebayashi, Ph.D.

教 授准教授

医学博士
博士（薬学）

中舘

西林

和真夫

The Laboratory of Molecular Genetics was established for developing various
molecular genetic techniques, spreading them to IMSUT investigators andsupporting security management related to experiments carried out using
recombinant DNA technologies. Since 2017, this laboratory has integrated theFrontier Research Unit for supporting selected investigators to challenge new fieldsof bio-medical sciences.

Frontier Research Unit
Protein phosphorylation and dephosphorylation
are among the most important intracellular signalingmechanisms, and are mediated, respectively, by protein kinases and protein phosphatases. We study various aspects of cellular signal transduction with a particular emphasis on the role and regulation of protein
phosphorylation and dephosphorylation in cellularstress responses, using yeast cells.
1.  Negative regulation of Pbs2 MAP2K by PP2C
phosphatases in the yeast HOG pathway.
Kazuo Tatebayashi
The budding yeast Saccharomyces cerevisiae survivegreatly fluctuating osmotic conditions in natural environment. To cope with an increased external osmolarity, yeast cells elicit a coordinated adaptive response. These adaptive responses are governed by
the Hog1 MAP kinase (MAPK), which is activated via
the High Osmolarity Glycerol (HOG) signaling pathway. The HOG pathway consists of a core module ofthree tiers of protein kinases termed a MAP kinase(MAPK), a MAPK kinase (MAPKK, MAP2K), and a
MAPKK kinase (MAPKKK, MAP3K). In addition,
the upstream part of the HOG pathway comprises thefunctionally redundant, but mechanistically distinct,
SHO1 and SLN1 branches. When yeast cells are exposed to extracellular high osmolarity, the osmosen-sors in the SHO1 and SLN1 branches independently
detect osmostress to activate cognate MAP3Ks. In theSHO1 branch, osmosensing complexes composed ofSho1, Opy2, Hkr1, and Msb2 activate the MAP3K
Ste11. In the SLN1 branch, the Sln1-Ypd1-Ssk1 phospho-relay system activates the functionally redundant MAP3Ks Ssk2 and Ssk22 (Ssk2/22). Activated
Ste11 and Ssk2/22 phosphorylate and activate theMAP2K Pbs2. Activated Pbs2, in turn, phosphorylates the MAPK Hog1 at T174 and Y176 in its activation loop for its activation.
Unregulated continuous activation of the HOG
pathway is deleterious to cell growth, probably by
preventing cell cycle progression. Therefore, a mechanism is needed that appropriately inactivates theHOG pathway. Two groups of the protein phosphatases are involved in the HOG pathway inactivation. The first group contains the members of the protein tyrosine phosphatases (PTP), namely, Ptp2 andPtp3, which dephosphorylate Hog1 at Y176. The second group contains the members of the serine/threonine protein phosphatase type 2 (PP2C), namely,Ptc1, Ptc2, Ptc3, and Ptc4. Of these, Ptc1, Ptc2, andPtc3 had been proposed as negative regulators of theHOG pathway, because their overexpression rescued
the lethality of the sln1Δ cell by inhibiting the constitutive activation of the HOG pathway. Furthermore,overexpression of either Ptc1 or Ptc2 inactivated Hog1
in vivo, and purified Ptc1 and Ptc2 dephosphorylated
T174 in vitro. In contrast, it was concluded that thesephosphatases did not inhibit Pbs2 in vivo, because

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overexpression of either Ptc1 or Ptc2 did not reduce
Hog1 phosphorylation at Y176, which served as anindicator of the Pbs2 activity. Thus, it had been proposed that these type 2C phosphatases inactivate
Hog1, but not Pbs2. The phosphatases that dephosphorylate Pbs2 had not been identified yet.
The results of overexpression experiments must
be regarded cautiously, because at high level of phosphatase expression, non-physiological substrates
might be sufficiently dephosphorylated. For that reason, we consider the gene inactivation experiments
more reliable. Furthermore, estimating Pbs2 activity
indirectly from the extent of Hog1 phosphorylation
might be difficult, as the efficiencies of Pbs2 phosphorylating at Hog1 T174 and Y176 may be significantlydifferent.
This year, we examined the effects of inactivating
the genes of type 2C phosphatases upon the phosphorylation status of Pbs2 at the activating phosphorylation sites Ser-514 and Thr-518 (S514 and T518) by directly measuring the levels of Pbs2 phosphorylationat S514 and T518 using the assay method we developed recently. We found that S514 and T518 are differentially dephosphorylated by different type 2Cserine/threonine phosphatases. Ptc1-Ptc4 collectively
regulate Pbs2 negatively, but each Ptc acts differently
to the two phosphorylation sites in Pbs2. T518 is predominantly dephosphorylated by Ptc1, whereas theeffect of Ptc2-Ptc4 could be seen only when Ptc1 is absent. On the other hand, S514 is more evenly dephosphorylated by Ptc1, Ptc2, Ptc3, and Ptc4. We also
show that Pbs2 dephosphorylation by Ptc1 requires
the adaptor protein Nbp2 that recruits Ptc1 to Pbs2.
2.  Osmostress enhances Pbs2 MAP2K phosphorylation by Ste11 MAP3K in the yeast osmo-regulatory HOG pathway
Kazuo Tatebayashi
The yeast MAPK Hog1 is activated by hyperosmotic stress through the High Osmolarity Glycerol(HOG) pathway, and orchestrates an array of osmo-adaptive changes in transcription, translation, cell
cycle, and metabolism. The current widely held model of the HOG pathway is as follows. The upstreamportion of the HOG pathway is comprised of thefunctionally redundant SHO1 and SLN1 branches. Inthe SHO1 branch, osmosensing complexes composedof Sho1, Opy2, Hkr1, and Msb2 activate the MAP3K
Ste11. In the SLN1 branch, the Sln1-Ypd1-Ssk1 phospho-relay mechanism is involved in activation of thefunctionally redundant MAP3Ks Ssk2 and Ssk22(Ssk2/22). Ste11 and Ssk2/22 phosphorylate theMAP2K Pbs2 at Ser-514 and/or Thr-518. Phosphorylated Pbs2 then activates Hog1. Recently, we foundthat osmostress not only activates membrane-associated osmosensors, but also enhances Hog1 phosphorylation by mono-phosphorylated Pbs2. The lack of
the osmotic enhancement of the Pbs2-Hog1 reaction
suppresses Hog1 activation by basal MAP3K activities and prevents pheromone-to-Hog1 crosstalk in theabsence of osmostress, which ensures the appropriate
Hog1 activation only under high osmolarity. It has
been unclear if osmostress acts on other phosphorylation steps in the MAP kinase cascade of the HOG
pathway.
This year, we examined if osmostress enhances
Pbs2 MAP2K phosphorylation by Ste11 MAP3K aswell as Hog1 phosphorylation. Ste11 is phosphorylated and activated by the Ste20/Cla4 kinases uponhigh osmolarity. The constitutively-active Ste11DDD mutant carries the substitution mutations of allactivating phosphorylation sites for Ste20/Cla4 tophosphomimic Asp (S281D, S285D, and T286D),
which circumvent the need for Ste20/Cla4 for Ste11activation. The endogenous-level expression of Ste11DDD did not induce the phosphorylation of Pbs2 under unstimulated condition, but induced it upon highosmolarity. These results strongly suggest that Pbs2
phosphorylation by activated Ste11 is osmotically-enhanced as Hog1 is. The underlying mechanism is under study.

Publication
Tatebayashi K, and Saito H. Two activating phosphorylation sites of Pbs2 MAP2K in the yeast HOG
pathway are differentially dephosphorylated byfour PP2C phosphatases Ptc1-Ptc4. J Biol Chem.
299(4):104569 (2023)