Hisashi Tatebe

Pom1 DYRK Regulates Localization of the Rga4 GAP to Ensure Bipolar Activation of Cdc42 in Fission Yeast

BACKGROUND In the fission yeast Schizosaccharomyces pombe, cell growth takes place exclusively at both ends of the cylindrical cell. During this highly polarized growth, microtubules are responsible for the placement of the cell-end marker proteins, the Tea1-Tea4/Wsh3 complex, which recruits the Pom1 DYRK-family protein kinase. Pom1 is required for proper positioning of growth sites, and the Deltapom1 mutation brings about monopolar cell growth. RESULTS Pom1 kinase physically interacts with Rga4, which has a GAP (GTPase-activating protein) domain for Rho-family GTPase. Genetic and biochemical evidence indicates that Rga4 functions as GAP for the Cdc42 GTPase, an evolutionarily conserved regulator of F-actin. CRIB (Cdc42/Rac interactive binding)-GFP microscopy has revealed that GTP-bound, active Cdc42 is concentrated to growing cell ends accompanied by developed F-actin structures, where the Rga4 GAP is excluded. The monopolar Deltapom1 mutant fails to eliminate Rga4 from the nongrowing cell end, resulting in monopolar distribution of GTP-Cdc42 to the growing cell end. However, mutational inactivation of Rga4 allows Cdc42 to be active at both ends of Deltapom1 cells, suggesting that mislocalization of Rga4 in the Deltapom1 mutant contributes to its monopolar phenotype. CONCLUSIONS Pom1 kinase recruited to cell ends by the Tea1-Tea4/Wsh3 complex is essential for proper localization of a GAP for Cdc42, Rga4, which ensures bipolar localization of GTP-bound, active Cdc42. Because of the established role of Cdc42 in F-actin formation, these observations provide a new insight into how the microtubule system achieves localized formation of F-actin to generate cell polarity.

Wsh3/Tea4 Is a Novel Cell-End Factor Essential for Bipolar Distribution of Tea1 and Protects Cell Polarity under Environmental Stress in S. pombe

BACKGROUND The fission yeast Schizosaccharomyces pombe has a cylindrical cell shape, for which growth is strictly limited to both ends, and serves as an excellent model system for genetic analysis of cell-polarity determination. Previous studies identified a cell-end marker protein, Tea1, that is transported by cytoplasmic microtubules to cell tips and recruits other cell-end factors, including the Dyrk-family Pom1 kinase. The deltatea1 mutant cells cannot grow in a bipolar fashion and show T-shaped morphology after heat shock. RESULTS We identified Wsh3/Tea4 as a novel protein that interacts with Win1 MAP kinase kinase kinase (MAPKKK) of the stress-activated MAP kinase cascade. Wsh3 forms a complex with Tea1 and is transported to cell tips by growing microtubules. The deltawsh3 mutant shows monopolar growth with abnormal Tea1 aggregate at the non-growing cell end; this abnormal aggregate fails to recruit Pom1 kinase. Consistent with the observed interaction between Win1 and Wsh3, cells lacking Wsh3 or Tea1 show more severe cell-polarity defects under osmolarity and heat-stress stimuli that are known to activate the stress MAPK cascade. Furthermore, mutants of the stress MAPK also exhibit cell-polarity defects when exposed to the same stress. CONCLUSIONS Wsh3/Tea4 is an essential component of the Tea1 cell-end complex. In addition to its role in bipolar growth during the normal cell cycle, the Wsh3-Tea1 complex, together with the stress-signaling MAPK cascade, contributes to cell-polarity maintenance under stress conditions.

Fission yeast TOR complex 2 activates the AGC-family Gad8 kinase essential for stress resistance and cell cycle control

Members of the mitogen-activated protein kinase (MAPK) subfamily responsive to environmental stress stimuli are known as SAPKs (stress-activated protein kinases), which are conserved from yeast to humans. In the fission yeast Schizosaccharomyces pombe, Spc1/Sty1 SAPK is activated by diverse forms of stress, such as osmostress, oxidative stress and heat shock, and induces gene expression through the Atf1 transcription factor. Sin1 (SAPK interacting protein 1) was originally isolated as a protein that interacts with Spc1, and its orthologs were also found in diverse eukaryotes. Here we report that Sin1 is not required for the stress gene expression regulated by Spc1 and Atf1, and that Sin1 is an essential component of TOR (target of rapamycin) complex 2 (TORC2). TORC2 is not essential for cell viability in S. pombe but plays important roles in cellular survival of stress conditions through phosphorylation and activation of an AGC-family protein kinase, Gad8. In addition, inactivation of Gad8 results in a synthetic growth defect with cdc25-22, a temperature-sensitive mutation of the Cdc25 phosphatase that activates Cdc2 kinase at G2/M. Gad8 also positively regulates expression of the CDK inhibitor gene rum1+, which is essential for cell cycle arrest in G1 after nitrogen starvation. These results strongly suggest that the TORC2–Gad8 pathway has multiple physiological functions in cellular stress resistance and cell cycle progression at both G1/S and G2/M transitions.

Rab-Family GTPase Regulates TOR Complex 2 Signaling in Fission Yeast

BACKGROUND From yeast to human, TOR (target of rapamycin) kinase plays pivotal roles in coupling extracellular stimuli to cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2), which phosphorylate and activate different AGC-family protein kinases. TORC1 is controlled by the small GTPase Rheb, but little is known about TORC2 regulators. RESULTS We have identified the Ryh1 GTPase, a human Rab6 ortholog, as an activator of TORC2 signaling in the fission yeast Schizosaccharomyces pombe. Mutational inactivation of Ryh1 or its guanine nucleotide exchange factor compromises the TORC2-dependent phosphorylation of the AGC-family Gad8 kinase. In addition, the effector domain of Ryh1 is important for its physical interaction with TORC2 and for stimulation of TORC2 signaling. Thus, GTP-bound Ryh1 is likely to be the active form stimulatory to TORC2-Gad8 signaling. Consistently, expression of the GTP-locked mutant Ryh1 is sufficient to promote interaction between TORC2 and Gad8 and to induce Gad8 hyperphosphorylation. The loss of functional Ryh1, TORC2, or Gad8 brings about similar vacuolar fragmentation and stress sensitivity, further corroborating their involvement in a common cellular process. Human Rab6 can substitute Ryh1 in S. pombe, and therefore Rab6 may be a potential activator of TORC2 in mammals. CONCLUSIONS In its GTP-bound form, Ryh1, an evolutionarily conserved Rab GTPase, activates TORC2 signaling to the AGC kinase Gad8. The Ryh1 GTPase and the TORC2-Gad8 pathway are required for vacuolar integrity and cellular stress resistance in S. pombe.

Identification of Cdc37 as a novel regulator of the stress-responsive mitogen-activated protein kinase.

ABSTRACT Eukaryotic cells utilize multiple mitogen-activated protein kinases (MAPKs) to transmit various extracellular stimuli to the nucleus. A subfamily of MAPKs that mediates environmental stress stimuli is also called stress-activated protein kinase (SAPK), which has crucial roles in cellular survival under stress conditions as well as inflammatory responses. Here we report that Cdc37, an evolutionarily conserved kinase-specific chaperone, is a positive regulator of Spc1 SAPK in the fission yeast Schizosaccharomyces pombe. Through a genetic screen, we have identified cdc37 as a mutation that compromises signaling through Spc1 SAPK. The Cdc37 protein physically interacts with Spc1, and the cdc37 mutation affects both the cellular level of the Spc1 protein and stress-induced Spc1 phosphorylation by Wis1 MAPK kinase (MAPKK). Consistently, expression of the stress response genes regulated by the Spc1 pathway is compromised in cdc37 mutant cells. On the other hand, a mutation in Hsp90, which often cooperates with Cdc37 in chaperoning protein kinases, does not affect Spc1 SAPK. These results suggest that Spc1 SAPK is a novel client protein for the Cdc37 chaperone, and the Cdc37 function is important to maintain the stability of the Spc1 protein and to facilitate stress signaling from Wis1 MAPKK to Spc1 SAPK.

Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose

The Target Of Rapamycin (TOR) is an evolutionarily conserved protein kinase that forms 2 distinct protein complexes referred to as TOR complex 1 (TORC1) and 2 (TORC2). Recent extensive studies have demonstrated that TORC1 is under the control of the small GTPases Rheb and Rag that funnel multiple input signals including those derived from nutritional sources; however, information is scarce as to the regulation of TORC2. A previous study using the model system provided by the fission yeast Schizosaccharomyces pombe identified Ryh1, a Rab-family GTPase, as an activator of TORC2. Here, we show that the nucleotide-binding state of Ryh1 is regulated in response to glucose, mediating this major nutrient signal to TORC2. In glucose-rich growth media, the GTP-bound form of Ryh1 induces TORC2-dependent phosphorylation of Gad8, a downstream target of TORC2 in fission yeast. Upon glucose deprivation, Ryh1 becomes inactive, which turns off the TORC2-Gad8 pathway. During glucose starvation, however, Gad8 phosphorylation by TORC2 gradually recovers independently of Ryh1, implying an additional TORC2 activator that is regulated negatively by glucose. The paired positive and negative regulatory mechanisms may allow fine-tuning of the TORC2-Gad8 pathway, which is essential for growth under glucose-limited environment.

Cyclodextrin complexed [60]fullerene derivatives with high levels of photodynamic activity by long wavelength excitation.

We have evaluated the photodynamic activities of C60 derivative·γ-cyclodextrin (γ-CDx) complexes and demonstrated that they were significantly higher than those of the pristine C60 and C70·γ-CDx complexes under photoirradiation at long wavelengths (610-720 nm), which represent the optimal wavelengths for photodynamic therapy (PDT). In particular, the cationic C60 derivative·γ-CDx complex had the highest photodynamic ability because the complex possessed the ability to generate high levels of (1)O2 and provided a higher level of intracellular uptake. The photodynamic activity of this complex was greater than that of photofrin, which is the most widely used of the known clinical photosensitizers. These findings therefore provide a significant level of information toward the optimization of molecular design strategies for the synthesis of fullerene derivatives for PDT.

Rab small GTPase emerges as a regulator of TOR complex 2

In diverse eukaryotic species from yeast to human, TOR (Target Of Rapamycin) protein kinase operates in signaling pathways that link extracellular stimuli to the control of cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2). While TORC1 is known to be under the control of the Ras-like small GTPase Rheb, our knowledge about TORC2 regulation is very limited. We thus set out to identify TORC2 activators through genetic approaches in the fission yeast Schizosaccharomyces pombe. Here we briefly review our study that has identified a Rab-family GTPase, Ryh1, and its GEF (guanine nucleotide exchange factor) as positive regulators of TORC2 signaling in S. pombe. Considering the evolutionary conservation of the TOR pathways, it is conceivable that Rab-family GTPases also play a role in the regulation of human TORC2 in cellular proliferation and insulin signaling.

Substrate specificity of TOR complex 2 is determined by a ubiquitin-fold domain of the Sin1 subunit

The target of rapamycin (TOR) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT. DOI: http://dx.doi.org/10.7554/eLife.19594.001

A photo-triggerable drug carrier based on cleavage of PEG lipids by photosensitiser-generated reactive singlet oxygen

To circumvent the limitations of polyethylene glycol (PEG) modified carriers, a photo-triggerable liposome was prepared which was modified by cholesterol derivatives via a cleavable vinyl ether linkage so that the PEGylated coating can be efficiently removed by a photoactivated fullerene. After the photocleavage of the PEG moiety, the intracellular uptake of the photo-triggerable liposome improved.