A yeast STE11 homologue CoMEKK1 is essential for pathogenesis-related morphogenesis in Colletotrichum orbiculare

Several signal transduction pathways, including mitogen-activated protein kinase (MAPK) pathways, are involved in appressorium development in Colletotrichum orbiculare, the causal agent of cucumber anthracnose disease. In this study, CoMEKK1, a yeast MAPK kinases (MAPKK) kinase STE11 homolog, was identified as a disrupted gene in an Agrobacterium tumefaciens-mediated transformation mutant. The phenotype of comekk1 disruptant was similar to that of cmk1, a Saccharomyces cerevisiae Fus3/Kss1 MAPK homolog mutant. Moreover, comekk1 and cmk1 mutants were sensitive to high osmotic and salinity stresses, indicating that Comekk1p/Cmk1p signal transduction is involved in stress tolerance. The transformants of the wild type and the comekk1 mutant expressing a constitutively active form of the CoMEKK1 showed slower hyphal growth and abnormal appressorium formation, whereas those of the cmk1 disruptant did not. A Cmk1p-green fluorescent protein (GFP) intracellular localization experiment indicated that nuclear localization of the Cmk1p-GFP fusion protein induced by salt stress was diminished in comekk1 mutants. These results indicate that Comekk1p functions upstream of Cmk1p.     

Cmk2, a novel serine/threonine kinase in fission yeast

The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin-dependent protein kinase family. The cmk2(+) gene is not essential for cell viability but overexpression of cmk2(+) blocks the cell cycle at G2 phase and this inhibition is cdc2-dependent. The Cmk2 is a cytoplasmic protein expressed in a cell cycle-dependent manner, peaking at the G1/S boundary. Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.     

H₂O₂ stress-specific regulation of S. pombe MAPK Sty1 by mitochondrial protein phosphatase Ptc4

In fission yeast, the stress-activated MAP kinase, Sty1, is activated via phosphorylation upon exposure to stress and orchestrates an appropriate response. Its activity is attenuated by either serine/threonine PP2C or tyrosine phosphatases. Here, we found that the PP2C phosphatase, Ptc4, plays an important role in inactivating Sty1 specifically upon oxidative stress. Sty1 activity remains high in a ptc4 deletion mutant upon H(2)O(2) but not under other types of stress. Surprisingly, Ptc4 localizes to the mitochondria and is targeted there by an N-terminal mitochondrial targeting sequence (MTS), which is cleaved upon import. A fraction of Sty1 also localizes to the mitochondria suggesting that Ptc4 attenuates the activity of a mitochondrial pool of this MAPK. Cleavage of the Ptc4 MTS is greatly reduced specifically upon H(2)O(2), resulting in the full-length form of the phosphatase; this displays a stronger interaction with Sty1, thus suggesting a novel mechanism by which the negative regulation of MAPK signalling is controlled and providing an explanation for the oxidative stress-specific nature of the regulation of Sty1 by Ptc4.     

ROCK1 phosphorylates and activates zipper-interacting protein kinase

Zipper-interacting protein kinase (ZIPK) regulates Ca(2+)-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.     

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.     

Phosphorylation of Tyr-176 of the yeast MAPK Hog1/p38 is not vital for Hog1 biological activity

Mitogen-activated protein kinases are crucial components in the life of eukaryotic cells. The current dogma for MAPK activation is that dual phosphorylation of neighboring Thr and Tyr residues at the phosphorylation lip is an absolute requirement for their catalytic and biological activity. In this study we addressed the role of Tyr and Thr phosphorylation in the yeast MAPK Hog1/p38. Taking advantage of the recently isolated hyperactive mutants, whose intrinsic basal activity is independent of upstream regulation, we demonstrate that Tyr-176 is not required for basal catalytic and biological activity but is essential for the salt-induced amplification of Hog1 catalysis. We show that intact Thr-174 is absolutely essential for biology and catalysis of the mutants but is mainly required for structural reasons and not as a phosphoacceptor. The roles of Thr-174 and Tyr-176 in wild type Hog1 molecules were also tested. Unexpectedly we found that Hog1(Y176F) is biologically active, capable of induction of Hog1 target genes and of rescuing hog1Delta cells from osmotic stress. Hog1(Y176F) was not able, however, to mediate growth arrest induced by constitutively active MAPK kinase/Pbs2. We propose that Thr-174 is essential for stabilizing the basal active conformation, whereas Tyr-176 is not. Tyr-176 serves as a regulatory element required for stimuli-induced amplification of kinase activity.     

Regulation of s6 kinase 1 activation by phosphorylation at ser-411

Ribosomal S6 kinase 1 (S6K1), as a key regulator of mRNA translation, plays an important role in cell cycle progression through the G(1) phase of proliferating cells and in the synaptic plasticity of terminally differentiated neurons. Activation of S6K1 involves the phosphorylation of its multiple Ser/Thr residues, including the proline-directed sites (Ser-411, Ser-418, Thr-421, and Ser-424) in the autoinhibitory domain near the C terminus. Phosphorylation at Thr-389 is also a crucial event in S6K1 activation. Here, we report that S6K1 phosphorylation at Ser-411 is required for the rapamycin-sensitive phosphorylation of Thr-389 and the subsequent activation of S6K1. Mutation of Ser-411 to Ala ablated insulin-induced Thr-389 phosphorylation and S6K1 activation, whereas mutation mimicking Ser-411 phosphorylation did not show any effect. Furthermore, phosphomimetic mutation of Thr-389 overcame the inhibitory effect of the mutation S411A. Thus, Ser-411 phosphorylation regulates S6K1 activation via the control of Thr-389 phosphorylation. In nervous system neurons, Cdk5-p35 kinase associates with S6K1 via the direct interaction between p35 and S6K1 and catalyzes S6K1 phosphorylation specifically at Ser-411. Inhibition of the Cdk5 activity or suppression of Cdk5 expression blocked S6K1 phosphorylation at Ser-411 and Thr-389, resulting in S6K1 inactivation. Similar results were obtained by treating asynchronous populations of proliferating cells with the CDK inhibitor compound roscovitine. Altogether, our findings suggest a novel mechanism by which the CDK-mediated phosphorylation regulates the activation of S6K1.     

TOR signalling regulates mitotic commitment through the stress MAP kinase pathway and the Polo and Cdc2 kinases

The coupling of growth to cell cycle progression allows eukaryotic cells to divide at particular sizes depending on nutrient availability. In fission yeast, this coupling involves the Spc1/Sty1 mitogen-activated protein kinase (MAPK) pathway working through Polo kinase recruitment to the spindle pole bodies (SPBs). Here we report that changes in nutrients influence TOR signalling, which modulates Spc1/Sty1 activity. Rapamycin-induced inhibition of TOR signalling advanced mitotic onset, mimicking the reduction in cell size at division seen after shifts to poor nitrogen sources. Gcn2, an effector of TOR signalling and modulator of translation, regulates the Pyp2 phosphatase that in turn modulates Spc1/Sty1 activity. Rapamycin- or nutrient-induced stimulation of Spc1/Sty1 activity promotes Polo kinase SPB recruitment and Cdc2 activation to advance mitotic onset. This advanced mitotic onset is abolished in cells depleted of Gcn2, Pyp2, or Spc1/Sty1 or on blockage of Spc1/Sty1-dependent Polo SPB recruitment. Therefore, TOR signalling modulates mitotic onset through the stress MAPK pathway via the Pyp2 phosphatase.     

The Srk1 protein kinase is a target for the Sty1 stress-activated MAPK in fission yeast

The fission yeast stress-activated Sty1/Spc1 MAPK pathway responds to a similar range of stresses as do the mammalian p38 and SAPK/JNK MAPK pathways. In addition, sty1(-) cells are sterile and exhibit a G(2) cell cycle delay, indicating additional roles of Sty1 in meiosis and cell cycle progression. To identify novel proteins involved in stress responses, a microarray analysis of the Schizosaccharomyces pombe genome was performed to find genes that are up-regulated following exposure to stress in a Sty1-dependent manner. One such gene identified, srk1(+) (Sty1-regulated kinase 1), encodes a putative serine/threonine kinase homologous to mammalian calmodulin kinases. At the C terminus of Srk1 is a putative MAPK binding motif similar to that in the p38 substrates, MAPK-activated protein kinases 2 and 3. Indeed, we find that Srk1 is present in a complex with the Sty1 MAPK and is directly phosphorylated by Sty1. Furthermore, upon stress, Srk1 translocates from the cytoplasm to the nucleus in a process that is dependent on the Sty1 MAPK. Finally, we show that Srk1 has a role in regulating meiosis in fission yeast; following nitrogen limitation, srk1(-) cells enter meiosis significantly faster than wild-type cells and overexpression of srk1(+) inhibits the nitrogen starvation-induced arrest in G(1).