Analysis of mitogen-activated protein kinase signaling specificity in response to hyperosmotic stress: use of an analog-sensitive HOG1 allele

When confronted with a marked increase in external osmolarity, budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high-osmolarity glycerol or HOG pathway) to elicit cellular responses necessary to permit continued growth. One input that stimulates the HOG pathway requires the integral membrane protein and putative osmosensor Sho1, which recruits and enables activation of the MAPK kinase kinase Ste11. In mutants that lack the downstream MAPK kinase (pbs2Delta) or the MAPK (hog1Delta) of the HOG pathway, Ste11 activated by hyperosmotic stress is able to inappropriately stimulate the pheromone response pathway. This loss of signaling specificity is known as cross talk. To determine whether it is the Hog1 polypeptide per se or its kinase activity that is necessary to prevent cross talk, we constructed a fully functional analog-sensitive allele of HOG1 to permit acute inhibition of this enzyme without other detectable perturbations of the cell. We found that the catalytic activity of Hog1 is required continuously to prevent cross talk between the HOG pathway and both the pheromone response and invasive growth pathways. Moreover, contrary to previous reports, we found that the kinase activity of Hog1 is necessary for its stress-induced nuclear import. Finally, our results demonstrate a role for active Hog1 in maintaining signaling specificity under conditions of persistently high external osmolarity.     

Phosphoproteomic analyses reveal novel cross‐modulation mechanisms between two signaling pathways in yeast

Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen‐activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time‐resolved phosphorylation site dynamics after pathway co‐stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone‐induced down‐regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways.     

Mechanisms regulating Raf-1 activity in signal transduction pathways

Raf-1 is a key protein involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Biochemical and genetic studies have demonstrated that Raf-1 functions downstream of activated tyrosine kinases and Ras and upstream of mitogen-activated protein kinase (MAPK) and MAPK kinase (MKK or MEK) in many signaling pathways. A major objective of our laboratory has been to determine how Raf-1 becomes activated in response to signaling events. Using mammalian, baculovirus, and Xenopus systems, we have examined the roles that phosphorylation and protein-protein interactions play in regulating the biological and biochemical activity of Raf-1. Our studies have provided evidence that the activity of Raf-1 can be modulated by both Ras-dependent and Ras-independent pathways. Recently, we reported that Arg89 of Raf-1 is a residue required for the association of Raf-1 and Ras. Mutation of this residue disrupted interaction with Ras and prevented Ras-mediated, but not protein kinase C-or tyrosine kinase-mediated, enzymatic activation of Raf-1 in the baculovirus expression system. Further analysis of this mutant demonstrated that kinase-defective Raf-1 proteins interfere with the propagation of proliferative and developmental signals by binding to Ras and blocking Ras function. Our findings have also shown that phosphorylation events play a role in regulating Raf-1. We have identified sites of in vivo phosphorylation that positively and negatively alter the biological and enzymatic activity of Raf-1. In addition, we have found that some of these phosphorylation sites are involved in mediating the interaction of Raf-1 with potential activators (Fyn and Src) and with other cellular proteins (14-3-3). Results from our work suggest that Raf-1 is regulated at multiple levels by several distinct mechanisms. © 1995 wiley-Liss, Inc.     

Complementation and characterization of the Pneumocystis carinii MAPK,PCM

Mitogen-activated protein kinase (MAPK) pathways transfer environmental signals into intracellular events such as proliferation and differentiation. Fungi utilize a specific pheromone-induced MAPK pathway to regulate conjugation, formation of an ascus, and entry into meiosis. We have previously identified a MAPK, PCM, from the fungal opportunist Pneumocystis, responsible for causing severe pneumonia in patients with AIDS. In order to gain insight into the function of PCM, we expressed it in Saccharomyces cerevisiae deficient in pheromone signaling and tested activation and inhibition of this MAPK pathway. PCM restored pheromone signaling in S. cerevisiae fus3Delta kss1Delta mutants with alpha-factor pheromone (six-fold increase) and was not activated by osmotic stress. Signaling through this pathway decreased 2.5-fold with 10 microM U0126, and was unaffected with SB203580. We evaluated the conditions for native PCM kinase activity isolated from Pneumocystis carinii organisms and found that 0.1 mM MgCl2, pH 6.5, temperature 30-35 degrees C, and 10 microM ATP were optimal. The activity of PCM is significantly elevated in P. carinii trophic forms compared to cysts, implicating a role for PCM in the life cycle transition of P. carinii from trophic forms to cysts.     

Hck is a key regulator of gene expression in alternatively activated human monocytes

IL-13 is a Th2 cytokine that promotes alternative activation (M2 polarization) in primary human monocytes. Our studies have characterized the functional IL-13 receptor complex and the downstream signaling events in response to IL-13 stimulation in alternatively activated monocytes/macrophages. In this report, we present evidence that IL-13 induces the activation of a Src family tyrosine kinase, which is required for IL-13 induction of M2 gene expression, including 15-lipoxygenase (15-LO). Our data show that Src kinase activity regulates IL-13-induced p38 MAPK tyrosine phosphorylation via the upstream kinases MKK3 or MKK6. Our findings also reveal that the IL-13 receptor-associated tyrosine kinase Jak2 is required for the activation of both Src kinase as well as p38 MAPK. Further, we found that Src tyrosine kinase-mediated activation of p38 MAPK is required for Stat1 and Stat3 serine 727 phosphorylation in alternatively activated monocytes/macrophages. Additional studies identify Hck as the specific Src family member, stimulated by IL-13 and involved in regulating both p38 MAPK activation and p38 MAPK-mediated 15-LO expression. Finally we show that the Hck regulates the expression of other alternative state (M2)-specific genes (Mannose receptor, MAO-A, and CD36) and therefore conclude that Hck acts as a key regulator controlling gene expression in alternatively activated monocytes/macrophages.     

Dynamic Phosphoproteome Profiling of Zebrafish Embryonic Fibroblasts during Cold Acclimation

Temperature affects almost all aspects of the fish life. To cope with low temperature, fish have evolved the ability of cold acclimation for survival. However, intracellular signaling events underlying cold acclimation in fish remain largely unknown. Here, the formation of cold acclimation in zebrafish embryonic fibroblasts (ZF4) is monitored and the phosphorylation events during the process are investigated through a large‐scale quantitative phosphoproteomic approach. In total, 11 474 phosphorylation sites are identified on 4066 proteins and quantified 5772 phosphosites on 2519 proteins. Serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation accounted for 85.5%, 13.3%, and 1.2% of total phosphosites, respectively. Among all phosphosites, 702 phosphosites on 510 proteins show differential regulation during cold acclimation of ZF4 cells. These phosphosites are divided into six clusters according to their dynamic changes during cold exposure. Kinase–substrate prediction reveals that mitogen‐activated protein kinase (MAPK) among the kinase groups is predominantly responsible for phosphorylation of these phosphosites. The differentially regulated phosphoproteins are functionally associated with various cellular processes such as regulation of actin cytoskeleton and MAPK signaling pathway. These data enrich the database of protein phosphorylation sites in zebrafish and provide key clues for the elucidation of intracellular signaling networks during cold acclimation of fish.     

Activated mutants of SHP-2 preferentially induce elongation of Xenopus animal caps

In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Previously, using a dominant negative mutant approach, we showed that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, we now have generated novel, activated mutants of SHP-2. Here, we show that expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. Our results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. Our activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems.     

Analysis of dual phosphorylation of Hog1 MAP kinase in Saccharomyces cerevisiae using quantitative mass spectrometry

The mitogen-activated protein kinase (MAPK) signaling pathway is activated in response to extracellular stimuli and regulates various activities in eukaryotic cells. Following exposure to stimuli, MAPK is known to be activated via dual phosphorylation at a conserved TxY motif in the activation loop; both threonine and tyrosine residues are phosphorylated by an upstream kinase. However, the mechanism underlying dual phosphorylation is not clearly understood. In the budding yeast Saccharomyces cerevisiae, the Hog1 MAPK mediates the high-osmolarity glycerol (HOG) signaling pathway. Tandem mass spectrometry and phosphospecific immunoblotting were performed to quantitatively monitor the dynamic changes occurring in the phosphorylation status of the TxY motif of Hog1 on exposure to osmotic stress. The results of our study suggest that the tyrosine residue is preferentially and dynamically phosphorylated following stimulation, and this in turn leads to the dual phosphorylation. The tyrosine residue was hyperphosphorylated in the absence of a threonine residue; this result suggests that the threonine residue is critical for the control of signaling noise and adaptation to osmotic stress.     

Quantitative profiling of dual phosphorylation of Fus3 MAP kinase in Saccharomyces cerevisiae

Mitogen-activated protein kinase (MAPK) signaling is a crucial component of eukaryotic cells; it plays an important role in responses to extracellular stimuli and in the regulation of various cellular activities. The signaling cascade is evolutionarily conserved in the eukaryotic kingdom from yeast to human. In response to a variety of extracellular signals, MAPK activity is known to be regulated via phosphorylation of a conserved TxY motif at the activation loop in which both threonine and tyrosine residues are phosphorylated by the upstream kinase. However, the mechanism by which both residues are phosphorylated continues to remain elusive. In the budding yeast, Saccharomyces cerevisiae, Fus3 MAPK is involved in the mating signaling pathway. In order to elucidate the functional mechanism of MAPK activation, we quantitatively profiled phosphorylation of the TxY motif in Fus3 using mass spectrometry (MS). We used synthetic heavy stable isotope-labeled phosphopeptides and nonphosphopeptides corresponding to the proteolytic TxY motif of Fus3 and accompanying data-dependent tandem MS to quantitatively monitor dynamic changes in the phosphorylation events of MAPK. Phosphospecific immunoblotting and the MS data suggested that the tyrosine residue is dynamically phosphorylated upon stimulation and that this leads to dual phosphorylation. In contrast, the magnitude of threonine phosphorylation did not change significantly. However, the absence of a threonine residue leads to hyperphosphorylation of the tyrosine residue in the unstimulated condition, suggesting that the threonine residue contributes to the control of signaling noise.     

Pheromone-induced phosphorylation of a G protein beta subunit in S. cerevisiae is associated with an adaptive response to mating pheromone

The mating pheromone response in S. cerevisiae is activated by a G protein-mediated signaling pathway in which G beta gamma is the active transducer of the signal. When exogenous pheromone is added to vegetatively growing cells, G beta is rapidly phosphorylated at several sites; phosphorylation does not require de novo protein synthesis. A mutation in G beta was constructed that eliminates signal-induced phosphorylation. This mutation leads to enhanced sensitivity to and impaired ability to recover from pheromone, but does not affect the ability of G beta gamma to transmit the mating signal. These phenotypes suggest that G protein phosphorylation mediates an adaptive response to pheromone-induced signaling. G beta phosphorylation does not require either the pheromone receptor C-terminus or the product of the SST2 gene, both of which mediate separate adaptive responses to pheromone. However, G beta phosphorylation is greatly facilitated by the presence of the G alpha subunit, which has also been shown to participate in an adaptation to pheromone.     

Effect of the MAPK cascade structure, nuclear translocation and regulation of transcription factors on gene expression

The mitogen activated protein kinase (MAP kinase) cascade system represents a highly conserved prototype of signal transduction by enzyme cascades. One of the best-studied properties of the MAPK system is its ability to convert graded input stimulus to switch-like all-or-none responses. Previous theoretical studies have centered on quantifying dual phosphorylated MAPK as a final output response and have not incorporated its influence on the regulation of gene expression. The main objective of the current work is to understand the regulatory effect of positive feedback loop embedded in the MAPK cascade, nuclear translocation of active MAPK, phosphorylation and activation of nuclear target proteins on the regulation of specific gene expression. To achieve this objective, we have simulated the MAPK cascade system, which resembles Hog1p activation pathway in yeast, at steady state. Thus, the input signal to the MAPK system is correlated with gene expression as a final system-level output response. The steady state simulation results suggest that other than regulating the signal propagation through cascades, the nuclear translocation of activated MAPK and subsequent regulation of gene expression represent one of the key modes to control the threshold level of response. This work proposes that, it is essential to consider the compartmental distributions of signaling species and the corresponding regulatory mechanisms of gene expression to study the system-level performance of signaling modules such as the MAPK cascade. Such an analysis will relate the extracellular cues to the final phenotypic response by capturing the mechanistic details of the signaling pathway.     

Integrated analysis of co-expressed MAP kinase substrates in Arabidopsis thaliana

MAP kinase (MAPK) signal transduction cascades are conserved eukaryotic pathways that modulate stress responses and developmental processes. In a recent report we have identified novel Arabidopsis MAPKK/MAPK/Substrate signaling pathways using microarrays containing 2,158 unique Arabidopsis proteins. Subsequently, several WRKY and TGA targets phosphorylated by MAPKs were verified in planta. We have also reported that specific MAPKK/MAPK modules expressed in Nicotiana benthamiana induced a cell death phenotype related to the immune response. We have generated a MAPK phosphorylation network based on our protein microarray experimental data. Here we further analyze our network by integrating phosphorylation and gene expression information to identify biologically relevant signaling modules. We have identified 108 phosphorylation events that occur among 96 annotated genes with highly similar pairwise expression profiles. Our analysis brings a new perspective on MAPK signaling by revealing new relationships between components of signaling pathways.Key words: MAPK, protein microarray, network, cell death, co-expression, signaling     

Oscillatory phosphorylation of yeast Fus3 MAP kinase controls periodic gene expression and morphogenesis

Signal-transduction networks can display complex dynamic behavior such as oscillations in the activity of key components [1-6], but it is often unclear whether such dynamic complexity is actually important for the network's regulatory functions [7, 8]. Here, we found that the mitogen-activated protein kinase (MAPK) Fus3, a key regulator of the yeast mating-pheromone response, undergoes sustained oscillations in its phosphorylation and activation state during continuous pheromone exposure. These MAPK activity oscillations led to corresponding oscillations in mating-gene expression. Oscillations in MAPK activity and gene expression required the negative regulator of G protein signaling Sst2 and partially required the MAPK phosphatase Msg5. Peaks in Fus3 activation correlated with periodic rounds of cell morphogenesis, with each peak preceding the formation of an additional mating projection. Preventing projection formation did not eliminate MAPK oscillation, but preventing MAPK oscillation blocked the formation of additional projections. A mathematical model was developed that reproduced several features of the observed oscillatory dynamics. These observations demonstrate a role for MAPK activity oscillation in driving a periodic downstream response and explain how the pheromone signaling pathway, previously thought to desensitize after 1-3 hr, controls morphology changes that continue for a much longer time.     

Checkpoints in a yeast differentiation pathway coordinate signaling during hyperosmotic stress

All eukaryotes have the ability to detect and respond to environmental and hormonal signals. In many cases these signals evoke cellular changes that are incompatible and must therefore be orchestrated by the responding cell. In the yeast Saccharomyces cerevisiae, hyperosmotic stress and mating pheromones initiate signaling cascades that each terminate with a MAP kinase, Hog1 and Fus3, respectively. Despite sharing components, these pathways are initiated by distinct inputs and produce distinct cellular behaviors. To understand how these responses are coordinated, we monitored the pheromone response during hyperosmotic conditions. We show that hyperosmotic stress limits pheromone signaling in at least three ways. First, stress delays the expression of pheromone-induced genes. Second, stress promotes the phosphorylation of a protein kinase, Rck2, and thereby inhibits pheromone-induced protein translation. Third, stress promotes the phosphorylation of a shared pathway component, Ste50, and thereby dampens pheromone-induced MAPK activation. Whereas all three mechanisms are dependent on an increase in osmolarity, only the phosphorylation events require Hog1. These findings reveal how an environmental stress signal is able to postpone responsiveness to a competing differentiation signal, by acting on multiple pathway components, in a coordinated manner.