Dynamics and organization of MAP kinase signal pathways

In the budding yeast, Saccharomyces cerevisiae, four separate but structurally related mitogen-activated protein kinase (MAPK) activation pathways are known. The best understood of these regulates mating. Pheromone binding to receptor informs cells of the proximity of a mating partner and induces differentiation to a mating competent state. The MARK activation cascade mediating this signal is made up of Ste 11 (a MEK kinase [MEKK]), Ste7 (a MAPK/ERK kinase [MEK]), and the redundant MAPK-related Fus3 and Kss1 enzymes. Another MAPK activation pathway is important for cell integrity and regulates cell wall construction. This cascade consists of Bck1 (a MEKK), the redundant Mkk1 and Mkk2 enzymes (MEKs), and Mpk1 (a MAPK). We exploited these two pathways to learn about the coordination and signal transmission fidelity of MAPK activation cascades. Two lines of evidence suggest that the activities of the mating and cell integrity pathways are coordinated during mating differentiation. First, cells deficient in Mpk1 are susceptible to lysis when they make a mating projection in response to pheromone. Second, Mpk1 activation during pheromone induction coincides with projection formation. The mechanism underlying this coordination is still unknown to us. Our working model is that projection formation generates a mobile second messenger for activation of the cell integrity pathway. Analysis of a STE7 mutation gave us some unanticipated but important insights into parameters important for fidelity of signal transmission. The Ste7 variant has a serine to proline substitution at position 368. Ste7-P³⁶⁸ has higher basal activity than the wild-type enzyme but still requires Ste 11 for its function. Additionally, the proline substitution enables the variant to transmit the signal from mammalian Raf expressed in yeast. This novel activity suggests that Ste7-P³⁶⁸ is inherently more permissive than Ste7 in its interactions with MEKKs. Yet, Ste7-P³⁶⁸ cross function in the cell integrity pathway occurs only when it is highly overproduced or when Ste5 is missing. This behavior suggests that Ste5, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7 specificity and fidelity of signal transmission. © 1995 wiley-Liss, Inc.     

When expressed in yeast, mammalian mitogen-activated protein kinases lose proper regulation and become spontaneously phosphorylated

MAPKs (mitogen-activated protein kinases) are key components in cell signalling pathways. Under optimal growth conditions, their activity is kept off, but in response to stimulation it is dramatically evoked. Because of the high degree of evolutionary conservation at the levels of sequence and mode of activation, MAPKs are believed to share similar regulatory mechanisms in all eukaryotes and to be functionally substitutable between them. To assess the reliability of this notion, we systematically analysed the activity, regulation and phenotypic effects of mammalian MAPKs in yeast. Unexpectedly, all mammalian MAPKs tested were spontaneously phosphorylated in yeast. JNKs (c-Jun N-terminal kinases) lost their phosphorylation in pbs2Delta cells, but p38s and ERKs (extracellular-signal-regulated kinases) maintained their spontaneous phosphorylation even in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Kinase-dead variants of ERKs and p38s were phosphorylated in strains lacking a single MEK (MAPK/ERK kinase), but not in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Thus, in yeast, p38 and ERKs are phosphorylated via a combined mechanism of autophosphorylation and MEK-mediated phosphorylation (any MEK). We further addressed the mechanism allowing mammalian MAPKs to exploit yeast MEKs in the absence of any activating signal. We suggest that mammalian MAPKs lost during evolution a C-terminal region that exists in some yeast MAPKs. Indeed, removal of this region from Hog1 and Mpk1 rendered them spontaneously and highly phosphorylated. It implies that MAPKs possess an efficient inherent autoposphorylation capability that is suppressed in yeast MAPKs via a C-terminal domain and in mammalian MAPKs via as yet unknown means.     

Ubiquitin‐dependent regulation of MEKK2/3‐MEK5‐ERK5 signaling module by XIAP and cIAP1

Mitogen‐activated protein kinases (MAPKs) are highly conserved protein kinase modules, and they control fundamental cellular processes. While the activation of MAPKs has been well studied, little is known on the mechanisms driving their inactivation. Here we uncover a role for ubiquitination in the inactivation of a MAPK module. Extracellular‐signal‐regulated kinase 5 (ERK5) is a unique, conserved member of the MAPK family and is activated in response to various stimuli through a three‐tier cascade constituting MEK5 and MEKK2/3. We reveal an unexpected role for Inhibitors of Apoptosis Proteins (IAPs) in the inactivation of ERK5 pathway in a bimodal manner involving direct interaction and ubiquitination. XIAP directly interacts with MEKK2/3 and competes with PB1 domain‐mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63‐linked ubiquitin chains to MEKK2 and MEKK3 which directly impede MEK5–ERK5 interaction in a trimeric complex leading to ERK5 inactivation. Consistently, loss of XIAP or cIAP1 by various strategies leads to hyperactivation of ERK5 in normal and tumorigenic cells. Loss of XIAP promotes differentiation of human primary skeletal myoblasts to myocytes in a MEKK2/3‐ERK5‐dependent manner. Our results reveal a novel, obligatory role for IAPs and ubiquitination in the physical and functional disassembly of ERK5‐MAPK module and human muscle cell differentiation.     

Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes

Cellular and genetic approaches were used to investigate the requirements for activation during spermatogenesis of the extracellular signal-regulated protein kinases (ERKs), more commonly known as the mitogen-activated protein kinases (MAPKs). The MAPKS and their activating kinases, the MEKs, are expressed in specific developmental patterns. The MAPKs and MEK2 are expressed in all premeiotic germ cells and spermatocytes, while MEK1 is not expressed abundantly in pachytene spermatocytes. Phosphorylated (active) variants of these kinases are diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid (OA), to induce transition from meiotic prophase to metaphase I (G2/MI), resulted in phosphorylation and enzymatic activation of ERK1/2. However, U0126, an inhibitor of the ERK-activating kinases, MEK1/2, did not inhibit OA-induced MAPK activation or chromosome condensation. Analysis of spermatocytes lacking MOS, a mitogen-activated protein kinase kinase kinase responsible for MEK and MAPK activation, revealed that MOS is not required for OA-induced activation of the MAPKs. OA-induced MAPK activation was inhibited by butyrolactone I, an inhibitor of cyclin-dependent kinases 1 and 2 (CDK1, CDK2); thus, these kinases may regulate MAPK activity. Additionally, spermatocytes lacking CDC25C condensed bivalent chromosomes and activated both MPF and MAPKs in response to OA treatment; therefore, there is a CDC25C-independent pathway for MPF and MAPK activation. These studies reveal that spermatocytes do not require either MOS or CDC25C for onset of the meiotic division phase or for activation of MPF and the MAPKs, thus implicating a novel pathway for activation of the ERK1/2 MAPKs in spermatocytes.     

Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7.

Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae. To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in the absence of any additional yeast proteins. These complexes were specific because neither Hog1 nor Mpk1 (two other yeast MAPKs), nor mammalian Erk2, was able to associate detectably with Ste7. Neither the kinase catalytic core of Ste7 nor the phosphoacceptor regions of Ste7 and Kss1 were necessary for complex formation. Ste7-Kss1 (and Ste7-Fus3) complexes were present in yeast cell extracts and were undiminished in extracts prepared from a ste5delta-ste11delta double mutant strain. In Ste7-Kss1 (or Ste7-Fus3) complexes isolated from naive or pheromone-treated cells, Ste7 phosphorylated Kss1 (or Fus3), and Kss1 (or Fus3) phosphorylated Ste7, in a pheromone-stimulated manner; dissociation of the high-affinity complex was shown to be required for either phosphorylation event. Deletions of Ste7 in the region required for its stable association with Kss1 and Fus3 in vitro significantly decreased (but did not eliminate) signaling in vivo. These findings suggest that the high-affinity and active site-independent binding observed in vitro facilitates signal transduction in vivo and suggest further that MEK-MAPK interactions may utilize a double-selection mechanism to ensure fidelity in signal transmission and to insulate one signaling pathway from another.     

Creation of a stress-activated p90 ribosomal S6 kinase. The carboxyl-terminal tail of the MAPK-activated protein kinases dictates the signal transduction pathway in which they function

Mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) lie immediately downstream of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38 MAPK. Although the family of MAPKAPKs shares sequence similarity, it demonstrates selectivity for the upstream activator. Here we demonstrate that each of the ERK- and p38 MAPK-regulated MAPKAPKs contains a MAPK docking site positioned distally to the residue(s) phosphorylated by MAPKs. The isolated MAPK docking sites show specificity for the upstream activator similar to that reported for the full-length proteins. Moreover, replacement of the ERK docking site of p90 ribosomal S6 kinase with the p38 MAPK docking site of MAPKAPK2 converts p90 ribosomal S6 kinase into a stress-activated kinase in vivo. It is apparent that mechanisms controlling events downstream of the proline-directed MAPKs involve specific MAPK docking sites within the carboxyl termini of the MAPKAPKs that determine the cascade in which the MAPKAPK functions.     

A docking site determining specificity of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules composed of three sequentially activated kinases (MAPKKK, MAPKK and MAPK). Because individual cells contain multiple MAPK cascades, mechanisms are required to ensure the fidelity of signal transmission. In yeast, external high osmolarity activates the HOG (high osmolarity glycerol) MAPK pathway, which consists of two upstream branches (SHO1 and SLN1) and common downstream elements including the Pbs2 MAPKK and the Hog1 MAPK. The Ssk2/Ssk22 MAPKKKs in the SLN1 branch, when activated, exclusively phosphorylate the Pbs2 MAPKK. We found that this was due to an Ssk2/Ssk22-specific docking site in the Pbs2 N-terminal region. The Pbs2 docking site constitutively bound the Ssk2/Ssk22 kinase domain. Docking site mutations drastically reduced the Pbs2-Ssk2/Ssk22 interaction and hampered Hog1 activation by the SLN1 branch. Fusion of the Pbs2 docking site to a different MAPKK, Ste7, allowed phosphorylation of Ste7 by Ssk2/Ssk22. Thus, the docking site contributes to both the efficiency and specificity of signaling. During these analyses, we also found a nuclear export signal and a possible nuclear localization signal in Pbs2.     

Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1

Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2 small middle dotMEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding alpha-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding alpha-helix G in the interaction of ERK2 with MEK1/2.     

How does the G protein,Gi2, transduce mitogenic signals?

Serpentine receptors coupled to the heterotrimeric G protein, G_i2, are capable of stimulating DNA synthesis in a variety of cell types. A common feature of the G_i2-coupled stimulation of DNA synthesis is the activation of the mitogen-activated protein kinases (MAPKs). The regulation of MAPK activation by the G_i2-coupled thrombin and acetylcholine muscarinic M₂ receptors occurs by a sequential activation of a network of protein kinases. The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation. MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK). Thus, Raf and MEKK converge at MEK to regulate MAPK. G_i2-coupled receptors are capable of activating MEK and MAPK by Raf-dependent and Raf-independent mechanisms. Pertussis toxin catalyzed ADP-ribosylation of α_i2 inhibits both the Raf-dependent and-independent pathways activated by G_i2-coupled receptors. The Raf-dependent pathway involves Ras activation, while the Raf-independent activation of MEK and MAPK does not involve Ras. The Raf-independent activation of MEK and MAPK most likely involves the activation of MEKK. The vertebrate MEKK is homologous to the Ste11 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The yeast Ste11 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins. MEKK appears to be conserved in the regulation of G protein-coupled signal pathways in yeast and vertebrates. Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system. Defining MEKK and Raf as a divergence in the MAPK regulatory network provides a mechanism for differential regulation of this system by G_i2-coupled receptors as well as other receptor systems, including the tyrosine kinases.     

Identification of Ste4 as a potential regulator of Byr2 in the sexual response pathway of Schizosaccharomyces pombe.

A conserved MAP kinase cascade is central to signal transduction in both simple and complex eukaryotes. In the yeast Schizosaccharomyces pombe, Byr2, a homolog of mammalian MAPK/ERK kinase kinase and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation. A screen for S. pombe proteins that interact with Byr2 in a two-hybrid system led to the isolation of Ste4, a protein that is known to be required for sexual function. Ste4 binds to the regulatory region of Byr2. This binding site is separable from the binding site for Ras1. Both Ste4 and Ras1 act upstream of Byr2 and act at least partially independently. Ste4 contains a leucine zipper and is capable of homotypic interaction. Ste4 has regions of homology with STE50, an S. cerevisiae protein required for sexual differentiation that we show can bind to STE11.     

The role of docking interactions in mediating signaling input, output, and discrimination in the yeast MAPK network

Cells use a network of mitogen-activated protein kinases (MAPKs) to coordinate responses to diverse extracellular signals. Here, we examine the role of docking interactions in determining connectivity of the yeast MAPKs Fus3 and Kss1. These closely related kinases are activated by the common upstream MAPK kinase Ste7 yet generate distinct output responses, mating and filamentous growth, respectively. We find that docking interactions are necessary for communication with the kinases and that they can encode subtle differences in pathway-specific input and output. The cell cycle arrest mediator Far1, a mating-specific substrate, has a docking motif that selectively binds Fus3. In contrast, the shared partner Ste7 has a promiscuous motif that binds both Fus3 and Kss1. Structural analysis reveals that Fus3 interacts with specific and promiscuous peptides in conformationally distinct modes. Induced fit recognition may allow docking peptides to achieve discrimination by exploiting subtle differences in kinase flexibility.     

Yeast MEK-dependent signal transduction: response thresholds and parameters affecting fidelity.

Ste7p and Mkk1p are MEK (MAPK/ERK kinase) family members that function in the mating and cell integrity signal transduction pathways in Saccharomyces cerevisiae. We selected STE7 and MKK1 mutations that stimulated their respective pathways in the absence of an inductive signal. Strikingly, serine-to-proline substitutions at analogous positions in Ste7p (position 368) and Mkk1p (position 386) were recovered by independent genetic screens. Such an outcome suggests that this substitution in other MEKs would exhibit similar properties. The Ste7p-P368 variant has higher basal enzymatic activity than Ste7p but still requires induction to reach full activation. The higher activity associated with Ste7p-P368 allows it to compensate for defects in the cell integrity pathway, but it does so only when it is overproduced or when Ste5p is missing. This behavior suggests that Ste5p, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7p specificity.     

赤散囊菌MAPKs超家族的鉴定及分析

本研究以赤散囊菌Eurotium rubrum全基因组序列为对象,利用HMMER软件构建隐马尔可夫模型（hidden markov models,HMM）结合BLAST的方法鉴定了促分裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）超家族。通过构建系统发育树对鉴定蛋白进行分析,并利用MEME软件进行了保守性基序的预测及活性位点注释。分析结果表明,赤散囊菌基因组包含了4个MAPK蛋白,分别属于Hog1-type、MpkC-type、Slt2-type和Fus3/Kss1-type类型;3个MAPK kinase（MAPKK）蛋白,分别属于MKK1-type、Pbs2-type和Ste7-type类型;3个MAPK kinase kinase（MAPKKK）蛋白,分别属于BCK1-type、Ste11-type和Ssk22-type类型。保守性基序分析及注释结果表明,MAPKs超家族蛋白都包含了蛋白激酶活性位点“-D[L/I/V]K-”以及保守性的ATP-binding标签序列。MAPK与MAPKK蛋白分别包含了“-TxY-”和“-SD[I/V]WS-”磷酸化位点,且MAPK蛋白还包含一个保守性的common docking基序（CD motif）,而MAPKKK蛋白则包含了一个功能不明的保守性基序,其一致性序列为“-GTPYWMAPEV-”。研究结果为揭示MAPKs信号途径在赤散囊菌中参与调控的生物学过程奠定了基础。