Multiple regulatory domains on the Byr2 protein kinase.

Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.     

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 14-3-3 proteins Rad24 and Rad25 negatively regulate Byr2 by affecting its localization in Schizosaccharomyces pombe

In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis. In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase [MAPK] kinase kinase). Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells. In contrast, the mating and sporulation efficiency of rad24- or rad25-null cells was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1(S214DT218D). Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined. The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation. Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants. In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein. Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation. Deletion of rad24 or rad25 fastened the timing of Byr2 translocation. Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.     

Solution structure of the Ras binding domain of the protein kinase Byr2 from Schizosaccharomyces pombe.

BACKGROUND: After activation, small GTPases such as Ras transfer the incoming signal to effectors by specifically interacting with the binding domain of these proteins. Structural details of the binding domain of different effectors determine which pathway is predominantly activated. Byr2 from fission yeast is a functional homolog of Raf, which is the direct downstream target of Ras in mammalians that initiates a protein kinase cascade. The amino acid sequence of Byr2's Ras binding domain is only weakly related to that of Raf, and Byr2's three-dimensional structure is unknown. RESULTS: We have solved the 3D structure of the Ras binding domain of Byr2 (Byr2RBD) from Schizosaccharomyces pombe in solution. The structure consists of three alpha helices and a mixed five-stranded beta pleated sheet arranged in the topology betabetaalphabetabetaalphabetaalpha with the first seven canonic secondary structure elements forming a ubiquitin superfold. 15N-(1)H-TROSY-HSQC spectroscopy of the complex of Byr2RBD with Ras*Mg(2+)*GppNHp reveals that the first and second beta strands and the first alpha helix of Byr2 are mainly involved in the protein-protein interaction as observed in other Ras binding domains. Although the putative interaction site of H-Ras from human and Ras1 from S. pombe are identical in sequence, binding to Byr2 leads to small but significant differences in the NMR spectra, indicating a slightly different binding mode. CONCLUSIONS: The ubiquitin superfold appears to be the general structural motif for Ras binding domains even in cases with vanishing sequence identity. However, details of the 3D structure and the interacting interface are different, thereby determining the specifity of the recognition of Ras and Ras-related proteins.     

Genes encoding farnesyl cysteine carboxyl methyltransferase in Schizosaccharomyces pombe and Xenopus laevis.

The mam4 mutation of Schizosaccharomyces pombe causes mating deficiency in h- cells but not in h+ cells. h- cells defective in mam4 do not secrete active mating pheromone M-factor. We cloned mam4 by complementation. The mam4 gene encodes a protein of 236 amino acids, with several potential membrane-spanning domains, which is 44% identical with farnesyl cysteine carboxyl methyltransferase encoded by STE14 and required for the modification of a-factor in Saccharomyces cerevisiae. Analysis of membrane fractions revealed that mam4 is responsible for the methyltransferase activity in S. pombe. Cells defective in mam4 produced farnesylated but unmethylated cysteine and small peptides but no intact M-factor. These observations strongly suggest that the mam4 gene product is farnesyl cysteine carboxyl methyltransferase that modifies M-factor. Furthermore, transcomplementation of S. pombe mam4 allowed us to isolate an apparent homolog of mam4 from Xenopus laevis (Xmam4). In addition to its sequence similarity to S. pombe mam4, the product of Xmam4 was shown to have a farnesyl cysteine carboxyl methyltransferase activity in S. pombe cells. The isolation of a vertebrate gene encoding farnesyl cysteine carboxyl methyltransferase opens the way to in-depth studies of the role of methylation in a large body of proteins, including Ras superfamily proteins.     

byr2, a Schizosaccharomyces pombe gene encoding a protein kinase capable of partial suppression of the ras1 mutant phenotype.

Schizosaccharomyces pombe contains a single gene, ras1, which is a homolog of the mammalian RAS genes. ras1 is required for conjugation, sporulation, and normal cell shape. ras1 has been previously identified as ste5. We report here a gene we call byr2 that can encode a predicted protein kinase and can partially suppress defects in ras1 mutants. ras1 mutant strains expressing high levels of byr2 can sporulate competently but are still defective in conjugation and abnormally round. byr2 mutants are viable and have normal shape but are absolutely defective in conjugation and sporulation. byr2 is probably identical to ste8. In many respects, byr2 resembles the byr1 gene, another suppressor of the ras1 mutation, which has been identified previously as ste1. Our data indicate that if ras1, byr2, and byr1 act along the same pathway, then the site of action for byr2 is between the sites for ras1 and byr1.     

The Ras-Byr2RBD complex: structural basis for Ras effector recognition in yeast.

BACKGROUND: The small GTP binding protein Ras has important roles in cellular growth and differentiation. Mutant Ras is permanently active and contributes to cancer development. In its activated form, Ras interacts with effector proteins, frequently initiating a kinase cascade. In the lower eukaryotic Schizosaccharomyces pombe, Byr2 kinase represents a Ras target that in terms of signal-transduction hierarchy can be considered a homolog of mammalian Raf-kinase. The activation mechanism of protein kinases by Ras is not understood, and there is no detailed structural information about Ras binding domains (RBDs) in nonmammalian organisms. RESULTS: The crystal structure of the Ras-Byr2RBD complex at 3 A resolution shows a complex architecture similar to that observed in mammalian homologous systems, with an interprotein beta sheet stabilized by predominantly polar interactions between the interacting components. The C-terminal half of the Ras switch I region contains most of the contact anchors, while on the Byr2 side, a number of residues from topologically distinct regions are involved in complex stabilization. A C-terminal helical segment, which is not present in the known mammalian homologous systems and which is part of the auto-inhibitory region, has an additional binding site outside the switch I region. CONCLUSIONS: The structure of the Ras-Byr2 complex confirms the Ras binding module as a communication element mediating Ras-effector interactions; the Ras-Byr2 complex is also conserved in a lower eukaryotic system like yeast, which is in contrast to other small GTPase families. The extra helical segment might be involved in kinase activation.     

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.     

Heterotrimeric G-protein subunit function in Candida albicans: both the alpha and beta subunits of the pheromone response G protein are required for mating

A pheromone-mediated signaling pathway that couples seven-transmembrane-domain (7-TMD) receptors to a mitogen-activated protein kinase module controls Candida albicans mating. 7-TMD receptors are typically connected to heterotrimeric G proteins whose activation regulates downstream effectors. Two Galpha subunits in C. albicans have been identified previously, both of which have been implicated in aspects of pheromone response. Cag1p was found to complement the mating pathway function of the pheromone receptor-coupled Galpha subunit in Saccharomyces cerevisiae, and Gpa2p was shown to have a role in the regulation of cyclic AMP signaling in C. albicans and to repress pheromone-mediated arrest. Here, we show that the disruption of CAG1 prevented mating, inactivated pheromone-mediated arrest and morphological changes, and blocked pheromone-mediated gene expression changes in opaque cells of C. albicans and that the overproduction of CAG1 suppressed the hyperactive cell cycle arrest exhibited by sst2 mutant cells. Because the disruption of the STE4 homolog constituting the only C. albicans gene for a heterotrimeric Gbeta subunit also blocked mating and pheromone response, it appears that in this fungal pathogen the Galpha and Gbeta subunits do not act antagonistically but, instead, are both required for the transmission of the mating signal.     

组蛋白H3K4甲基转移酶复合物亚基Ash2参与裂殖酵母产孢

在氮源缺乏及信息素存在的条件下,裂殖酵母(Schizosaccharomyces pombe)进行减数分裂并完成产孢。在此过程中,信息素介导的MAPK(Mitogen-activated protein kinases)信号通路调控减数分裂相关基因的表达。Spk1是MAPK通路的核心成员,通过蛋白磷酸化的方式激活转录因子Ste11,从而激活mei2⁺、mam2⁺和map3⁺等减数分裂相关基因的表达。尽管组蛋白H3K4甲基化参与基因转录激活、染色质重塑等诸多生物学过程,但其在裂殖酵母产孢过程中的作用并不清楚。文章通过序列比对,发现裂殖酵母Ash2作为H3K4甲基转移酶复合物COMPASS的亚基具有两个保守的结构域,定位于细胞核内参与H3K4的甲基化修饰。ash2⁺的缺失引起裂殖酵母在氮源缺乏时产孢过程的延迟及产孢率下降。ChIP、定量PCR分析结果显示,ash2⁺的缺失降低了spk1⁺编码区H3K4的二甲基化水平,造成spk1⁺mRNA水平的明显下调。在ash2Δ细胞中,虽然ste11⁺的转录水平没有变化,但Ste11的靶基因mei2⁺、mam2⁺和map3⁺的转录水平明显下降。在裂殖酵母中,组蛋白H3K4甲基转移酶复合物COMPASS的亚基Ash2通过调控二甲基化水平修饰从而调节MAPK信号通路,参与裂殖酵母的有性生殖,为建立表观遗传修饰与减数分裂之间的联系提供了新的线索。     

SNT1/FRS2 mediates germinal vesicle breakdown induced by an activated FGF receptor1 in Xenopus oocytes

The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from the fibroblast growth factor receptor (FGFR), which plays vital roles during embryogenesis. Activating FGFR mutations cause several craniosynostoses and dwarfism syndromes in humans. Here we show that the Xenopus homolog of mammalian FRS-2 (XFRS2) is essential for the induction of oocyte maturation by an XFGFR1 harboring an activating mutation (XFGFR1act). Using a dominant-negative form of kinase suppressor of Ras, we show the Mek activity is required for germinal vesicle breakdown (GVBD) induced by co-expression of XFGFR1act and XFRS2, but this activity is not required for progesterone-induced GVBD. Furthermore, Mek/MAPK activity is critical for the induction and/or maintenance of H1 kinase activity at metaphase of meiosis II in progesterone-treated oocytes. An activated XFGFR1 containing a mutation in the phospholipase Cgamma binding site (XFGFR1actY672F) displayed a reduced ability to induce cell-cycle progression in oocytes, suggesting phospholipase Cgamma may not be necessary but that it augments XFGFR signaling in this system. Oocytes co-expressing XFGFR1act and XFRS2 showed substantial H1 kinase activity, but this activity was blocked when the oocytes were treated with the phosphatidylinositol 3-kinase inhibitor LY294002. Although phosphatidylinositol 3-kinase activity is essential for XFGFR1act/XFRS2-induced oocyte maturation, this activity is not required for maturation induced by progesterone. Finally, ectopic expression of Xspry2, a negative regulator of XFGFR signaling, greatly reduced MAPK activation and GVBD induced by the expression of either XFGFR1act plus XFRS2 or activated Ras (H-RasV12). In contrast, Xspry2 did not prevent GVBD induced by an activated form of Raf1, suggesting that Xspry2 exerts its inhibitory function upstream or parallel to Raf and downstream of Ras.     

SAM domains can utilize similar surfaces for the formation of polymers and closed oligomers

The mitogen-activated protein kinase (MAPK) Byr2 and its activator Ste4 are involved in the mating pheromone response pathway of Schizosaccharomyces pombe and interact via their SAM domains. SAM domains can self-associate to form higher-order structures, including dimers, polymers and closed oligomers. Ste4-SAM is adjacent to a trimeric leucine zipper domain and we have shown previously that the two domains together (Ste4-LZ-SAM) bind to a monomeric Byr2-SAM with high affinity (Kd approximately 20 nM), forming a 3:1 complex. Here, we map the surfaces of Byr2-SAM and Ste4-SAM that is involved the interaction. A set of 38 mutants of Byr2-SAM and 33 mutants of Ste4-SAM were prepared, covering most of the protein surfaces. These mutants were purified and screened for binding, yielding a map of residues that are required for binding and a complementary map of residues that are not required. We find that the interface maps to regions of the SAM domains that are known to be important for the formation of SAM polymers. These results indicate that SAM domains can create a variety of oligomeric architectures utilizing common binding surfaces.     

A gene encoding a protein with seven zinc finger domains acts on the sexual differentiation pathways of Schizosaccharomyces pombe.

Byr3 was selected as a multicopy suppressor of the sporulation defects of diploid Schizosaccharomyces pombe cells that lack ras1. Like cells mutant at byr1 and byr2, two genes that encode putative protein kinases and that in multiple copies are also suppressors of the sporulation defects of ras1 null diploid cells, cells mutant at byr3 are viable but defective in conjugation. Nucleic acid sequence indicates byr3 has the capacity to encode a protein with seven zinc finger binding domains, similar in structure to the cellular nucleic acid binding protein (CNBP), a human protein that was identified on the basis of its ability to bind DNA. Expression of CNBP in yeast can partially suppress conjugation defects of cells lacking byr3.     

Two ras pathways in fission yeast are differentially regulated by two ras guanine nucleotide exchange factors

How a given Ras prreotein coordinates multiple signaling inputs and outputs is a fundamental issue of signaling specificity. Schizosaccharomyces pombe contains one Ras, Ras1, that has two distinct outputs. Ras1 activates Scd1, a presumptive guanine nucleotide exchange factor (GEF) for Cdc42, to control morphogenesis and chromosome segregation, and Byr2, a component of a mitogen-activated protein kinase cascade, to control mating. So far there is only one established Ras1 GEF, Ste6. Paradoxically, ste6 null (ste6 Delta) mutants are sterile but normal in cell morphology. This suggests that Ste6 specifically activates the Ras1-Byr2 pathway and that there is another GEF capable of activating the Scd1 pathway. We thereby characterized a potential GEF, Efc25. Genetic data place Efc25 upstream of the Ras1-Scd1, but not the Ras1-Byr2, pathway. Like ras1 Delta and scd1 Delta, efc25 Delta is synthetically lethal with a deletion in tea1, a critical element for cell polarity control. Using truncated proteins, we showed that the C-terminal GEF domain of Efc25 is essential for function and regulated by the N terminus. We conclude that Efc25 acts as a Ras1 GEF specific for the Scd1 pathway. While ste6 expression is induced during mating, efc25 expression is constitutive. Moreover, Efc25 overexpression renders cells hyperelongated and sterile; the latter can be rescued by activated Ras1. This suggests that Efc25 can recruit Ras1 to selectively activate Scd1 at the expense of Byr2. Reciprocally, Ste6 overexpression can block Scd1 activation. We propose that external signals can partly segregate two Ras1 pathways by modulating GEF expression and that GEFs can influence how Ras is coupled to specific effectors.     

A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae

MATa cells carrying an sst2 mutation are unable to recover from the G1-specific cell cycle arrest induced by the mating pheromone alpha factor. The KSS1 gene, when overexpressed, suppresses this adaptation defect. KSS1 overexpression also suppresses the recovery defect manifested by cells expressing an alpha factor receptor lacking its 136 amino acid cytoplasmic tail. Because SST2 product and the receptor tail contribute independently to events that allow recovery from pheromone-induced growth arrest, KSS1 function defines a third independent process that promotes desensitization. The KSS1 gene encodes an apparent protein kinase homologous to the CDC28 (S. cerevisiae) and cdc2+ (S. pombe) gene products. The recovery-promoting activity of the KSS1 gene requires a functional WHI1 gene, which encodes a yeast homolog to animal cyclins, suggesting that the KSS1 and WHI1 proteins act in the same growth control pathway.