Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

RASSF1A disrupts the NOTCH signaling axis via SNURF/RNF4-mediated ubiquitination of HES1.

RASSF1A promoter methylation has been correlated with tumor dedifferentiation and aggressive oncogenic behavior. Nevertheless, the underlying mechanism of RASSF1A-dependent tumor dedifferentiation remains elusive. Here, we show that RASSF1A directly uncouples the NOTCH-HES1 axis, a key suppressor of differentiation. Interestingly, the crosstalk of RASSF1A with HES1 occurs independently from the signaling route connecting RASSF1A with the Hippo pathway. At the molecular level, we demonstrate that RASSF1A acts as a scaffold essential for the SUMO-targeted E3 ligase SNURF/RNF4 to target HES1 for degradation. The reciprocal relationship between RASSF1A and HES1 is evident across a wide range of human tumors, highlighting the clinical significance of the identified pathway. We show that HES1 upregulation in a RASSF1A-depleted environment renders cells non-responsive to the downstream effects of γ-secretase inhibitors (GSIs) which restrict signaling at the level of the NOTCH receptor. Taken together, we report a mechanism through which RASSF1A exerts autonomous regulation of the critical Notch effector HES1, thus classifying RASSF1A expression as an integral determinant of the clinical effectiveness of Notch inhibitors.

The Complex Interplay of TGF-ß and Notch Signaling in the Pathogenesis of Fibrosis.

Fibrosis is a major medical challenge, as it leads to irreversible tissue remodeling and organ dysfunction. Its progression contributes significantly to morbidity and mortality worldwide, with limited therapeutic options available. Extensive research on the molecular mechanisms of fibrosis has revealed numerous factors and signaling pathways involved. However, the interactions between these pathways remain unclear. A comprehensive understanding of the entire signaling network that drives fibrosis is still missing. The TGF-ß and Notch signaling pathways play a key role in fibrogenesis, and this review focuses on their functional interplay and molecular mechanisms. Studies have shown synergy between TGF-ß and Notch cascades in fibrosis, but antagonistic interactions can also occur, especially in cardiac fibrosis. The molecular mechanisms of these interactions vary depending on the cell context. Understanding these complex and context-dependent interactions is crucial for developing effective strategies for treating fibrosis.

Phytochrome-interacting factor PIF3 integrates phytochrome B and UV-B signaling pathways to regulate gibberellin- and auxin-dependent growth in cucumber hypocotyls.

In Arabidopsis, the photoreceptors phytochrome B (PhyB) and UV-B resistance 8 (UVR8) mediate light responses that play a major role in regulating photomorphogenic hypocotyl growth, but how they crosstalk to coordinate this process is not well understood. Here we report map-based cloning and functional characterization of an ultraviolet (UV)-B-insensitive, long-hypocotyl mutant, lh1, and a wild-type-like mutant, lh2, in cucumber (Cucumis sativus), which show defective CsPhyB and GA oxidase2 (CsGA20ox-2), a key gibberellic acid (GA) biosynthesis enzyme, respectively. The lh2 mutation was epistatic to lh1 and partly suppressed the long-hypocotyl phenotype in the lh1lh2 double mutant. We identified phytochrome interacting factor (PIF) CsPIF3 as playing a critical role in integrating the red/far-red and UV-B light responses for hypocotyl growth. We show that two modules, CsPhyB-CsPIF3-CsGA20ox-2-DELLA and CsPIF3-auxin response factor 18 (CsARF18), mediate CsPhyB-regulated hypocotyl elongation through GA and auxin pathways, respectively, in which CsPIF3 binds to the G/E-box motifs in the promoters of CsGA20ox-2 and CsARF18 to regulate their expression. We also identified a new physical interaction between CsPIF3 and CsUVR8 mediating CsPhyB-dependent, UV-B-induced hypocotyl growth inhibition. Our work suggests that hypocotyl growth in cucumber involves a complex interplay of multiple photoreceptor- and phytohormone-mediated signaling pathways that show both conservation with and divergence from those in Arabidopsis.

Tiny Drosophila intestinal stem cells, big power.

The signaling pathways are highly conserved between Drosophila and mammals concerning intestinal development, regeneration, and disease. The powerful genetic tools of Drosophila make it a valuable and convenient alternative to answer basic biological questions that can not be addressed using mammalian models. In this review, we discuss recent advances in how we use fly midgut to answer the following key questions: (1) How intestine stem cell niches are established; (2) which factors control asymmetric division of stem cells; (3) how intestinal cells interact with environmental factors, such as tissue damage, microbiota, and diet; (4) how to screen aging/cancer-related factors or drugs by fly intestine stem cells.

A tale of two players: the role of phosphate in iron and zinc homeostatic interactions.

MAIN CONCLUSION: This minireview details the impact of iron-phosphate and zinc-phosphate interactions in plants and provides perspectives for further areas of research regarding nutrient homeostasis. Iron (Fe) and zinc (Zn) are among the most important micronutrients for plant growth and have numerous implications for human health and agriculture. While plants have developed efficient uptake and transport mechanisms for Fe and Zn, emerging research has shown that the availability of other nutrients in the environment influences the homeostasis of Fe and Zn within plants. In this minireview, we present the current knowledge regarding homeostatic interactions of Fe and Zn with the macronutrient phosphorous (P) and the resulting physiological responses to combined deficiencies of these nutrients. Fe and P interactions have been shown to influence root development, photosynthesis, and biological processes aiding Fe uptake. Zn and P interactions also influence root growth, and coordination of Zn-dependent transcriptional regulation contributes to phosphate (Pi) transport in the plant. Understanding homeostatic interactions among these different nutrients is of critical importance to obtain a more complete understanding of plant nutrition in complex soil environments.

Auxin/Cytokinin Antagonistic Control of the Shoot/Root Growth Ratio and Its Relevance for Adaptation to Drought and Nutrient Deficiency Stresses.

The hormones auxin and cytokinin regulate numerous aspects of plant development and often act as an antagonistic hormone pair. One of the more striking examples of the auxin/cytokinin antagonism involves regulation of the shoot/root growth ratio in which cytokinin promotes shoot and inhibits root growth, whereas auxin does the opposite. Control of the shoot/root growth ratio is essential for the survival of terrestrial plants because it allows growth adaptations to water and mineral nutrient availability in the soil. Because a decrease in shoot growth combined with an increase in root growth leads to survival under drought stress and nutrient limiting conditions, it was not surprising to find that auxin promotes, while cytokinin reduces, drought stress tolerance and nutrient uptake. Recent data show that drought stress and nutrient availability also alter the cytokinin and auxin signaling and biosynthesis pathways and that this stress-induced regulation affects cytokinin and auxin in the opposite manner. These antagonistic effects of cytokinin and auxin suggested that each hormone directly and negatively regulates biosynthesis or signaling of the other. However, a growing body of evidence supports unidirectional regulation, with auxin emerging as the primary regulatory component. This master regulatory role of auxin may not come as a surprise when viewed from an evolutionary perspective.

Coordinated homeostasis of essential mineral nutrients: a focus on iron.

In plants, iron (Fe) transport and homeostasis are highly regulated processes. Fe deficiency or excess dramatically limits plant and algal productivity. Interestingly, complex and unexpected interconnections between Fe and various macro- and micronutrient homeostatic networks, supposedly maintaining general ionic equilibrium and balanced nutrition, are currently being uncovered. Although these interactions have profound consequences for our understanding of Fe homeostasis and its regulation, their molecular bases and biological significance remain poorly understood. Here, we review recent knowledge gained on how Fe interacts with micronutrient (e.g. zinc, manganese) and macronutrient (e.g. sulfur, phosphate) homeostasis, and on how these interactions affect Fe uptake and trafficking. Finally, we highlight the importance of developing an improved model of how Fe signaling pathways are integrated into functional networks to control plant growth and development in response to fluctuating environments.

Plant resilience to phosphate limitation: current knowledge and future challenges.

Phosphorus (P) is an essential macronutrient for all living organisms. Importantly, plants require a large amount of P to grow, and P deficiency causes huge losses in plant production. Although this issue can be mitigated by the appropriate use of phosphate (Pi) rock-derived P fertilizers, phosphate rock is a finite natural resource. Moreover, the increased demand for food as a result of our growing global population is another factor contributing to a prospective P crisis. While creating crops that are resilient to Pi deficiency presents great scientific challenge, the current progress in our understanding of how plants regulate Pi homeostasis offers some opportunities for further study. In this review, we present the published research supporting these opportunities, which are based on the molecular mechanisms that plants have evolved to respond to P deficiency. First, we focus on recent advances in P sensing and signaling pathways in the regulation of root system architecture. Next, we describe the mechanisms that regulate Pi transport and accumulation, in a Pi- (or other nutrient) dependent manner. Integrating these data will help to design an innovative strategy for improving Pi nutrition in plants. In addition, this will help with Pi scarcity, one of the challenges facing agriculture in the twenty first century.

The BMP Pathway in Blood Vessel and Lymphatic Vessel Biology.

Bone morphogenetic proteins (BMPs) were originally identified as the active components in bone extracts that can induce ectopic bone formation. In recent decades, their key role has broadly expanded beyond bone physiology and pathology. Nowadays, the BMP pathway is considered an important player in vascular signaling. Indeed, mutations in genes encoding different components of the BMP pathway cause various severe vascular diseases. Their signaling contributes to the morphological, functional and molecular heterogeneity among endothelial cells in different vessel types such as arteries, veins, lymphatic vessels and capillaries within different organs. The BMP pathway is a remarkably fine-tuned pathway. As a result, its signaling output in the vessel wall critically depends on the cellular context, which includes flow hemodynamics, interplay with other vascular signaling cascades and the interaction of endothelial cells with peri-endothelial cells and the surrounding matrix. In this review, the emerging role of BMP signaling in lymphatic vessel biology will be highlighted within the framework of BMP signaling in the circulatory vasculature.

Hormonal Regulation and Crosstalk of Auxin/Cytokinin Signaling Pathways in Potatoes In Vitro and in Relation to Vegetation or Tuberization Stages.

Auxins and cytokinins create versatile regulatory network controlling virtually all aspects of plant growth and development. These hormonal systems act in close contact, synergistically or antagonistically, determining plant phenotype, resistance and productivity. However, the current knowledge about molecular interactions of these systems is still scarce. Our study with potato plants aimed at deciphering potential interactions between auxin and cytokinin signaling pathways at the level of respective gene expression. Potato plants grown on sterile medium with 1.5% (vegetation) or 5% (tuberization) sucrose were treated for 1 h with auxin or cytokinin. Effects of these two hormones on expression profiles of genes belonging to main signaling pathways of auxin and cytokinin were quantified by RT-qPCR. As a result, several signaling genes were found to respond to auxin and/or cytokinin by up- or down-regulation. The observed effects were largely organ-specific and depended on sucrose content. Auxin strongly reduced cytokinin perception apparatus while reciprocal cytokinin effect was ambiguous and sucrose-dependent. In many cases, functional clustering of genes of the same family was observed. Promoters in some clusters are enriched with canonic hormone-response cis-elements supporting their direct sensitivity to hormones. Collectively, our data shed new light on the crosstalk between auxin- and cytokinin signaling pathways.

Signaling Networks That Control Cellular Plasticity in Pancreatic Tumorigenesis, Progression, and Metastasis.

Pancreatic ductal adenocarcinoma is one of the deadliest cancers, and its incidence on the rise. The major challenges in overcoming the poor prognosis with this disease include late detection and the aggressive biology of the disease. Intratumoral heterogeneity; presence of a robust, reactive, and desmoplastic stroma; and the crosstalk between the different tumor components require complete understanding of the pancreatic tumor biology to better understand the therapeutic challenges posed by this disease. In this review, we discuss the processes involved during tumorigenesis encompassing the inherent plasticity of the transformed cells, development of tumor stroma crosstalk, and enrichment of cancer stem cell population during tumorigenesis.

The Ve-resistance locus, a plant signaling intercept.

MAIN CONCLUSION: The Ve-resistance locus in tomato and potato affects both stress/defense and growth, consistent with a signaling intercept and a competitive regulatory mechanism. Acting in an antagonistic fashion, the two genes comprising the tomato Ve-resistance locus have been shown to influence both the defense/stress cascade, which causes wilt symptoms, and plant growth (Nazar et al. in Planta 247:1339-1350, 2018c); in contrast, both have been reported to elevate wilt resistance in potato or Arabidopsis. In a further examination of this influence in potato transformed with the Ve1 gene, effects are again demonstrated with respect to both disease resistance and crop productivity consistent with the Ve locus being a signaling intercept and the antagonistic effects, previously observed in tomato. The results support a competitive model in which the tomato Ve1 and Ve2 proteins act to reduce the detrimental effects of the defense/stress cascade and energy transfers to the developing potato tubers.

Future perspective: high-throughput construction of new ultrasensitive cytokine and virion liquid chips for high-throughput screening (HTS) of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases.

Pathogen-host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane-protein interactions were found to mediate E. coli invasion of the blood-brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)-host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca2+]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract.

Structural basis of jasmonate-amido synthetase FIN219 in complex with glutathione S-transferase FIP1 during the JA signal regulation.

Far-red (FR) light-coupled jasmonate (JA) signaling is necessary for plant defense and development. FR insensitive 219 (FIN219) is a member of the Gretchen Hagen 3 (GH3) family of proteins in Arabidopsis and belongs to the adenylate-forming family of enzymes. It directly controls biosynthesis of jasmonoyl-isoleucine in JA-mediated defense responses and interacts with FIN219-interacting protein 1 (FIP1) under FR light conditions. FIN219 and FIP1 are involved in FR light signaling and are regulators of the interplay between light and JA signaling. However, how their interactions affect plant physiological functions remains unclear. Here, we demonstrate the crystal structures of FIN219-FIP1 while binding with substrates at atomic resolution. Our results show an unexpected FIN219 conformation and demonstrate various differences between this protein and other members of the GH3 family. We show that the rotated C-terminal domain of FIN219 alters ATP binding and the core structure of the active site. We further demonstrate that this unique FIN219-FIP1 structure is crucial for increasing FIN219 activity and determines the priority of substrate binding. We suggest that the increased FIN219 activity resulting from the complex form, a conformation for domain switching, allows FIN219 to switch to its high-affinity mode and thereby enhances JA signaling under continuous FR light conditions.

GRK2-Mediated Crosstalk Between ß-Adrenergic and Angiotensin II Receptors Enhances Adrenocortical Aldosterone Production In Vitro and In Vivo.

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone via both G proteins and the adapter protein ßarrestin1, which binds the receptor following its phosphorylation by GPCR-kinases (GRKs) to initiate G protein-independent signaling. ß-adrenergic receptors (ARs) also induce aldosterone production in AZG cells. Herein, we investigated whether GRK2 or GRK5, the two major adrenal GRKs, is involved in the catecholaminergic regulation of AngII-dependent aldosterone production. In human AZG (H295R) cells in vitro, the ßAR agonist isoproterenol significantly augmented both AngII-dependent aldosterone secretion and synthesis, as measured by the steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA inductions. Importantly, GRK2, but not GRK5, was indispensable for the ßAR-mediated enhancement of aldosterone in response to AngII. Specifically, GRK2 inhibition with Cmpd101 abolished isoproterenol's effects on AngII-induced aldosterone synthesis/secretion, whereas the GRK5 knockout via CRISPR/Cas9 had no effect. It is worth noting that these findings were confirmed in vivo, since rats overexpressing GRK2, but not GRK5, in their adrenals had elevated circulating aldosterone levels compared to the control animals. However, treatment with the ß-blocker propranolol prevented hyperaldosteronism in the adrenal GRK2-overexpressing rats. In conclusion, GRK2 mediates a ßAR-AT1R signaling crosstalk in the adrenal cortex leading to elevated aldosterone production. This suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems at the level of the adrenal cortex and that its inhibition might be therapeutically advantageous in hyperaldosteronism-related conditions.

Mapping signaling pathway cross-talk in Drosophila cells.

During development and homeostasis, cells integrate multiple signals originating either from neighboring cells or systemically. In turn, responding cells can produce signals that act in an autocrine, paracrine, or endocrine manner. Although the nature of the signals and pathways used in cell-cell communication are well characterized, we lack, in most cases, an integrative view of signaling describing the spatial and temporal interactions between pathways (e.g., whether the signals are processed sequentially or concomitantly when two pathways are required for a specific outcome). To address the extent of cross-talk between the major metazoan signaling pathways, we characterized immediate transcriptional responses to either single- or multiple pathway stimulations in homogeneous Drosophila cell lines. Our study, focusing on seven core pathways, epidermal growth factor receptor (EGFR), bone morphogenetic protein (BMP), Jun kinase (JNK), JAK/STAT, Notch, Insulin, and Wnt, revealed that many ligands and receptors are primary targets of signaling pathways, highlighting that transcriptional regulation of genes encoding pathway components is a major level of signaling cross-talk. In addition, we found that ligands and receptors can integrate multiple pathway activities and adjust their transcriptional responses accordingly.

From Crosstalk between Immune and Bone Cells to Bone Erosion in Infection.

Bone infection and inflammation leads to the infiltration of immune cells at the site of infection, where they modulate the differentiation and function of osteoclasts and osteoblasts by the secretion of various cytokines and signal mediators. In recent years, there has been a tremendous effort to understand the cells involved in these interactions and the complex pathways of signal transduction and their ultimate effect on bone metabolism. These crosstalk mechanisms between the bone and immune system finally emerged, forming a new field of research called osteoimmunology. Diseases falling into the category of osteoimmunology, such as osteoporosis, periodontitis, and bone infections are considered to have a significant implication in mortality and morbidity of patients, along with affecting their quality of life. There is a much-needed research focus in this new field, as the reported data on the immunomodulation of immune cells and their signaling pathways seems to have promising therapeutic benefits for patients.

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells. METHODS: In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF this has already been defined above cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC-MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells. RESULTS: We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication. CONCLUSIONS: Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

Cpp1 phosphatase mediated signaling crosstalk between Hog1 and Cek1 mitogen-activated protein kinases is involved in the phenotypic transition in Candida albicans.

Cellular signaling pathways involved in cell growth and differentiation mediated by mitogen-activated protein kinase (MAPK) cascades have been well characterized in fungi. However, the mechanisms of signaling crosstalk between MAPKs to ensure signaling specificity are largely unknown. Previous work showed that activation of the Candida albicans Cek1 MAPK pathway resulted in opaque cell formation and filamentation, which mirrored the phenotypes to hog1Δ. Additionally, deleting the HOG1 gene stimulated Cek1p. Thus, we hypothesized that an unknown factor could act as a bridge between these two MAPKs. In Saccharomyces cerevisiae, the dual-specificity phosphatase (DSP) Msg5 specifically dephosphorylates Fus3p/Kss1p. C. albicans Cpp1, an ortholog of Msg5, has been shown to be important in regulating Cek1p. Compared with the wild-type strain, hog1Δ shows a ∼40% reduction in CPP1 expression. Consistent with previous reports, CPP1 deletion also resulted in Cek1 hyperphosphorylation, implicating Cpp1 as a regulator of the Hog1 and Cek1 cascades. Interestingly, both cpp1Δ and hog1Δ induced 100% opaque colony formation in MTL-homozygous strains grown on N-acetylglucosamine (NAG) plates, whereas the wild-type and complemented strains exhibited 80.9% and 77.1% white-to-opaque switching rates, respectively. CPP1 gene deletion also caused hyperfilamentous phenotypes in both white and opaque cells. These phenomena may be due to highly phosphorylated Cek1p, as deleting CEK1 in the cpp1Δ background generated nonfilamentous strains and reduced opaque colony formation. Taken together, we conclude that cpp1Δ and hog1Δ exhibited comparable phenotypes, and both are involved in regulating Cek1 phosphorylation, implicating Cpp1 phosphatase as a key intermediary between the Hog1 and Cek1 signal transduction pathways.