Global Regulation of a Differentiation MAPK Pathway in Yeast

Cell differentiation requires different pathways to act in concert to produce a specialized cell type. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth in response to nutrient limitation. Differentiation to the filamentous cell type requires multiple signaling pathways, including a mitogen-activated protein kinase (MAPK) pathway. To identify new regulators of the filamentous growth MAPK pathway, a genetic screen was performed with a collection of 4072 nonessential deletion mutants constructed in the filamentous (Σ1278b) strain background. The screen, in combination with directed gene-deletion analysis, uncovered 97 new regulators of the filamentous growth MAPK pathway comprising 40% of the major regulators of filamentous growth. Functional classification extended known connections to the pathway and identified new connections. One function for the extensive regulatory network was to adjust the activity of the filamentous growth MAPK pathway to the activity of other pathways that regulate the response. In support of this idea, an unregulated filamentous growth MAPK pathway led to an uncoordinated response. Many of the pathways that regulate filamentous growth also regulated each other’s targets, which brings to light an integrated signaling network that regulates the differentiation response. The regulatory network characterized here provides a template for understanding MAPK-dependent differentiation that may extend to other systems, including fungal pathogens and metazoans.     

The Signaling Mucins Msb2 and Hkr1 Differentially Regulate the Filamentation Mitogen-activated Protein Kinase Pathway and Contribute to a Multimodal Response

A central question in the area of signal transduction is why pathways utilize common components. In the budding yeast Saccharomyces cerevisiae, the HOG and filamentous growth (FG) MAPK pathways require overlapping components but are thought to be induced by different stimuli and specify distinct outputs. To better understand the regulation of the FG pathway, we examined FG in one of yeast''s native environments, the grape-producing plant Vitis vinifera. In this setting, different aspects of FG were induced in a temporal manner coupled to the nutrient cycle, which uncovered a multimodal feature of FG pathway signaling. FG pathway activity was modulated by the HOG pathway, which led to the finding that the signaling mucins Msb2p and Hkr1p, which operate at the head of the HOG pathway, differentially regulate the FG pathway. The two mucins exhibited different expression and secretion patterns, and their overproduction induced nonoverlapping sets of target genes. Moreover, Msb2p had a function in cell polarization through the adaptor protein Sho1p that Hkr1p did not. Differential MAPK activation by signaling mucins brings to light a new point of discrimination between MAPK pathways.     

Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway

Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other''s activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways.     

The regulation of filamentous growth in yeast

Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host-cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basis of the response. Filamentous growth is regulated by evolutionarily conserved signaling pathways. One of these pathways is a mitogen activated protein kinase (MAPK) pathway. A remarkable feature of the filamentous growth MAPK pathway is that it is composed of factors that also function in other pathways. An intriguing challenge therefore has been to understand how pathways that share components establish and maintain their identity. Other canonical signaling pathways-rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), and target of rapamycin (TOR)-also regulate filamentous growth, which raises the question of how signals from multiple pathways become integrated into a coordinated response. Together, these pathways regulate cell differentiation to the filamentous type, which is characterized by changes in cell adhesion, cell polarity, and cell shape. How these changes are accomplished is also discussed. High-throughput genomics approaches have recently uncovered new connections to filamentous growth regulation. These connections suggest that filamentous growth is a more complex and globally regulated behavior than is currently appreciated, which may help to pave the way for future investigations into this eukaryotic cell differentiation behavior.     

Comparative Analysis of Transmembrane Regulators of the Filamentous Growth Mitogen-Activated Protein Kinase Pathway Uncovers Functional and Regulatory Differences

Filamentous growth is a microbial differentiation response that involves the concerted action of multiple signaling pathways. In budding yeast, one pathway that regulates filamentous growth is a Cdc42p-dependent mitogen-activated protein kinase (MAPK) pathway. Several transmembrane (TM) proteins regulate the filamentous growth pathway, including the signaling mucin Msb2p, the tetraspan osmosensor Sho1p, and an adaptor Opy2p. The TM proteins were compared to identify common and unique features. Msb2p, Sho1p, and Opy2p associated by coimmunoprecipitation analysis but showed predominantly different localization patterns. The different localization patterns of the proteins resulted in part from different rates of turnover from the plasma membrane (PM). In particular, Msb2p (and Opy2p) were turned over rapidly compared to Sho1p. Msb2p signaled from the PM, and its turnover was a rate-limiting step in MAPK signaling. Genetic analysis identified unique phenotypes of cells overexpressing the TM proteins. Therefore, each TM regulator of the filamentous growth pathway has its own regulatory pattern and specific function in regulating filamentous growth. This specialization may be important for fine-tuning and potentially diversifying the filamentation response.     

Drosophila cyfip Regulates Synaptic Development and Endocytosis by Suppressing Filamentous Actin Assembly

The formation of synapses and the proper construction of neural circuits depend on signaling pathways that regulate cytoskeletal structure and dynamics. After the mutual recognition of a growing axon and its target, multiple signaling pathways are activated that regulate cytoskeletal dynamics to determine the morphology and strength of the connection. By analyzing Drosophila mutations in the cytoplasmic FMRP interacting protein Cyfip, we demonstrate that this component of the WAVE complex inhibits the assembly of filamentous actin (F-actin) and thereby regulates key aspects of synaptogenesis. Cyfip regulates the distribution of F-actin filaments in presynaptic neuromuscular junction (NMJ) terminals. At cyfip mutant NMJs, F-actin assembly was accelerated, resulting in shorter NMJs, more numerous satellite boutons, and reduced quantal content. Increased synaptic vesicle size and failure to maintain excitatory junctional potential amplitudes under high-frequency stimulation in cyfip mutants indicated an endocytic defect. cyfip mutants exhibited upregulated bone morphogenetic protein (BMP) signaling, a major growth-promoting pathway known to be attenuated by endocytosis at the Drosophila NMJ. We propose that Cyfip regulates synapse development and endocytosis by inhibiting actin assembly.     

Distinct Patterns of DNA Damage Response and Apoptosis Correlate with Jak/Stat and PI3Kinase Response Profiles in Human Acute Myelogenous Leukemia

Background

Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML) disease subtypes based on survival, DNA damage response and apoptosis pathways.

Methodology and Principal Findings

Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct “DNA damage response (DDR)/apoptosis” profiles: 1) AML blasts with a defective DDR and failure to undergo apoptosis; 2) AML blasts with proficient DDR and failure to undergo apoptosis; 3) AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the “DDR/apoptosis” proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents.

Conclusions and Significance

Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in combination with chemotherapy.     

Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules

Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs.     

Stimulus Bias Provides Evidence for Conformational Constraints in the Structure of a G Protein-coupled Receptor

A key characteristic of G protein-coupled receptors (GPCRs) is that they activate a plethora of signaling pathways. It is now clear that a GPCR coupling to these pathways can be regulated selectively by ligands that differentially drive signaling down one pathway in preference to another. This concept, termed stimulus bias, is revolutionizing receptor biology and drug discovery by providing a means of selectively targeting receptor signaling pathways that have therapeutic impact. Herein, we utilized a novel quantitative method that determines stimulus bias of synthetic GPCR ligands in a manner that nullifies the impact of both the cellular background and the “natural bias” of the endogenous ligand. By applying this method to the M₂ muscarinic acetylcholine receptor, a prototypical GPCR, we found that mutation of key residues (Tyr-80^2.61 and Trp-99^3.28) in an allosteric binding pocket introduces stimulus bias in response to the atypical ligands AC-42 (4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)piperidine HCl) and 77-LH-28-1 (1-(3-(4-butyl-1-piperidinyl)propyl)- 3,3-dihydro-2(1H)-quinolinone). By comparing stimulus bias factors among receptor internalization, G protein activation, extracellular-regulated protein kinase 1/2 (ERK1/2) signaling, and receptor phosphorylation, we provide evidence that Tyr-80^2.61 and Trp-99^3.28 act either as molecular switches or as gatekeeper residues that introduce constraints limiting the active conformation of the M₂ muscarinic acetylcholine receptor and thereby regulate stimulus bias. Furthermore, we provide evidence that downstream signaling pathways previously considered to be related to each other (i.e. receptor phosphorylation, internalization, and activation of ERK1/2) can act independently.