Large-scale functional analysis of the roles of phosphorylation in yeast metabolic pathways.

Protein phosphorylation is a widespread posttranslational modification that regulates almost all cellular functions. To investigate the large number of phosphorylation events with unknown functions, we monitored the concentrations of several hundred intracellular metabolites in Saccharomyces cerevisiae yeast strains with deletions of 118 kinases or phosphatases. Whereas most deletion strains had no detectable difference in growth compared to wild-type yeast, two-thirds of deletion strains had alterations in metabolic profiles. For about half of the kinases and phosphatases encoded by the deleted genes, we inferred specific regulatory roles on the basis of knowledge about the affected metabolic pathways. We demonstrated that the phosphatase Ppq1 was required for metal homeostasis. Combining metabolomic data with published phosphoproteomic data in a stoichiometric model enabled us to predict functions for phosphorylation in the regulation of 47 enzymes. Overall, we provided insights and testable predictions covering greater than twice the number of known phosphorylated enzymes in yeast, suggesting extensive phosphorylation-dependent regulation of yeast metabolism.

A sentinel protein assay for simultaneously quantifying cellular processes.

We describe a proteomic screening approach based on the concept of sentinel proteins, biological markers whose change in abundance characterizes the activation state of a given cellular process. Our sentinel assay simultaneously probed 188 biological processes in Saccharomyces cerevisiae exposed to a set of environmental perturbations. The approach can be applied to analyze responses to large sets of uncharacterized perturbations in high throughput.

The calcineurin signaling network evolves via conserved kinase-phosphatase modules that transcend substrate identity.

To define a functional network for calcineurin, the conserved Ca(2+)/calmodulin-regulated phosphatase, we systematically identified its substrates in S. cerevisiae using phosphoproteomics and bioinformatics, followed by copurification and dephosphorylation assays. This study establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution. Analyses of closely related yeasts show that many proteins were recently recruited to the network by acquiring a calcineurin-recognition motif. Calcineurin substrates in yeast and mammals are distinct due to network rewiring but, surprisingly, are phosphorylated by similar kinases. We postulate that corecognition of conserved substrate features, including phosphorylation and docking motifs, preserves calcineurin-kinase opposition during evolution. One example we document is a composite docking site that confers substrate recognition by both calcineurin and MAPK. We propose that conserved kinase-phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop that establish common regulatory motifs in signaling networks.

Measuring adhesion forces in powder collectives by inertial detachment.

One way of measuring adhesion forces in fine powders is to place the particles on a surface, retract the surface with a high acceleration, and observe their detachment due to their inertia. To induce detachment of micrometer-sized particles, an acceleration in the order of 500,000g is required. We developed a device in which such high acceleration is provided by a Hopkinson bar and measured via laser vibrometry. Using a Hopkinson bar, the fundamental limit of mechanically possible accelerations is reached, since higher values cause material failure. Particle detachment is detected by optical video microscopy. With subsequent automated data evaluation a statistical distribution of adhesion forces is obtained. To validate the method, adhesion forces for ensembles of single polystyrene and silica particles on a polystyrene coated steel surface were measured under ambient conditions. We were able to investigate more than 150 individual particles in one experiment and obtained adhesion values of particles in a diameter range of 3-13 µm. Measured adhesion forces of small particles agreed with values from colloidal probe measurements and theoretical predictions. However, we observe a stronger increase of adhesion for particles with a diameter larger than roughly 7-10 µm. We suggest that this discrepancy is caused by surface roughness and heterogeneity. Large particles adjust and find a stable position on the surface due to their inertia while small particles tend to remain at the position of first contact. The new device will be applicable to study a broad variety of different particle-surface combinations on a routine basis, including strongly cohesive powders like pharmaceutical drugs for treatment of lung diseases.

Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling.

Cytosolic compartmentalization through liquid-liquid unmixing, such as the formation of RNA granules, is involved in many cellular processes and might be used to regulate signal transduction. However, specific molecular mechanisms by which liquid-liquid unmixing and signal transduction are coupled remain unknown. Here, we show that during cellular stress the dual specificity kinase DYRK3 regulates the stability of P-granule-like structures and mTORC1 signaling. DYRK3 displays a cyclic partitioning mechanism between stress granules and the cytosol via a low-complexity domain in its N terminus and its kinase activity. When DYRK3 is inactive, it prevents stress granule dissolution and the release of sequestered mTORC1. When DYRK3 is active, it allows stress granule dissolution, releasing mTORC1 for signaling and promoting its activity by directly phosphorylating the mTORC1 inhibitor PRAS40. This mechanism links cytoplasmic compartmentalization via liquid phase transitions with cellular signaling.

Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery-and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis.

The target of rapamycin complex 1 (TORC1) is an essential multiprotein complex conserved from yeast to humans. Under favorable growth conditions, and in the absence of the macrolide rapamycin, TORC1 is active, and influences virtually all aspects of cell growth. Although two direct effectors of yeast TORC1 have been reported (Tap42, a regulator of PP2A phosphatases and Sch9, an AGC family kinase), the signaling pathways that couple TORC1 to its distal effectors were not well understood. To elucidate these pathways we developed and employed a quantitative, label-free mass spectrometry approach. Analyses of the rapamycin-sensitive phosphoproteomes in various genetic backgrounds revealed both documented and novel TORC1 effectors and allowed us to partition phosphorylation events between Tap42 and Sch9. Follow-up detailed characterization shows that Sch9 regulates RNA polymerases I and III, the latter via Maf1, in addition to translation initiation and the expression of ribosomal protein and ribosome biogenesis genes. This demonstrates that Sch9 is a master regulator of protein synthesis.