Marcus B. Smolka

A Multidimensional Chromatography Technology for In-depth Phosphoproteome Analysis

Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its application to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phosphopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that ∼50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.

Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases

Understanding the role of DNA damage checkpoint kinases in the cellular response to genotoxic stress requires the knowledge of their substrates. Here, we report the use of quantitative phosphoproteomics to identify in vivo kinase substrates of the yeast DNA damage checkpoint kinases Mec1, Tel1, and Rad53 (orthologs of human ATR, ATM, and CHK2, respectively). By analyzing 2,689 phosphorylation sites in wild-type and various kinase-null cells, 62 phosphorylation sites from 55 proteins were found to be controlled by the DNA damage checkpoint. Examination of the dependency of each phosphorylation on Mec1 and Tel1 or Rad53, combined with sequence and biochemical analysis, revealed that many of the identified targets are likely direct substrates of these kinases. In addition to several known targets, 50 previously undescribed targets of the DNA damage checkpoint were identified, suggesting that a wide range of cellular processes is likely regulated by Mec1, Tel1, and Rad53.

TORC1 regulates endocytosis via Npr1-mediated phosphoinhibition of a ubiquitin ligase adaptor.

The TORC1 kinase signaling complex is a key determinant of cell growth that senses nutritional status and responds by coordinating diverse cellular processes including transcription, translation, and autophagy. Here, we demonstrate that TORC1 modulates the composition of plasma membrane (PM) proteins by regulating ubiquitin-mediated endocytosis. The mechanism involves the Npr1 kinase, a negative regulator of endocytosis that is itself negatively regulated by TORC1. We show that Npr1 inhibits the activity of Art1, an arrestin-like adaptor protein that promotes endocytosis by targeting the Rsp5 ubiquitin ligase to specific PM cargoes. Npr1 antagonizes Art1-mediated endocytosis via N-terminal phosphorylation, a modification that prevents Art1 association with the PM. Thus, our study adds ubiquitin ligase targeting and control of endocytosis to the known effector mechanisms of TORC1, underscoring how TORC1 coordinates ubiquitin-mediated endocytosis with protein synthesis and autophagy in order to regulate cell growth.

Quantitative protein profiling using two-dimensional gel electrophoresis, isotope coded affinity tag labeling and mass spectrometry

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.

Checkpoint proteins control morphogenetic events during DNA replication stress in Saccharomyces cerevisiae

In response to DNA replication stress in Saccharomyces cerevisiae, the DNA replication checkpoint maintains replication fork stability, prevents precocious chromosome segregation, and causes cells to arrest as large-budded cells. The checkpoint kinases Mec1 and Rad53 act in this checkpoint. Treatment of mec1 or rad53Δ mutants with replication inhibitors results in replication fork collapse and inappropriate partitioning of partially replicated chromosomes, leading to cell death. We describe a previously unappreciated function of various replication stress checkpoint proteins, including Rad53, in the control of cell morphology. Checkpoint mutants have aberrant cell morphology and cell walls, and show defective bud site selection. Rad53 shows genetic interactions with septin ring pathway components, and, along with other checkpoint proteins, controls the timely degradation of Swe1 during replication stress, thereby facilitating proper bud growth. Thus, checkpoint proteins play an important role in coordinating morphogenetic events with DNA replication during replication stress.

The ALS/FTLD associated protein C9orf72 associates with SMCR8 and WDR41 to regulate the autophagy-lysosome pathway.

Hexanucleotide repeat expansion in the C9orf72 gene is a leading cause of frontotemporal lobar degeneration (FTLD) with amyotrophic lateral sclerosis (ALS). Reduced expression of C9orf72 has been proposed as a possible disease mechanism. However, the cellular function of C9orf72 remains to be characterized. Here we report the identification of two binding partners of C9orf72: SMCR8 and WDR41. We show that WDR41 interacts with the C9orf72/SMCR8 heterodimer and WDR41 is tightly associated with the Golgi complex. We further demonstrate that C9orf72/SMCR8/WDR41 associates with the FIP200/Ulk1 complex, which is essential for autophagy initiation. C9orf72 deficient mice, generated using the CRISPR/Cas9 system, show severe inflammation in multiple organs, including lymph node, spleen and liver. Lymph node enlargement and severe splenomegaly are accompanied with macrophage infiltration. Increased levels of autophagy and lysosomal proteins and autophagy defects were detected in both the spleen and liver of C9orf72 deficient mice, supporting an in vivo role of C9orf72 in regulating the autophagy/lysosome pathway. In summary, our study elucidates potential physiological functions of C9orf72 and disease mechanisms of ALS/FTLD.

An FHA domain-mediated protein interaction network of Rad53 reveals its role in polarized cell growth

The DNA damage checkpoint kinase Rad53 is important for the survival of budding yeast under genotoxic stresses. We performed a biochemical screen to identify proteins with specific affinity for the two Forkhead associated (FHA) domains of Rad53. The N-terminal FHA1 domain was found to coordinate a complex protein interaction network, which includes nuclear proteins involved in DNA damage checkpoints and transcriptional regulation. Unexpectedly, cytosolic proteins involved in cytokinesis, including septins, were also found as FHA1 binding proteins. Consistent with this interaction, a Rad53 mutant defective in its nuclear localization was found to localize to the bud neck. Abnormal morphology was observed in cells overexpressing the FHA1 domain and in rad53Δ cells under DNA replication stress. Further, septin Shs1 appears to have an important role in the response to DNA replication stress. Collectively, the results suggest a novel function of Rad53 in the regulation of polarized cell growth in response to DNA replication stress.

Dynamic Changes in Protein-Protein Interaction and Protein Phosphorylation Probed with Amine-reactive Isotope Tag

We present an approach for quantitative analysis of changes in the composition and phosphorylation of protein complexes by MS. It is based on a new class of stable isotope-labeling reagent, the amine-reactive isotope tag (N-isotag), for specific and quantitative labeling of peptides following proteolytic digestion of proteins. Application of the N-isotag method to the analysis of Rad53, a DNA damage checkpoint kinase in Saccharomyces cerevisiae, led to the identification of dynamic associations between Rad53 and the nuclear transport machinery, histones, and chromatin assembly proteins in response to DNA damage. Over 30 phosphorylation sites of Rad53 and its associated proteins were identified and quantified, and they showed different changes in phosphorylation in response to DNA damage. Interestingly, Ser789 of Rad53 was found to be a major initial phosphorylation site, and its phosphorylation regulates the Rad53 abundance in response to DNA damage. Collectively, these results demonstrate that N-isotag-based quantitative MS is generally applicable to study dynamic changes in the composition of protein complexes and their phosphorylation patterns in a site-specific manner in response to different cell stimuli.

DNA Damage Signaling Recruits the Rtt107-Slx4 Scaffolds via Dpb11 to Mediate Replication Stress Response

The DNA damage checkpoint kinase Mec1(ATR) is critical for maintaining the integrity of replication forks. Though it has been proposed to promote fork repair, the mechanisms by which Mec1 regulates DNA repair factors remain unclear. Here, we found that Mec1 mediates a key interaction between the fork protein Dpb11 and the DNA repair scaffolds Slx4-Rtt107 to regulate replication stress response. Dissection of the molecular basis of the interaction reveals that Slx4 and Rtt107 jointly bind Dpb11 and that Slx4 phosphorylation is required. Mutation of Mec1 phosphorylation sites in Slx4 disrupts its interaction with Dpb11 and compromises the cellular response to replisomes blocked by DNA alkylation. Multiple fork repair factors associate with Rtt107 or Slx4, supporting that Mec1-dependent assembly of the Rtt107-Slx4-Dpb11 complex functions to coordinate fork repair. Our results unveil how Mec1 regulates the Slx4 and Rtt107 scaffolds and establish a mechanistic link between DNA damage signaling and fork repair.

DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9

In response to genotoxic stress, a transient arrest in cell-cycle progression enforced by the DNA-damage checkpoint (DDC) signalling pathway positively contributes to genome maintenance. Because hyperactivated DDC signalling can lead to a persistent and detrimental cell-cycle arrest, cells must tightly regulate the activity of the kinases involved in this pathway. Despite their importance, the mechanisms for monitoring and modulating DDC signalling are not fully understood. Here we show that the DNA-repair scaffolding proteins Slx4 and Rtt107 prevent the aberrant hyperactivation of DDC signalling by lesions that are generated during DNA replication in Saccharomyces cerevisiae. On replication stress, cells lacking Slx4 or Rtt107 show hyperactivation of the downstream DDC kinase Rad53, whereas activation of the upstream DDC kinase Mec1 remains normal. An Slx4–Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phosphorylated histone H2A, two positive regulators of Rad9-dependent Rad53 activation. A decrease in DDC signalling results from hypomorphic mutations in RAD53 and H2A and rescues the hypersensitivity to replication stress of cells lacking Slx4 or Rtt107. We propose that the Slx4–Rtt107 complex modulates Rad53 activation by a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings show that DDC signalling is monitored and modulated through the direct action of DNA-repair factors.