Global Identification of Small Ubiquitin-related Modifier (SUMO) Substrates Reveals Crosstalk between SUMOylation and Phosphorylation Promotes Cell Migration.

Proteomics studies have revealed that SUMOylation is a widely used post-translational modification (PTM) in eukaryotes. However, how SUMO E1/2/3 complexes use different SUMO isoforms and recognize substrates remains largely unknown. Using a human proteome microarray-based activity screen, we identified over 2500 proteins that undergo SUMO E3-dependent SUMOylation. We next constructed a SUMO isoform- and E3 ligase-dependent enzyme-substrate relationship network. Protein kinases were significantly enriched among SUMOylation substrates, suggesting crosstalk between phosphorylation and SUMOylation. Cell-based analyses of tyrosine kinase, PYK2, revealed that SUMOylation at four lysine residues promoted PYK2 autophosphorylation at tyrosine 402, which in turn enhanced its interaction with SRC and full activation of the SRC-PYK2 complex. SUMOylation on WT but not the 4KR mutant of PYK2 further elevated phosphorylation of the downstream components in the focal adhesion pathway, such as paxillin and Erk1/2, leading to significantly enhanced cell migration during wound healing. These studies illustrate how our SUMO E3 ligase-substrate network can be used to explore crosstalk between SUMOylation and other PTMs in many biological processes.

Global Analysis of SUMO-Binding Proteins Identifies SUMOylation as a Key Regulator of the INO80 Chromatin Remodeling Complex.

SUMOylation is a critical regulator of a broad range of cellular processes, and is thought to do so in part by modulation of protein interaction. To comprehensively identify human proteins whose interaction is modulated by SUMOylation, we developed an in vitro binding assay using human proteome microarrays to identify targets of SUMO1 and SUMO2. We then integrated these results with protein SUMOylation and protein-protein interaction data to perform network motif analysis. We focused on a single network motif we termed a SUMOmodPPI (SUMO-modulated Protein-Protein Interaction) that included the INO80 chromatin remodeling complex subunits TFPT and INO80E. We validated the SUMO-binding activity of INO80E, and showed that TFPT is a SUMO substrate both in vitro and in vivo We then demonstrated a key role for SUMOylation in mediating the interaction between these two proteins, both in vitro and in vivo By demonstrating a key role for SUMOylation in regulating the INO80 chromatin remodeling complex, this work illustrates the power of bioinformatics analysis of large data sets in predicting novel biological phenomena.

Activation of diverse signalling pathways by oncogenic PIK3CA mutations.

The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing 'driver' oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signalling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K-enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signalling events and for discovering novel signalling molecules as readouts of pathway activation or potential therapeutic targets.

Bcl2-associated athanogene 3 interactome analysis reveals a new role in modulating proteasome activity.

Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach.

Profiling the human protein-DNA interactome reveals ERK2 as a transcriptional repressor of interferon signaling.

Protein-DNA interactions (PDIs) mediate a broad range of functions essential for cellular differentiation, function, and survival. However, it is still a daunting task to comprehensively identify and profile sequence-specific PDIs in complex genomes. Here, we have used a combined bioinformatics and protein microarray-based strategy to systematically characterize the human protein-DNA interactome. We identified 17,718 PDIs between 460 DNA motifs predicted to regulate transcription and 4,191 human proteins of various functional classes. Among them, we recovered many known PDIs for transcription factors (TFs). We identified a large number of unanticipated PDIs for known TFs, as well as for previously uncharacterized TFs. We also found that over three hundred unconventional DNA-binding proteins (uDBPs)--which include RNA-binding proteins, mitochondrial proteins, and protein kinases--showed sequence-specific PDIs. One such uDBP, ERK2, acts as a transcriptional repressor for interferon gamma-induced genes, suggesting important biological roles for such proteins.

Fungal biology reaps the benefit of genomics.

A report on the 23rd Fungal Genetics Conference, Pacific Grove, USA, 15-20 March 2005.

The rice blast pathosystem as a case study for the development of new tools and raw materials for genome analysis of fungal plant pathogens.

Fungi have an astounding and diverse impact on this planet. While they are agents of human diseases and the cause of allergic reactions, factories for the conversion of carbon in environmental and industrially adapted systems, and potential biological weapons, their importance as plant pathogens is unparalleled. In plants alone, fungi cause tens of thousands of different diseases and are responsible for massive losses of food, fiber and forestry at an estimated annual cost of hundreds of billions of dollars. These losses are not only realized in the incomes of individual farmers and state economies, but contribute significantly to world hunger problems and issues relating to safeguarding a global food supply. Our collective understanding of how fungi, particularly plant pathogens, grow, reproduce, identify a host and cause disease is still at a formative stage. There is an equal lack of detailed knowledge about how a plant recognizes that it is being attacked and then mounts an adequate defense response. The advent of genomic technologies has given researchers an unprecedented opportunity to address these mysteries in a powerful and more holistic manner. Where the genetic revolution of only a few years ago allowed for the characterization of single genes, today's genomic technologies are facilitating the evaluation of the entire complement of genes in an organism and the discovery of the suites of genes that act during any one time or particular condition. This review will describe the recent development of tools for whole or partial genome analysis and multigenome comparisons. Th discussion focuses on the rice blast pathosystem as a case study.