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.

Characterization of Protein-Protein Interactions Using Protein Microarrays.

Functional protein microarrays allow fast, straightforward, and efficient high-throughput screening of protein-protein interactions. The microarray approach has outpaced other interaction methods, such as yeast two-hybrid screens, in part because of the vast amounts of information that can be obtained during a single assay. This protocol describes how to perform a binding assay for a protein of interest using a proteome microarray composed of thousands of functional, recombinant proteins adhered to a microchip.

Protein Microarrays: Flexible Tools for Scientific Innovation.

Protein microarrays have emerged as a powerful tool for the scientific community, and their greatest advantage lies in the fact that thousands of reactions can be performed in a parallel and unbiased manner. The first high-density protein microarray, dubbed the "yeast proteome array," consisted of approximately 5800 full-length yeast proteins and was initially used to identify protein-lipid interactions. Further assays were subsequently developed to allow measurement of protein-DNA, protein-RNA, and protein-protein interactions, as well as four well-known posttranslational modifications: phosphorylation, acetylation, ubiquitylation, and SUMOylation. In this introduction, we describe the advent of high-density protein microarrays, as well as current methods for assessing a wide variety of protein interactions and posttranslational modifications.

Structural and biochemical characterization of the bilin lyase CpcS from Thermosynechococcus elongatus.

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded ß barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.