PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse

Abstract

PhosphoSitePlus (http://www.phosphosite.org) is an open, comprehensive, manually curated and interactive resource for studying experimentally observed post-translational modifications, primarily of human and mouse proteins. It encompasses 1,30,000 non-redundant modification sites, primarily phosphorylation, ubiquitinylation and acetylation. The interface is designed for clarity and ease of navigation. From the home page, users can launch simple or complex searches and browse high-throughput data sets by disease, tissue or cell line. Searches can be restricted by specific treatments, protein types, domains, cellular components, disease, cell types, cell lines, tissue and sequences or motifs. A few clicks of the mouse will take users to substrate pages or protein pages with sites, sequences, domain diagrams and molecular visualization of side-chains known to be modified; to site pages with information about how the modified site relates to the functions of specific proteins and cellular processes and to curated information pages summarizing the details from one record. PyMOL and Chimera scripts that colorize reactive groups on residues that are modified can be downloaded. Features designed to facilitate proteomic analyses include downloads of modification sites, kinase-substrate data sets, sequence logo generators, a Cytoscape plugin and BioPAX download to enable pathway visualization of the kinase-substrate interactions in PhosphoSitePlus®.

Figure 1.

Informational content of PhosphoSite® curated from the literature from its launch in 2003 through 2011. Phosphorylation sites (blue), proteins (red) and references (gray). The approximate dates that descriptions of three other resources were published are indicated by black arrows: HPRD (19), phospho.ELM (20) and PHOSIDA (21). Inflection points indicate the publication of large MS data sets; a few are marked with red arrowheads.

Figure 2.

Overview of the navigational flow and content of PSP.

Figure 3.

The Downloads, Links and Applications section provides (1) multiple modification site data sets from PhosphoSitePlus, the complete archive of current reference proteins (Phosphosite_seq), and the extensive Kinase–Substrate data set in three formats: the Kinase_Substrate_Data set is a tab-delimited txt file, a Cytoscape Plugin (phosphositeClient_jar), and BioPAX format (37); (2) a logo generator with which users can analyze uploaded data sets; (3) ATM substrate sequences extracted from the kinase-substrate data set were segregated into in vitro and in vivo reactions and (4) analyzed using the PSP Logo Generator and (5) a subset of the data extracted from PSP and visualized using Cytoscape (36).

Figure 4.

Protein Page overview section for YAP1. (lower left) The linked Hippo Pathway from CST, with links to PSP proteins, can be opened and downloaded from this page. (lower right) Selected PDF files, in this case 3KYS (46), can be opened in the Viewer window with the OpenAstexViewer (41).

Figure 5.

Protein Page sections 3 and 4 for YAP1. (A) The Modification Sites and Domains section. (B) Modification Sites in Parent Protein, Orthologs, and Isoforms section. The two left most columns outlined in red show the total number of references using SS or MS to characterize the site group.

Figure 6.

The locations of four protein posttranslational modifications relative to Pfam-A domains. The locations of 4 modification types were scored as either inside or outside of Pfam-A domains. pS/T, phospho-Ser/-Thr; pY, phospho-Tyr; acK, acetyl-Lys; ubK, ubiquitinyl-Lys.

Figure 7.

The topology of modified residues from PSP on the small G protein RAN visualized with the PDB file for 3CH4. (A) The table of modification sites on the Ran (human) Protein Page. (B) PDB 3CH5 was opened in the Astex viewer. Tyr-147 and -155 are marked. (C) The PSP ChimeraX script was downloaded and opened in Chimera. Tyr-147 and -155 are marked, and the tyrosyl hydroxyls that can be phosphorylated are colored red. (D) Chimera model of 3CH4 in space-filling mode. The acetylated ε-amino group of Lys-71 is colored green.