Update: 2011 February 21

About PhosphoNET …

PhosphoNET is an open-access, online resource developed by Kinexus Bioinformatics Corporation to foster the study of cell signalling systems to advance biomedical research in academia and industry. PhosphoNET is the world’s largest repository of known and predicted information on human phosphorylation sites, their evolutionary conservation and the identities of protein kinases that may target these sites. PhosphoNET presently holds data on over 650,000 known and putative phosphorylation sites (P-sites) in over 23,000 human proteins that have been collected from the scientific literature and other reputable websites. Over 14% of these phospho-sites have been experimentally validated. The rest have been predicted with a novel P-Site Predictor algorithm developed at Kinexus with academic partners at the University of British Columbia and Simon Fraser University.

With the PhosphoNET Evolution module, this website also provides information about cognate proteins in over 20 other species that may share these human phospho-sites. This helps to define the most functionally important phospho-sites as these are expected to be highly conserved in nature.

With the Kinase Predictor module, listings are provided for the top 50 human protein kinases that are likely to phosphorylate each of these phospho-sites using another proprietary kinase substrate prediction algorithm developed at Kinexus. Our kinase substrate predictions are based on deduced consensus phosphorylation site amino acid frequency scoring matrices that we have determined for each of ~500 different human protein kinases. The specificity matrices are generated directly from the primary amino acid sequences of the catalytic domains of these kinases, and when available, have proven to correlate strongly with substrate prediction matrices based on alignment of known substrates of these kinases. The higher the score, the better the prospect that a kinase will phosphorylate a given site. Over 30 million kinase-substrate phospho-site pairs are quantified in PhosphoNET. Kinexus Bioinformatics Corporation has the capability to test most of these putative interactions in vitro for our clients.

Role of Phosphorylation …

Protein phosphorylation is well recognized as the premier mode of post-translational regulation of proteins. The human genome encodes over 23,000 proteins, and more than two thirds of these proteins have already been shown to be subject to phosphorylation. Research at Kinexus has revealed the vast majority of proteins are in fact phosphorylatable, and there may be very few exceptions. Based on the number of experimentally confirmed tyrosine phosphorylated sites in PhosphoNET (i.e. >18,900) and the percentage of tyrosine phosphorylation sites relative to the total number of phospho-sites discovered in randomized studies (i.e. ~4%), we originally estimated that there are at least 500,000 human phospho-sites. With our proprietary P-site Predictor algorithm, we have been able to identify over 560,000 new putative human phospho-sites. Of the ~92,500 phospho-sites that have been demonstrated, functional information is available for only about 2%. Only about 0.85% of these phosphorylations have been correlated with an increase in enzymatic activity or a gain of function, whereas only 0.3% result in a decrease in enzymatic activity or a reduction of function. We believe that only a small percentage of the human phosphorylation sites directly regulate activities of proteins and control protein-protein interactions. It would appear that the major role of hyperphosphorylation of proteins is actually to facilitate their degradation by proteases. The challenge for the biomedical research community is to identify the most critical phosphorylation sites that regulate protein functions and serve as biomarkers of human disease. Kinexus is committed to the identification of these important phospho-sites and the development of tools to permit their analysis in experimental model and clinical specimens. PhosphoNET is a free Internet resource that has been developed to facilitate this important endeavor.

Instructions …

PhosphoNET is designed to be fast and simple to navigate. You can search for target protein if you know its UniProt ID, International Protein ID, or one of its common alias names. You can also search PhosphoNET with the complete 15 amino acid sequence (in single letter code) surrounding a phospho-site (the phosphorylated amino acid should be defined in the 8th position). A list of possible options for proteins are generated by typing at least three letters of their name or identification number, and waiting for a few seconds for a complete list to appear. Once a unique phosphoprotein webpage is created, you can hover your cursor over the coloured buttons to the right of the phospho-site sequences to get more information that will appear next to the button. If the button is clicked, this information will appear in the white “Info Box”. Clicking on the orange buttons activates links to additional webpages. PhosphoNET Evolution and PhosphoNET Kinase Predictor are accessed by clicking on the orange buttons under the “Evol.” and “Kinase Pred.” Headings, respectively. Dark grey buttons are presently inactive. The optimum web browsers for using PhosphoNET are Internet Explorer, Firefox and Safari.

Due to the large size of the PhosphoNET Knowledgebase, it may take 15-30 seconds for the PhosphoNET Evolution and PhosphoNET Kinase Predictor modules webpages to load.

Coming Soon…

This website is a work in progress with very limited financial assistance. At this juncture, PhosphoNET features three modules that lists basic information about human phospho-sites, their evolutionary conservation and the identities of protein kinases that may target these phospho-sites. Our goal is to consolidate this information and use it to predict the architecture of key cell signalling networks. Later this year, we are aiming to launch a fourth module of PhosphoNET that predicts SH2- and PTB-domain containing proteins that most likely target these phospho-sites. Quantitative information on several hundred different phosphorylation sites in hundreds of diverse cells and tissues is also available freely through our KiNET DataBank . PhosphoNET is the first of several planned on-line knowledgebases that will become available from Kinexus Bioinformatics Corporation as part of our SigNET KnowledgeBank to accelerate signal transduction research. Additional knowledgebases are in development for human protein kinases, phosphatases, adapter, stress proteins and transcription factors.

Funding and Advertising Opportunities …

To develop and sustain this free resource, we are seeking sponsorship from foundations and corporations. We gratefully acknowledge financial grant support for PhosphoNET from the National Research Council of Canada Industrial Assistance Program and the Natural Sciences and Engineering Research Council of Canada.

It is possible for commercial vendors of signal transduction reagents and services to advertise their relevant products and services on this site. Interested parties should contact our Sales and Marketing Department at info@kinexus.ca or call toll free in North America 1-866-KINEXUS.

Some PhosphoNET Website Statistics …

• Total number of phosphosites: 657,391

• Total number of experimentally-confirmed phosphosites: 92,449

• Percent serine phosphosites: 54.6%

• Percent threonine phosphosites: 31.3%

• Percent tyrosine phosphosites: 14.1%

• Total number of proteins represented: 23,469

• Average number phosphosites per protein: 28

• Number of phosphosites with some functional information: 1553

• Number of phosphosites that are activatory: 631

• Number of phosphosites that are inhibitory: 229

• Average Mr of phosphoproteins: 61,520 Da

• Median Mr of phosphoproteins: 46,000 Da

• Average number amino acids per phosphoprotein: 552

• Median number amino acids per phosphoprotein: 410