The NetworKIN database can be accessed in several different ways. In the following, we will explain the various features of the web interface, using the tumour suppressor 53BP1 as an example. For large-scale analysis or visualization, most users will probably prefer to download the complete set of predictions for human phosphoproteins, which is available in tab-separated and Cytoscape format.
For all other users, the primary entry point to NetworKIN is its search interface shown in A. The user can select a specific substrate and/or kinase to view the corresponding subset of predictions; in our example, we query for 53BP1 as the substrate and use the wildcard * to obtain predictions for all kinases. The web interface also offers an advanced search form, which enables the user to pose much more refined queries. In either case, the search results will be presented as a table in which each row shows a predicted relation between a kinase and a specific phosphorylation site in a substrate. In case of 53BP1, we get a list of 78 predictions for 39 sites and 12 kinases; the first 10 of these predictions are shown in B. For each prediction we list two scores, namely the context score and the motif score, both of which should preferably be high. It should be noted that the motif scores for different kinase families are not comparable; in particular, motif scores from NetPhosK should not be compared with motif scores from Scansite. For this reason, the predictions for a given phosphorylation site are sorted by their context score. As the results of a single query may be extensive, the results can also be downloaded in the formats mentioned previously.
Figure 2. Using NetworKIN. A researcher is interested in the 53BP1 tumour surpressor. From the homepage of NetworKIN, this protein is chosen as the substrate protein to query the database for predictions relating any kinase to specific phosphorylation sites within (more ...)
Furthermore, the user can investigate the predictions in greater detail via the web interface. For each substrate, we link to Phospho.ELM or PhosphoSite where the user can find manually curated information on in vivo phosphorylation sites including, when known, the kinase(s) involved (C). To allow the user to investigate how a specific prediction was made by NetworKIN, we provide a link to the string network viewer, in which the most probable path connecting the kinase and the substrate will be highlighted (D). Alternatively, the user can select multiple predictions and display the network context for all the proteins involved. From the network viewer, the evidence underlying each individual association can be inspected in further detail. This ability to thoroughly investigate individual predictions is particularly useful for interpreting non-obvious cases, which are often based on indirect links between the kinase and the substrate.
Although Phospho.ELM, PhosphoSite and hence NetworKIN are kept up-to-date with new published phosphorylation sites, many researchers will be interested in predictions for their own, unpublished sites. We thus allow users to submit protein sequences and a corresponding set of phosphorylation sites for analysis; although possible, we discourage submitting sequences without prior knowledge on phosphorylation. After uploading the data, the user will be presented with a confirmation page where potential data entry errors can be detected and fixed. The final predictions will be presented in a tabular format similar to the one used when querying the precomputed results in the database.
Many users are interested in specific kinases or substrates; however, others may want to get an overview of the complete phosphorylation network. To facilitate this, the resource offers a global map of all predictions currently in the database. All kinases and substrates are shown using a colour scale to signify their connectivity, namely the number of substrates for a given kinase or the number of kinases for a given substrate. By selecting one or more kinases, all corresponding substrates are highlighted and vice versa. Deselecting one kinase will deselect only the substrates specific for that kinase, keeping the other ones. We find this approach to be an intuitive way to gain insight into pleiotropic properties of kinases. Similar to the search interface, map selections can be visualized in their network context.