PhosphoLink User Guide

PhosphoLink is a literature-based database of kinase and phosphatase regulatory relationships. It helps researchers explore which kinases or phosphatases have been reported to regulate which substrate proteins, what evidence supports each relationship, and where phosphorylation or dephosphorylation sites are reported or mapped.

Use this guide to understand the main data types in PhosphoLink, navigate the website, interpret evidence records, and explore regulatory networks.

Integrated databases, reference resources, and open-source software used by PhosphoLink are listed in Acknowledgements on the About page.

What PhosphoLink contains

PhosphoLink focuses on experimentally discussed or curated regulatory relationships from the scientific literature.

Each regulatory relationship is represented as a directed link:

Enzyme → Substrate

The enzyme side may be:

• A protein kinase
• A protein phosphatase
• A kinase or phosphatase family, when the source describes regulation at the family level

The substrate side is a regulated protein.

Each relationship includes:

• The reaction type: phosphorylation or dephosphorylation
• The enzyme or enzyme family
• The substrate protein
• Supporting source literature
• Evidence text extracted or curated from the source
• Database comments for that relationship
• Site information when available
• An annotation score summarizing support for the pair

PhosphoLink is not intended to be a prediction-only resource. Relationships are included when they are supported by source literature or curated database evidence.

Main things you can do

Regulatory relationship

A regulatory relationship is a reported kinase-to-substrate or phosphatase-to-substrate link.

Examples:

• Kinase → substrate: phosphorylation
• Phosphatase → substrate: dephosphorylation
• Kinase family → substrate: family-level phosphorylation evidence
• Phosphatase family → substrate: family-level dephosphorylation evidence

A single enzyme-substrate pair may be supported by multiple publications.

Evidence

Evidence is the source text that supports a relationship.

On a regulatory relationship page, Evidence usually contains sentences from the publication that describe the enzyme, substrate, regulation, or site information. Relevant phrases may be highlighted in the abstract.

Use Evidence when you want to check what the paper says.

Comment

Comment is PhosphoLink’s note for a specific relationship and source.

It may summarize how the relationship is described in the database, clarify family-level wording, or provide additional context about the curated entry.

Comment is not the same as Evidence, and it is not user feedback.

Annotation score

Annotation score is a pair-level score for the enzyme-substrate relationship.

It summarizes support across all sources linked to that pair. It is shown at the relationship level, not as a score for a single abstract.

Use Annotation score as a quick summary of database support, but open the relationship page to read the underlying Evidence and Comment.

In the website and the browse tables, Annotation score is shown on a five-point scale. The underlying stored value is normalized to 0 to 1, then displayed as 1 to 5.

Reported site and mapped site

PhosphoLink stores phosphorylation or dephosphorylation site information when available.

Reported site is the site wording as described in the source. It may refer to a specific isoform, residue, or position used in the paper.

Mapped site is the residue and position mapped onto the sequence view shown in PhosphoLink. This may be the canonical protein sequence or a selected isoform.

Use reported sites to understand the original publication wording. Use mapped sites to inspect sequence and structure positions in PhosphoLink.

PhosphoLink groups data into a small set of record types and views. Use this page as a map; each area is described in the guide sections that follow.

Home, search, and Browse Database

The Home page links to the User Guide, Network, Browse Database, and Download.

The Download page provides files for the full edge table, the agent-extracted subset, and the four Browse Database lists. See Downloads for a short guide to each file.

The top search bar finds proteins, families, regulatory relationships, and source literature by UniProt accession, gene symbol, protein name, family name, PMID, or publication keywords. Results are grouped by record type.

Use UniProt accessions or PMIDs for the most specific matches.

Browse Database opens sortable tables when you want to scan or filter full lists rather than open a known record directly.

A protein record contains the main information PhosphoLink has for one UniProt accession. Open records from search, Browse Database, or links on family, relationship, and literature pages.

Basic information

The Basic Info section may include:

• UniProt accession
• Recommended protein name
• Gene symbol
• Organism
• Role labels
• Family membership
• Function (from UniProt)
• For substrates: phosphorylation sites, sequence, and structure (when available)

Role labels indicate how the protein appears in PhosphoLink. A single protein may have both kinase and substrate roles.

For example, one protein may be both a kinase and a substrate.

Phosphorylation sites

For substrate proteins, the Sites as Substrate table on the Basic Info tab lists phosphorylation and dephosphorylation sites linked to that accession.

Each row summarizes one reported site and the literature behind it. The table may include:

• Source PMIDs — publications that mention the site
• Modification — phosphorylation or dephosphorylation
• Reported site — residue position as stated in the source
• Mapped site — position on the sequence currently selected in PhosphoLink

The reported site follows the source wording. The mapped site is the coordinate PhosphoLink uses on the displayed sequence. When canonical and isoform views are available, switching the sequence view can update mapped coordinates; the reported site text usually stays unchanged because it reflects the original source.

The mapped positions in this table are also highlighted on the sequence and structure views described in the following sections.

Sequence view

The Sequence row on the Basic Info tab shows the canonical or isoform amino acid sequence for the accession.

Amino acids are shown in blocks with position labels above each group. Mapped sites from the Sites table are highlighted on the sequence at the corresponding residues. Use the sequence selector in the Sites or Sequence header to switch between canonical and isoform views when isoforms are available.

Structure view

When a cached AlphaFold prediction exists for the accession, the Structure row shows an interactive 3D panel.

• Structure data: Models come from AlphaFold DB (EBI). PhosphoLink displays the AlphaFold PDB associated with the UniProt accession on the page. Cartoon colors follow AlphaFold model confidence (pLDDT). PhosphoLink does not generate these structures.
• 3D viewer: Rendering uses 3Dmol.js.
• Mapped sites: Mapped positions from the Sites table can be highlighted on the structure at the corresponding residues, using the same canonical or isoform-aware mapping as the sequence view.

If no structure is cached for that protein, the page states that no AlphaFold structure is available.

Regulatory relationships tab

The Regulatory Relationships tab lists all regulatory links involving the protein.

This table may include:

• Enzyme
• Substrate
• Reaction type
• Annotation score
• Literature count
• Link to the relationship page

Open a regulatory relationship page to inspect Evidence and Comment for each supporting source.

Related source literature tab

The Related Source Literature tab lists publications connected to the protein through its regulatory relationships.

Use this tab to move from a protein-centered view to the papers supporting its regulatory links.

Family records describe kinase or phosphatase groups represented in PhosphoLink.

A family page may include:

• Family name
• Member proteins
• Family-level regulatory relationships
• Related source literature

Family-level relationships are used when the evidence supports regulation by a group rather than a single protein accession.

In the Network view, a family may appear as one node representing the group.

A regulatory relationship page is the main place to evaluate support for one enzyme-substrate pair.

Pair summary

The top of the page summarizes the relationship.

For complete phosphorylation site tables, open the substrate protein record.

Source blocks

Each supporting source is shown in its own block.

A source block may include:

Evidence

Sentences or curated text supporting the relationship. Abstract phrases may be highlighted.

Comment

PhosphoLink’s database note for that pair and source.

Annotation confidence

When shown, this is a source-level confidence display. It should not be confused with the pair-level Annotation score shown at the top of the page.

How to read a relationship page

Use this order:

1. Check the enzyme, substrate, and reaction type.
2. Review the site summary.
3. Check the annotation score and literature count.
4. Read each Evidence block.
5. Read the Comment for database context.
6. Open PMIDs for source-level context.
7. Open the substrate protein page for complete site information.

A source literature page organizes PhosphoLink content around one publication.

Use this page when you want to know which relationships a paper supports.

On the detail page you will see:

• PMID (in the page heading)
• Title
• Link to PubMed
• Publication year, when available
• Abstract
• A table of regulatory relationships supported by the paper (reaction type, enzyme or family, substrate, annotation confidence)

The Browse Database literature list shows PMID, year, title, and the number of linked regulatory relationships.

Each relationship row links to the corresponding regulatory relationship page, where Evidence and Comment are shown for that pair.

The Network page is an interactive workspace for exploring regulatory relationships between proteins. Build a focused network around proteins or regulator families of interest, inspect the supporting evidence, and download the results for further analysis.

Read the network

Nodes represent proteins or kinase/phosphatase families. Arrows represent regulatory relationships and point toward the regulated substrate. Family nodes are drawn larger than single-protein nodes.

The colour scale in the Guide card on the Network page (bottom-right) indicates how common each node type is within the displayed network. A node with more than one role may use multiple colours.

The toolbar

The tools live in a vertical toolbar along the left edge of the workspace. Each button shows its name beside its icon. From top to bottom: Undo, Redo, Search and Add, Manage Network, Regulatory Router, and PhosphoLink Agent.

Manage Network, Regulatory Router, and PhosphoLink Agent are unavailable (grayed out) until at least one node is on the graph.

The workspace can show up to 300 nodes at once.

Build a network

Click Search and Add in the left toolbar to open the search panel, then search by gene name, UniProt ID, or family name. Select a result to add it to the workspace. When proteins with known relationships are present, the connecting arrows appear automatically.

To grow the network from an existing node:

1. Right-click the node.
2. Choose a relationship category.
3. Pick Add all or Search and add.

Right-click a node and select Delete to remove it from the workspace.

Manage Network

Click Manage Network to open a panel with three groups of actions:

Complete regulatory relationships draws missing regulatory edges between proteins already on the graph. These are connections that exist in PhosphoLink but are not shown yet. Use Complete missing relationships in selected region after selecting two or more nodes, or Complete missing relationships in entire network for the whole workspace. The button is disabled when no missing edges remain in that scope.

Download exports the visible network or a selected region as Excel. The file includes Proteins, Relationship, and Literature sheets. Only regulatory edges currently drawn on the graph are exported.

The Relationship sheet lists each exported enzyme–substrate pair with reaction type, protein names, sites, PMIDs, and Verified annotation (Yes / No). This column uses the same rule as the green checkmark beside Annotation score on browse pages: Yes when at least one supporting annotation for that pair is curator-verified.

Delete removes part of the graph or clears the workspace. You can Keep only selected region (delete the rest), Delete selected region, or Delete entire network.

The same complete, download, and delete actions are also available from region context menus. Select multiple nodes with Ctrl+click or Shift+drag, then right-click the selection.

Inspect nodes and relationships

Select a node to open its information panel. The panel summarizes the protein or family and provides a link to its full record.

Select an arrow to view the relationship, available evidence, source literature, and links to the connected records. Double-click a node to open its full protein or family page.

Right-click an arrow to delete that edge from the workspace.

Use the mouse or trackpad to move around the workspace:

Undo and redo

Use the Undo and Redo buttons at the top of the left toolbar, or press Ctrl+Z / Ctrl+Y ( Cmd+Z / Cmd+Y on macOS). Each stack keeps up to five recent steps.

Undo and redo cover node additions and removals and related layout updates made during the current session.

Regulatory Router

Regulatory Router finds regulatory paths between two nodes already on the workspace, using the full curated link set behind PhosphoLink.

Open Regulatory Router with the left toolbar button. Choose From and To from the dropdown lists, or select a node on the graph and use Use selected beside either field. Click Find paths to search.

Results are listed shortest to longest. Click a path to select it, then use Add to graph to add every protein along the selected paths to the workspace. Open Settings (gear icon) to adjust Max result length (hop limit) and Results shown.

Changing From or To, or removing either endpoint from the graph, clears the current path results. Minimizing Router keeps your results for the current session; refreshing the page starts a new session.

PhosphoLink Agent

The Network page includes PhosphoLink Agent, a natural-language assistant for questions about proteins, regulatory relationships, source literature, and the network currently displayed.

Open the Agent with the PhosphoLink Agent button in the left toolbar. You can ask questions such as which proteins are connected, what evidence supports a relationship, or what is shown in the current network. Use the trash icon to clear the chat history. Minimize the panel with the header control; reopen it from the same button.

To view the complete Evidence and Comment sections, site tables, and PMID lists, please open the linked structured record pages from the agent's reply.

The Download page provides detailed evidence-record datasets and four summary views of the complete PhosphoLink database.

Evidence-level datasets

PhosphoLink_Literature_Derived_Regulatory_Evidence_Records_*.xlsx contains literature-derived records with annotated original-sentence Evidence and a detailed Comment describing the regulatory relationship.

PhosphoLink_All_Regulatory_Evidence_Records_*.xlsx contains all regulatory evidence records and additionally includes records integrated from external databases. External-only records provide provenance in Comment, but do not have original-sentence Evidence.

The remaining four files summarize the complete database from protein, family, regulatory relationship, and publication perspectives. Use them when you do not need one row per evidence record.

Files

File details

PhosphoLink_Literature_Derived_Regulatory_Evidence_Records_*.xlsx

This file contains literature-derived regulatory evidence records. Each row includes annotated original-sentence Evidence and a detailed Comment describing the literature-derived regulatory relationship.

Choose the complete file when analyzing both phosphorylation and dephosphorylation, or use one of its reaction-specific subsets:

• PhosphoLink_Literature_Derived_Phosphorylation_Evidence_Records_*.xlsx
• PhosphoLink_Literature_Derived_Dephosphorylation_Evidence_Records_*.xlsx

PhosphoLink_All_Regulatory_Evidence_Records_*.xlsx

This is the complete PhosphoLink database at evidence-record level. Each row records one kinase-, phosphatase-, or regulator-substrate relationship evidence record from the literature or an integrated external database.

Choose the complete file when analyzing both phosphorylation and dephosphorylation, or use one of its reaction-specific subsets:

• PhosphoLink_All_Phosphorylation_Evidence_Records_*.xlsx
• PhosphoLink_All_Dephosphorylation_Evidence_Records_*.xlsx

• enzyme_gene_name - Gene symbol of the kinase, phosphatase, or regulator.
• enzyme_protein_name - Full protein name of the kinase, phosphatase, or regulator.
• enzyme_id - UniProt accession of the kinase, phosphatase, or regulator, or a family ID.
• enzyme_type - Regulatory role (kinase, phosphatase, phosphorylation regulator, or dephosphorylation regulator).
• enzyme_organism - Organism of the kinase, phosphatase, or regulator.
• substrate_gene_name - Gene symbol of the substrate protein.
• substrate_protein_name - Full protein name of the substrate.
• substrate_id - UniProt accession of the substrate.
• substrate_organism - Organism of the substrate.
• pmid - Supporting PubMed ID.
• year - Publication year for the supporting PubMed record.
• evidence - Evidence text supporting the relationship.
• comment - Extraction or curation notes.
• enzyme_name_extract - Raw extracted kinase/phosphatase/regulator name from the source record.
• substrate_name_extract - Raw extracted substrate name from the source TSV.
• annotation_score - Five-point display score for the relationship. This matches the website display.
• annotation_score_raw - Underlying normalized score in the 0 to 1 range.
• annotation_verified - Yes if this evidence record is curator-verified; otherwise No. Matches the Verified badge on individual source-literature annotations.
• report_site - Site as reported in source.
• canonical_name - Canonical substrate identifier.
• canonical_seq - Canonical substrate sequence.
• mapped_site_in_canonical - Mapped site on canonical sequence.
• isoform_name - Isoform used for mapping.
• isoform_seq - Isoform sequence.
• mapped_site_in_isoform - Mapped site on isoform sequence.

PhosphoLink_Protein_Entries_*.xlsx

This is the protein-centered summary view of the complete PhosphoLink database.

It corresponds to the Protein Entries database page.

• UniProt ID - Protein accession.
• Name - Displayed protein or gene name.
• Type - Protein role category.
• Family - Associated kinase/phosphatase family name.
• Organism - Source organism.

PhosphoLink_Kinase_Phosphatase_Families_*.xlsx

This is the kinase- and phosphatase-family-centered summary view of the complete PhosphoLink database.

It corresponds to the Kinase/Phosphatase Family database page.

• Family name - Family name shown in browse view.
• Type - Kinase or phosphatase family type.
• Members - Comma-separated UniProt IDs of member proteins.

PhosphoLink_Regulatory_Relationships_*.xlsx

This is the regulatory-relationship-centered summary view of the complete PhosphoLink database.

It corresponds to the Regulatory Relationships database page.

• Type - Phosphorylation or dephosphorylation.
• Kinase / Phosphatase / Regulator - Displayed name of the regulatory protein.
• Kinase / Phosphatase / Regulator (UniProt ID) - UniProt ID of the kinase, phosphatase, or regulator.
• Substrate - Displayed substrate name.
• Substrate UniProt ID - UniProt ID of the substrate.
• Site - Observed site labels.
• Source Literature - PMID list supporting the relationship.
• Annotation score - Five-point display score shown in browse view.
• Annotation score (raw) - Underlying normalized score in the 0 to 1 range.
• Verified annotation - Yes if at least one supporting annotation for this pair is curator-verified; otherwise No. Matches the green checkmark beside Annotation score on browse pages.

PhosphoLink_Source_Literature_*.xlsx

This is the publication-centered summary view of the complete PhosphoLink database.

It corresponds to the Source Literature database page.

• PMID - PubMed identifier.
• Year - Publication year.
• Title - Article title.
• MeSH - MeSH terms as stored in PhosphoLink (original PubMed heading string).
• Regulatory Relationships - Comma-separated source-target relationship list.

Use Report Error on a protein, family, regulatory relationship, or source literature detail page to suggest a correction.