Phosphosites

Phosphosites are specific amino acid residues within proteins that can be modified by phosphorylation. In eukaryotes, the most common sites are serine, threonine, and tyrosine residues, where a phosphate group is attached to a hydroxyl group. In many bacteria and other organisms, phosphorylation also occurs on histidine or aspartate in two-component signaling systems. Phosphorylation is a reversible post-translational modification that regulates protein activity, interaction networks, subcellular localization, and stability, and it plays a central role in cellular signaling.

Kinases catalyze the transfer of a phosphate group from ATP to the target residue, while phosphatases remove it. Kinases often recognize short sequence motifs or structural contexts, leading to large networks of phosphorylation events that relay and coordinate cellular responses. The functional outcome of phosphorylation is highly context dependent and can include enzyme activation or inhibition, creation or disruption of protein interaction sites, and changes in cellular localization.

Phosphoproteomics uses mass spectrometry to identify and quantify phosphosites on a proteome scale. Phosphopeptides are typically enriched by methods such as immobilized metal affinity chromatography or titanium dioxide prior to MS analysis. Data are curated in resources such as PhosphoSitePlus and UniProt, and researchers report the protein, residue, organism, experimental conditions, and often estimates of occupancy or dynamics.

Phosphosites are dynamically regulated, often in response to stimuli, cell cycle cues, or developmental signals. They participate in signaling networks and cross-talk with other post-translational modifications. Conservation of specific phosphosites across species can indicate functional importance, though many sites are lineage-specific or conditionally modified. In prokaryotes, histidine and aspartate phosphorylation underlie signaling networks distinct from the eukaryotic serine/threonine/tyrosine phosphorylation.

Limitations include substoichiometric occupancy, transient modifications, and biases in detection methods, which together require careful interpretation and integrative analyses.