The aminopeptidase B (Ap-B) is phosphorylated in HEK293 cells.

Proteolysis is a post-translational modification (PTM) that affects the whole proteome. First regarded as only destructive, it is more precise than expected. It is finely regulated by other PTMs like phosphorylation. Aminopeptidase B (Ap-B), a M1 metallopeptidase, hydrolyses the peptide bond on the carbonyl side of basic residues at the NH 2 -terminus of peptides. 2D electrophoresis (2DE) was used to show that Ap-B is modified by phosphorylation. Detection of Ap-B by western blot after 2DE reveals several isoforms with different isoelectric points. Using alkaline phosphatase, Pro-Q Diamond phosphorylation-specific dye and kinase-specific inhibitors, we confirmed that Ap-B is phosphorylated. Phosphorylation can alter the structure of proteins leading to changes in their activity, localization, stability and association with other interacting molecules. We showed that Ap-B phosphorylation might delay its turnover. Our study illustrates the central role of the crosstalk between kinases and proteases in the regulation of many biological processes. • The metallopeptidase, hydrolyses the peptide bond on the carbonyl side of basic residues at the NH 2 -terminus of peptides. • Phosphorylation is responsible for the regulation of many proteases. • After 2D electrophoresis, Ap-B presents a "train of spots" signature which is specific of phosphorylation. • Specific dyes and kinase inhibitors were used to show that Ap-B is phosphorylated on serine, threonine and tyrosine residues. • Ap-B phosphorylation seems to delay its turnover. [ABSTRACT FROM AUTHOR]