Phosphorylation of meprin β controls its cell surface abundance and subsequently diminishes ectodomain shedding

Meprin beta is a zinc-dependent metalloprotease exhibiting a unique cleavage specificity with strong preference for acidic amino acids at the cleavage site. Proteomic studies revealed a diverse substrate pool of meprin beta including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprin beta is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprin beta including interactors, sheddases, and activators has been intensively investigated while intracellular regulation has been barely addressed in the literature. This study aimed to analyze C-terminal phosphorylation of meprin beta with regard to cell surface expression and proteolytic activity. By immunoprecipitation of endogenous meprin beta from the colon cancer cell line Colo320 and subsequent LC-MS analysis, we identified several phosphorylation sites in its C-terminal region. Here, T694 in the C-terminus of meprin beta was the most preferred residue after phorbol 12-myristate 13-acetate (PMA) stimulation. We further demonstrated the role of protein kinase C (PKC) isoforms for meprin beta phosphorylation and identified the involvement of PKC-alpha and PKC-beta. As a result of phosphorylation, the meprin beta activity at the cell surface is reduced and, consequently, the extent of substrate cleavage is diminished. Our data indicate that this decrease of the surface activity is caused by the internalization and degradation of meprin beta.

Automated summary

Meprin beta is a zinc-dependent metalloprotease with a unique cleavage specificity for acidic amino acids at the cleavage site. Phosphorylation of meprin beta affects its cell surface activity and substrate cleavage extent, leading to internalization and degradation. The involvement of PKC isoforms in meprin beta phosphorylation was also demonstrated in this study.