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

Abstract

Meprinβ 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 meprinβ including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprinβ is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprinβ 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 meprinβ with regard to cell surface expression and proteolytic activity. By immunoprecipitation of endogenous meprinβ 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 meprinβ 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 meprinβ phosphorylation and identified the involvement of PKC-α and PKC-β. As a result of phosphorylation, the meprinβ 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 meprinβ.