Trypsin is a digestive enzyme that is widely used for cell detachment, which is the first stage of tumor metastasis. Recent studies show that adhesion-related kinases are involved in cell detachment. Proline-rich tyrosine kinase 2 (Pyk2) is a crucial kinase in the regulation of cell adhesion and detachment. However, the effect of Pyk2 on cell detachment is controversial. In the present study, we found that Pyk2 expression was rapidly decreased after trypsin treatment in gastric cancer, breast cancer, colon cancer, lung cancer, and human gastric epithelial cells. Knockdown of Pyk2 accelerated cell detachment. Furthermore, lysosome inhibitor NH4CL suppressed cell detachment and increased ubiquitination of Pyk2. Cbl-b is a type of E3 ubiquitin ligase that interacted with Pyk2, reduced the expression of Pyk2, and promoted trypsin-induced degradation of Pyk2. These findings suggest that Cbl-b promoted cell detachment through mono-ubiquitination of Pyk2. Our data provide a new insight into the role of Cbl-b in cell detachment.
Apoptosis Blotting, Western