2022 Volume 66 Issue 1 Pages 13-20
Sixty-eight human tyrosine kinases were cloned into the Escherichia coli expression vector pET21a(+) and 48 of them were successfully expressed in the BL21(DE3) strain. Among them, 18 kinases were activated in the bacterial cells by autophosphorylation and they phosphorylated endogenous proteins. Since E. coli expression systems do not usually enable post-translational modifications, the in vivo phosphorylation should be due to the tyrosine kinase expressed. In this study, we attempted to use the bacterial expression system as a site for the kinase reaction to determine the substrate sequence preference of a certain kinase, namely, a phosphorylation motif. As a first example, phosphoproteomic analysis of E. coli expressing active bone marrow tyrosine kinase gene in chromosome X protein (BMX) was performed to extract the phosphorylation motifs. The motifs were compared with that obtained from Kinapple, the database of in vitro human kinome profiling, and common features rich in acidic amino acids were found. Furthermore, the phosphorylation state of expressed BMX was analyzed. Phos-tag SDS-PAGE in conjunction with alanine scans of the phosphorylation sites found by mass spectrometry revealed that Y224, Y234, and Y566 are predominantly phosphorylated. Moreover, phosphorylation at S453 and T572 was implicated in activation in E. coli, whereas these sites did not affect activity in 293 cells. These results suggest that, although the bacterial system does not fully reflect the physiological properties of the kinase, it is a simple experimental method that can infer the substrate preference of tyrosine kinases similar to that observed with substrates of human origin.
