Characterizing serine 354 in the Bcr protein: Implications in the negative regulation of the Bcr-Abl oncoprotein.

摘要

The Bcr 64-413 truncated protein, Delta Bcr, can inhibit the tyrosine kinase and transformation activities of the BCR-ABL oncogene. Bcr-Abl co-expressed with Delta Bcr in COS 1 cells has decreased tyrosine phosphorylation and decreased in vitro autophosphorylation activity. Similarly, Bcr-Abl protein derived from Bcr-Abl-positive leukemia cells has inhibited in vitro autophosphorylation activity when incubated with the Delta Bcr protein or peptide derived from Delta Bcr phosphorylated on serine 354. Delta Bcr can be expressed in Bcr-Abl positive leukemia cells using the adenovirus system and can inhibit the growth and survival of these cells. This effect is Bcr-Abl-specific, because Delta Bcr expression has no effect on Bcr-Abl-negative leukemia cells.; This thesis begins to verify the significance of phosphorylated serine 354 in the Bcr protein. Replacing serine 354 for alanine decreases the inhibitory effects of Delta Bcr in the Bcr-Abl-positive leukemic cell line K562. K562 cells infected with Adv/Delta BCR had decreased viability compared to mock infected or Adv/GAL infected K562 cells. There was also a significant difference in viability compared to Adv/Delta Bcr S354A infected K562 cells, indicating that Serine 354 contributes to the cytotoxic function of Delta Bcr in Bcr-Abl positive leukemia cells.; The Delta Bcr protein migrates as two distinct species (p45 and p55/60) on denaturing SDS-PAGE while Delta Bcr S3 54A migrates as only one species of 45 kDa. The higher molecular weight species of Delta Bcr, which is absent in Delta Bcr S354A, specifically binds to the Abl SH2 domain in in vitro pull down assays with the GST-Abl SH2 fusion protein.; I propose that Delta Bcr exists inside of cells as two species and that the p55/p60 form is the active inhibitor of Bcr-Abl. By interacting with the Abl SH2 domain Delta Bcr is a novel Bcr-Abl inhibitor that may compete with the SH2 binding partners of Bcr-Abl as well as hinder Bcr-Abl's access to substrates.