Quantities of proteins in cells are balanced by protein synthesis and degradation. Protein ubiquitination is an important adenosine-triphosphate dependent proteolytic pathway for 'short-lived' proteins. We show that soluble steel-factor (SLF) stimulation at 37°C rapidly induced polyubiquitination of c-kit protein in growth-factor-dependent human-myeloid cell line M07e, resulting in smeared, retarded migration of c-kit protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the molecular weight region of 145 kD. Receptor ubiquitination was almost completely absent when cells were treated with SLF at 4°C or at 37°C in the presence of 0.2% sodium azide, or when the cells were pretreated with anti-c-kit monoclonal antibody or genistein, a tyrosine-kinase inhibitor. This suggested that c- kit ubiquitination was ligand dependent and appeared to require intrinsic tyrosine-kinase activation of the c-kit protein. Flow-cytometric analysis of c-kit expression on the cell surface of M07e cells showed down modulation of c-kit within 5 minutes after soluble-SLF treatment at 37°C. However, rapid receptor down modulation was almost completely suppressed when cells were treated with SLF at 4°C or at 37°C in the presence of 0.2% sodium azide, conditions that concomitantly suppressed polyubiquitination of c-kit protein. In addition, these conditions almost completely suppressed radiolabeled SLF (125I-SLF) internalization after ligand-receptor interaction. Pulse-chase studies of 35S-methionine-labeled c-kit protein showed that SLF stimulation at 37°C strikingly enhanced c-kit degradation (T 1/2 ; ~20 minutes) compared with that in cells stimulated with SLF at 4°C or at 37°C with 0.2% sodium azide. However, in the presence of chloroquine, which blocks lysosomal degradation, this ligand-induced c-kit degradation at 37°C was only suppressed in part. These data suggest that SLF-induced polyubiquitination of the c-kit receptor protein may play a role in regulation of c-kit-encoded protein-receptor expression in M07e cells.
ASJC Scopus subject areas
- Cell Biology