title: Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells.
abstract: 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 degrees 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 degrees C or at 37 degrees 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 degrees C. However, rapid receptor down modulation was almost completely suppressed when cells were treated with SLF at 4 degrees C or at 37 degrees 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 degrees C strikingly enhanced c-kit degradation (T1/2; approximately 20 minutes) compared with that in cells stimulated with SLF at 4 degrees C or at 37 degrees C with 0.2% sodium azide. However, in the presence of chloroquine, which blocks lysosomal degradation, this ligand-induced c-kit degradation at 37 degrees 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.
authors.affiliation: First Department of Internal Medicine (Hematology/Oncology), Tokyo Medical College, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Miyazawa
authors.forename: K
authors.initials: K
authors.lastname: Toyama
authors.forename: A
authors.initials: A
authors.lastname: Gotoh
authors.forename: P C
authors.initials: PC
authors.lastname: Hendrie
authors.forename: C
authors.initials: C
authors.lastname: Mantel
authors.forename: H E
authors.initials: HE
authors.lastname: Broxmeyer
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001386:Azides / Q000494:pharmacology; D002460:Cell Line; D015536:Down-Regulation; D019833:Genistein; D016298:Hematopoietic Cell Growth Factors / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D008969:Molecular Sequence Data; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D016184:Receptors, Colony-Stimulating Factor / Q000378:metabolism*; D019810:Sodium Azide; D019089:Stem Cell Factor; D013696:Temperature; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D001386:Azides; D016298:Hematopoietic Cell Growth Factors; D007529:Isoflavones; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D019089:Stem Cell Factor; D014452:Ubiquitins; D019810:Sodium Azide; D019833:Genistein; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases