Ligand-dependent polyubiquitination of c-kit gene product: A possible mechanism of receptor down modulation in M07e cells

Keisuke Miyazawa, Keisuke Toyama, Akihiko Gotoh, Paul C. Hendrie, Charlie Mantel, Hal Broxmeyer

Research output: Contribution to journalArticle

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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°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.

Original languageEnglish
Pages (from-to)137-145
Number of pages9
JournalBlood
Volume83
Issue number1
StatePublished - Jan 1 1994

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Stem Cell Factor
Proto-Oncogene Proteins c-kit
Genes
Modulation
Ligands
Sodium Azide
Ubiquitination
Degradation
Proteins
Protein-Tyrosine Kinases
Genistein
Chloroquine
Electrophoresis
Sodium Dodecyl Sulfate
Myeloid Cells
Methionine
Intercellular Signaling Peptides and Proteins
Proteolysis
Polyacrylamide Gel Electrophoresis
Adenosine Triphosphate

ASJC Scopus subject areas

  • Hematology

Cite this

Ligand-dependent polyubiquitination of c-kit gene product : A possible mechanism of receptor down modulation in M07e cells. / Miyazawa, Keisuke; Toyama, Keisuke; Gotoh, Akihiko; Hendrie, Paul C.; Mantel, Charlie; Broxmeyer, Hal.

In: Blood, Vol. 83, No. 1, 01.01.1994, p. 137-145.

Research output: Contribution to journalArticle

Miyazawa, K, Toyama, K, Gotoh, A, Hendrie, PC, Mantel, C & Broxmeyer, H 1994, 'Ligand-dependent polyubiquitination of c-kit gene product: A possible mechanism of receptor down modulation in M07e cells', Blood, vol. 83, no. 1, pp. 137-145.
Miyazawa, Keisuke ; Toyama, Keisuke ; Gotoh, Akihiko ; Hendrie, Paul C. ; Mantel, Charlie ; Broxmeyer, Hal. / Ligand-dependent polyubiquitination of c-kit gene product : A possible mechanism of receptor down modulation in M07e cells. In: Blood. 1994 ; Vol. 83, No. 1. pp. 137-145.
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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°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.",
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N2 - 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.

AB - 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.

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