Site-specific ubiquitination determines lysosomal sorting and signal attenuation of the granulocyte colony-stimulating factor receptor.
Ubiquitination of cytokine receptors controls intracellular receptor routing and signal duration, but the underlying molecular determinants are unclear. The suppressor of cytokine signaling protein SOCS3 drives lysosomal degradation of the granulocyte colony-stimulating factor receptor (G-CSFR), depending on SOCS3-mediated ubiquitination of a specific lysine located in a conserved juxtamembrane motif. Here, we show that, despite ubiquitination of other lysines, positioning of a lysine within the membrane-proximal region is indispensable for this process. Neither reallocation of the motif nor fusion of ubiquitin to the C-terminus of the G-CSFR could drive lysosomal routing. However, within this region, the lysine could be shifted 12 amino acids toward the C-terminus without losing its function, arguing against the existence of a linear sorting motif and demonstrating that positioning of the lysine relative to the SOCS3 docking site is flexible. G-CSFR ubiquitination peaked after endocytosis, was inhibited by methyl-beta-cyclodextrin as well as hyperosmotic sucrose and severely reduced in internalization-defective G-CSFR mutants, indicating that ubiquitination mainly occurs at endosomes. Apart from elucidating structural and spatio-temporal aspects of SOCS3-mediated ubiquitination, these findings have implications for the abnormal signaling function of G-CSFR mutants found in severe congenital neutropenia, a hematopoietic disorder with a high leukemia risk.
Wölfler
Albert
A
Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands.
Irandoust
Mahban
M
Meenhuis
Annemarie
A
Gits
Judith
J
Roovers
Onno
O
Touw
Ivo P
IP
D000595:Amino Acid Sequence; D000818:Animals; D004252:DNA Mutational Analysis; D004705:Endocytosis / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000502:physiology*; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000378:metabolism; D054875:Ubiquitination
D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; C106644:SOCS3 protein, human; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D008239:Lysine