Mechanism of endocytosis of CD33/Siglec-3: role of ITIMs, tyrosine phosphorylation, and monoubiquitylation
Walter, Roland Bruno, 1970-
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The sialic acid-binding immunoglobulin-like lectin, CD33/Siglec-3, is a founding member of a subgroup of highly related Siglecs with differential expression in the innate immune system. Common structural features include a conserved proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a distal ITIM-like motif, which, when phosphorylated, can bind and activate the tyrosine phosphatases, SHP-1 and SHP-2, suggesting an inhibitory function of these Siglecs. Despite its unknown function, CD33 has gained importance as target for immunotherapy as it is expressed on the majority of acute myeloid leukemias. Specifically, some clinical success has been achieved with gemtuzumab ozogamicin (GO; Mylotarg(TM)), an immunoconjugate that utilizes an anti-CD33 antibody to facilitate uptake of a toxic calicheamicin-y1 derivative. We have now studied principles underlying endocytosis of CD33, in particular when bound to antibody, in detail. We found that the tyrosine motifs of CD33 control both CD33 internalization and GO-induced cytotoxicity. Pervanadate significantly increased CD33 uptake in an ITIM-dependent manner; this effect was prevented by co-treatment with PP2, suggesting that ITIM phosphorylation is regulated by Src-family kinase activity and critically controls CD33 uptake. We identified several proteins, including SHP-1, SHP-2, and Syk, which differentially bound to phosphorylated wild-type and mutant CD33. Depletion of myeloid cells from SHP-1 and SHP-2 by short interfering RNA resulted in increased CD33 internalization in TF-1 and ML-1 but not in HL-60 or NB4 cells, suggesting significant differences with regard to cellular responses to antibody ligation. Syk was dispensable for CD33 internalization and GO-induced cytotoxicity. Finally, we show that CD33 can undergo ITIM- and Src-family kinase-dependent monoubiquitylation, a process that may include Cbl family proteins as E3 ligases. CD33 internalization was abrogated in HEK293T cells expressing CD33 bearing lysine-to-arginine mutations, a phenotype that was rescued by fusion of ubiquitin to CD33. When lysine-to arginine mutants were expressed in myeloid cells, surface CD33 levels were increased while antibody-internalization was slightly reduced, whereas fusion with ubiquitin reduced surface CD33 display. Together, we demonstrate the role of the ITIMs and tyrosine phosphorylation for endocytosis of CD33, and identify ITIM-dependent monoubiquitylation as a novel post-translational modification involved in receptor downregulation and endocytosis.
- Pathology