Negative Regulation of Lck by Cbl Ubiquitin Ligase

• Published in: Proceedings of the National Academy of Sciences - PNAS 2002-03, Vol.99 (6), p.3794-3799
• Main Authors: Rao, Navin, Miyake, Sachiko, Reddi, Alagarsamy Lakku, Douillard, Patrice, Ghosh, Amiya K., Dodge, Ingrid L., Zhou, Pengcheng, Fernandes, Norvin D., Band, Hamid
• Format: Article
• Language: English
• Subjects: Antibodies; Biological Sciences; Cbl protein; CD4 Antigens - metabolism; Cell Line; Cell lines; Enzyme Activation; Gene Deletion; Genes, Reporter - genetics; Humans; Immunoblotting; Immunology; Jurkat Cells; Lck protein; Ligases - chemistry; Ligases - deficiency; Ligases - genetics; Ligases - metabolism; Lymphatic system; Lymphocyte Activation; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - antagonists & inhibitors; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - chemistry; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - genetics; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - metabolism; Physiological regulation; Plasmids; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Protein-Tyrosine Kinases - metabolism; Proteins; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins - deficiency; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-cbl; Receptors; Receptors, Antigen, T-Cell - metabolism; src Homology Domains; T cell antigen receptors; T lymphocytes; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; Ubiquitin - metabolism; Ubiquitin-Protein Ligases; ubiquitination; Ubiquitins; ZAP-70 protein; ZAP-70 Protein-Tyrosine Kinase
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.062055999

The Cbl-family ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent targeting to lysosomes. Cbl associates with the lymphoid-restricted nonreceptor tyrosine kinase Lck, but the functional relevance of this interaction remains unknown. Here, we demonstrate that T cell receptor and CD4 coligation on human T cells results in enhanced association between Cbl and Lck, together with Lck ubiquitination and degradation. A Cbl-/-T cell line showed a marked deficiency in Lck ubiquitination and increased levels of kinase-active Lck. Coexpression in 293T cells demonstrated that Lck kinase activity and Cbl ubiquitin ligase activity were essential for Lck ubiquitination and negative regulation of Lck-dependent serum response element-luciferase reporter activity. The Lck SH3 domain was pivotal for Cbl-Lck association and Cbl-mediated Lck degradation, with a smaller role for interactions mediated by the Cbl tyrosine kinase-binding domain. Finally, analysis of a ZAP-70-deficient T cell line revealed that Cbl inhibited Lck-dependent mitogen-activated protein kinase activation, and an intact Cbl RING finger domain was required for this functional effect. Our results demonstrate a direct, ubiquitination-dependent, negative regulatory role of Cbl for Lck in T cells, independent of Cbl-mediated regulation of ZAP-70.