Negative regulation of Lck by Cbl ubiquitin ligase

Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3794-9. doi: 10.1073/pnas.062055999.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • CD4 Antigens / metabolism
  • Cell Line
  • Enzyme Activation
  • Gene Deletion
  • Genes, Reporter / genetics
  • Humans
  • Jurkat Cells
  • Ligases / chemistry
  • Ligases / deficiency
  • Ligases / genetics
  • Ligases / metabolism*
  • 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*
  • Protein Binding
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Protein-Tyrosine Kinases / metabolism
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / deficiency
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-cbl
  • Receptors, Antigen, T-Cell / metabolism
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases
  • ZAP-70 Protein-Tyrosine Kinase
  • src Homology Domains

Substances

  • CD4 Antigens
  • Proto-Oncogene Proteins
  • Receptors, Antigen, T-Cell
  • Ubiquitin
  • Proto-Oncogene Proteins c-cbl
  • Ubiquitin-Protein Ligases
  • Protein-Tyrosine Kinases
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
  • ZAP-70 Protein-Tyrosine Kinase
  • ZAP70 protein, human
  • Ligases
  • CBL protein, human