Regulation of the Src family kinase Lck by Hsp90 and ubiquitination.
Regulation of the Src-related tyrosine kinase Lck is crucial to the outcome of T-cell receptor (TCR) stimulation. It was previously shown that the stability of the constitutively active mutant LckY505F is controlled by Hsp90 (M. J. Bijlmakers and M. Marsh, Mol. Biol. Cell. 11:1585-1595, 2000). Here we establish that following TCR stimulation, endogenous activated Lck in T cells is also degraded in the presence of the Hsp90 inhibitor geldanamycin. Using Lck constructs expressed in COS-7 cells, we show that the presence of activating Lck mutations results not only in the enhanced dependence on Hsp90 but also in enhanced ubiquitination of Lck. Although both processes were induced by mutations Y505F and W97A that release the SH2 and SH3 inhibitory intramolecular interactions, respectively, neither process required Lck kinase activity or activation-dependent phosphorylation at serines 42 and 59 or tyrosine 394. By binding to the ATP-binding site, the Src family inhibitor PP2 reduced ubiquitination and overcame the need for Hsp90 monitoring of active Lck. We conclude that the levels of active Lck are influenced by two opposing processes, targeting for degradation by ubiquitination and rescue from degradation by Hsp90 monitoring. Based on the PP2 result, we propose that activation-induced conformational changes of the Lck kinase domain instigate both regulatory processes.
Giannini
Ana
A
Department of Immunobiology, Guy's Hospital, King's College London, United Kingdom.
Bijlmakers
Marie-José
MJ
D000818:Animals; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D006801:Humans; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D010766:Phosphorylation; D011485:Protein Binding; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D025801:Ubiquitin; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)