Regulation of the Src family kinase Lck by Hsp90 and ubiquitination

Regulation of the Src-related tyrosine kinase Lek is crucial to the outcome of T-cell receptor (TCR) stimulation. It was previously shown that the stability of the constitutively active mutant LekY505F is controlled by Hsp90 (M. J. Bijimakers and M. Marsh, Mol. Biol. Cell. 11:1585-1595, 2000). Here we establish that following TCR stimulation, endogenous activated Lek in T cells is also degraded in the presence of the Hsp90 inhibitor geldanamycin. Using Lek constructs expressed in COS-7 cells, we show that the presence of activating Lek mutations results not only in the enhanced dependence on Hsp90 but also in enhanced ubiquitination of Lek. Although both processes were induced by mutations Y505F and W97A that release the SH2 and SH3 inhibitory intramolecular interactions, respectively, neither process required Lek 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 Lek. We conclude that the levels of active Lek 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 Lek kinase domain instigate both regulatory processes.