Biological Function of the Phosphorylation of Serine 59 in the Tyrosine Kinase Lck

Abstract

Lck is a member of the Src-family of protein tyrosine kinases. It is required for the activation of T lymphocytes (T cells). Lck interacts both with proteins through its SH2 and SH3 domains, and also with the SH2 and SH3 domains of other proteins. The unique region of Lck contains a proline rich region surrounding serine 59. Serine 59 becomes phosphorylated following ligation of the T cell antigen receptor (TCR), however a role for serine 59 phosphorylation is still emerging. The SCANSITE computer program identified the serine 59 containing proline rich region as a putative binding site for the SH3 domain of a 47 kDa adaptor protein Nck. Nck consist solely of four binding domains: one SH2 domain and three SH3 domains and links receptor tyrosine kinases with several binding partners. If Lck and Nck indeed interact, phosphorylation of Lck on serine 59 would be predicted to influence this interaction, due to its proximity to the proline rich region. Nck may serve as a binding partner for Lck in resting cells and potentially act as a bridge to locate Lck in close proximity to subunits of the TCR. This would represent a novel function for Nck and a new mechanism for Lck localization to the TCR. In order to test this hypothesis several GST containing constructs including full length Nck or individual Nck SH3 domains were expressed in bacteria, purified and used in pulldown experiments. Interaction between Lck and full length Nck was observed. A stronger interaction is observed between Lck and Nck when the second SH3 domain of Nck is used in pulldown experiments. Using a purified Lck protein we were able to show that the interaction between Lck and Nck is direct and independent of other proteins. This project was supported by NIH grants GM 48099 (MLH) and CA 37372 (RLG)