Role of the Lck Src Homology 2 and 3 Domains in Protein Tyrosine Phosphorylation

Kay K Lee-Fruman,Tassie L Collins,Steven J Burakoff

doi:10.1074/jbc.271.40.25003

Kay K Lee-Fruman, Tassie L Collins + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.40.25003

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Abstract

Many protein tyrosine phosphorylation events that occur as a result of T cell receptor (TCR) stimulation are enhanced when CD4 is co-cross-linked with the TCR, and this increased phosphorylation is thought to be a mechanism by which T cell functions are augmented by CD4. Such enhanced tyrosine phosphorylation was originally attributed to the kinase activity of the CD4-associated tyrosine kinase Lck. However, it has been shown that CD4-associated Lck lacking the catalytic domain can enhance T cell functions, suggesting that the noncatalytic domains of Lck are also important in CD4 signaling. Using T cells expressing various CD4-Lck chimeric molecules, we assessed the role of different Lck domains in early T cell signaling. Following TCR-CD4 co-cross-linking, cells expressing a CD4-Lck full-length chimera showed enhanced tyrosine phosphorylation of many cellular proteins in a CD4-dependent manner. Surprisingly, cells expressing a CD4-Lck chimera lacking the catalytic domain (termed CD4-N32) also showed enhanced phosphorylation. This enhancement of phosphorylation required both the Src homology 2 (SH2) and SH3 domains of Lck. Lck has been postulated to dimerize through the SH2 and SH3 domains. In this way CD4-N32 may interact with endogenous Lck, and although it lacks intrinsic kinase activity, it may be capable of enhancing phosphorylation through the associated full-length Lck. Consistent with this model, when CD4-Lck chimeric molecules were expressed in J. CaM1.6 cells lacking endogenous Lck, CD4-N32 failed to enhance tyrosine phosphorylation. Moreover, a Lck SH2 and SH3 domain fragment expressed as a glutathione S-transferase fusion protein associated with Lck when incubated with activated Jurkat T cell lysates, suggesting that the SH2 and SH3 domains of Lck can associate with endogenous full-length Lck upon activation. Thus, our data suggest that dimerization is an important mechanism of Lck function in T cell activation.

Highlights

Lck is a 56-kDa protein tyrosine kinase that is predominantly expressed in T lymphocytes
Expression of CD4-Lck Chimeric Molecules in a T Cell Hybridoma—BY155.16 cells [14], a murine T cell hybridoma line that lacks CD4, was transfected with chimeric CD4-Lck molecules containing the extracellular and transmembrane domains of CD4 ligated to the full-length Lck (CD4-FL); the N, SH3, and Src homology 2 (SH2) domains of Lck (CD4-N32); the N and SH3 domains (CD4-N3); the N and SH2 domains (CD4-N2); or the N domain of Lck (CD4-N) (Fig. 1A)
Protein tyrosine kinase activity was observed when CD4-FL was immunoprecipitated from cells and an in vitro kinase assay was performed, whereas the CD4-Lck chimeras lacking the catalytic domain did not demonstrate kinase activity as measured by autophosphorylation

Summary

Introduction

Lck is a 56-kDa protein tyrosine kinase that is predominantly expressed in T lymphocytes. Following TCR-CD4 co-cross-linking, cells expressing a CD4-Lck full-length chimera showed enhanced tyrosine phosphorylation of many cellular proteins in a CD4-dependent manner.

Results

Conclusion