Biological consequences of the phosphorylation of serine 59 on the tyrosine kinase Lck

Maria Luz Vazquez, Purdue University

Abstract

Lck is a member of the Src-family of protein tyrosine kinases and is required for the activation of T cells. The structure of Lck is typical of members of the Src-family and includes: an N-terminal, membrane targeting Src-homology 4 (SH4) domain, which contains sites for lipid modifications that target the protein to the membrane; a unique domain; a Src-homology 3 (SH3) domain, which binds proline rich regions in interacting proteins; a Src-homology 2 (SH2) domain, which binds to tyrosine phosphorylated residues, a catalytic domain, and finally a short C-terminal regulatory domain. Lck interacts with cellular proteins through its SH2 and SH3 domains, but it can also interact with the SH2 and SH3 domains of other proteins. The unique region of Lck contains a proline rich region surrounding serine 59. The serine/threonine, proline-directed ERK MAP kinase was identified as the kinase responsible for the phosphorylation of serine 59 following TCR ligation. The SCANSITE computer program identified this proline rich region as a potential binding site for the SH3 domain of a 47kDa adaptor protein Nck. Nck consists solely of four binding domains: one SH2 domain and three SH3 domains. Nck links receptor tyrosine kinases to downstream proteins, and it is involved in actin polymerization. It was also reported in T cells that Nck binds to the CD3ϵ subunit of the T cell antigen receptor (TCR) following TCR engagement. I hypothesized that if Nck interacted with the proline rich regions of Lck surrounding serine 59, phosphorylation of serine 59 might disrupt this interaction. Pulldown experiments from LSTRA and Jurkat T cell lysates using full length GST-tagged Nck expressed in bacteria demonstrated an interaction between Lck and Nck. Using purified Lck protein, I found that the interaction between Lck and Nck was direct, independent of other proteins. Using a variety of approaches, I demonstrated that the phosphorylation of serine 59 disrupted the Lck/Nck interaction. In order to address the physiological function of serine 59 phosphorylation, wildtype (WTLck) and S59DLck were stably transfected into Lck-deficient JCaM1.6 cells. Stimulation of cells expressing S59DLck resulted in differences in the activation of proximal versus distal signaling events compared to cells expressing WTLck. Taken together all the results showed that Nck is a new interacting partner for Lck, and that serine 59 phosphorylation disrupts the interaction between Lck and Nck. I also demonstrated that serine 59 might play a role in the phosphorylation of the tyrosine kinase ZAP-70 without changing the activation of the downstream MAP kinase pathway.

Degree

Ph.D.

Advisors

Vazquez, Purdue University.

Subject Area

Biochemistry

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