Regulation of *Raf-1 signaling by the tyrosine kinase Lck in T lymphocytes

Manqing Li, Purdue University

Abstract

The engagement of T cell receptors (TCRs) leads to the activation of the Ras/Raf/MEK/ERK mitogen-activated protein kinase pathway. The Src-family kinase Lck and Fyn are the first kinases activated following antigen binding. The subsequent phosphorylation of tyrosine residues within the ITAM motifs of the TCR subunits initiates the signaling cascade leading to the activation of Ras. A loss of function point mutation in the SH3 domain of Lck (W97ALck) rendered Lck incapable of supporting ERK activation, despite the fact that expression of W97ALck resulted in an enhanced activation of signaling events immediately downstream of the TCR. A systematic investigation into the steps leading to ERK activation identified a role for Lck in the complex activation mechanism of Raf-1. Stable cell lines expressing either wild type Lck (WTLck) or W97ALck were established in the Lck-deficient T cell line, J.CaM1.6. Compared to WTLck, the expression of W97ALck resulted in the constitutive tyrosine phosphorylation of the TCR ζ-subunits, the constitutive tyrosine phosphorylation of the tyrosine kinase ZAP-70, and the elevated tyrosine phosphorylation of PLC-γ1. Following TCR ligation W97ALck expressing cells supported the full activation of Ras, but showed a defective activation of MEK1/2 and ERK1/2. The W97A mutation in Lck and mutation of tyrosines 340/341 in Raf-1 each resulted in a failure to support the full activation of Raf-1 following TCR ligation. However, the partial activation of the YY340/341FFRaf-1 was not further decreased by the W97A mutation in Lck. This suggested that the failure of W97ALck to fully support the TCR-initiated activation of Raf-1 resulted from an inability of this mutant to phosphorylate tyrosine residues 340/341 in Raf-1. Further, it was found that prior phosphorylation of tyrosines 340/341 was needed for the phosphorylation of serine 338 in Raf-I, which is required for Raf-1 full activation. These results support a model whereby after Lck initiates TCR signaling leading to the activation of Ras and the recruitment of Raf-1 to the plasma membrane, membrane associated Lck directly participates in the activation of Raf-1 by phosphorylating tyrosines 340/341. The ability of Lck to phosphorylate Raf-1 is critically dependent on the presence of a functional SH3 domain.

Degree

Ph.D.

Advisors

Harrison, Purdue University.

Subject Area

Biochemistry|Immunology

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