pp56Lck mediates T cell receptor-z=ζ chain binding to the microfilament cytoskeleton1

The T cell receptor z=ζ-chain (z=ζ) on mature murine T lymphocytes binds to the microfilament cytoskeleton in response to antigen receptor ligation (Rozdzial et al., Immunity Vol.3, 1995). Using a cell-free system, we show here that binding of z=ζ to actin has a specific requirement for ATP and divalent cations with an apparent Km for ATP in the millimolar range. This binding can be disrupted by either EDTA or cytochalasin D. suggesting that microfilaments provide the structural framework for an active process involving cellular kinases. Indeed, tyrosine phosphorylated z=ζ is a predominant form of the z=ζ-chain bound to polymerized actin. Challenge with alkaline phosphatase prevents z=ζ-chain association in solution and releases z=ζ-chain from the bound state. Phosphorylated Src-family kinase p56Lck (Lck) also associates with membrane skeleton upon TCR engagement and is a component of the reconstituted cytoskeletal pellet. In contrast to pp59Fyn, z=ζ-chain phosphorylation and z=ζ-cytoskeletal binding are abrogated in cell lysates depleted of Lck and in activated murine T cells dificient in Lck, implicating Lck as the kinase involved in z=ζ-chain tyrosine phosphorylation and z=ζ-cytoskeletal binding. Finally, a recombinant SH2 peptide inhibits reconstituted z=ζ-cytoskeleton association in a concentration-dependent manner, suggesting that z=ζ-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in z=ζ-chain activation motifs and the SH2 domain of a Src family tyrosine kinase.