pp56Lck mediates TCR zeta-chain binding to the microfilament cytoskeleton.
The TCR zeta-chain (zeta) on mature murine T lymphocytes binds to the microfilament cytoskeleton in response to Ag receptor ligation. Here, we report the role of Src family kinases in zeta-cytoskeletal binding, using mutant mice and a cell-free model system. Binding of zeta to actin in the cell-free system has a specific requirement for ATP and divalent cations, with an apparent Michaelis-Menton constant for ATP in the millimolar range, and can be disrupted by either EDTA or the microfilament poison, cytochalasin D, suggesting that microfilaments provide the structural framework for an active process involving cellular kinases. Indeed, tyrosine-phosphorylated zeta is a predominant form of the zeta-chain bound to polymerized actin, while challenge with alkaline phosphatase prevents zeta-chain association in solution and releases zeta-chain from the bound state. Phosphorylated Src-family kinase pp56Lck also associates with membrane skeleton upon TCR engagement and is a component of the reconstituted cytoskeletal pellet. Zeta-chain phosphorylation and zeta-cytoskeletal binding are abrogated in cell lysates with reduced levels of pp56Lck and in activated mutant murine T cells lacking pp56Lck, implicating pp56Lck as the kinase involved in zeta-chain tyrosine phosphorylation and zeta-cytoskeletal binding. Finally, recombinant Lck Src homology 2 domain preferentially inhibits reconstituted zeta-cytoskeleton association, suggesting that zeta-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in zeta-chain activation motifs and the Src homology 2 domain of the Lck protein tyrosine kinase.
Rozdzial
M M
MM
Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA.
Pleiman
C M
CM
Cambier
J C
JC
Finkel
T H
TH
D008841:Actin Cytoskeleton / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002413:Cations, Divalent; D002474:Cell-Free System; D003599:Cytoskeleton / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000235:genetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009363:Neoplasm Proteins; D010766:Phosphorylation; D011485:Protein Binding / Q000276:immunology; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains / Q000276:immunology; D019061:src-Family Kinases
D002413:Cations, Divalent; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; C490512:Frk protein, mouse; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases