Nocodazole inhibits signal transduction by the T cell antigen receptor.
The potential role of the cytoskeleton in signaling via the T cell antigen receptor (TCR) was investigated using pharmacological agents. In Jurkat T cells, disruption of the actin-based cytoskeleton with cytochalasin D or disruption of the microtubules with colchicine did not affect TCR induction of proximal signaling events triggered by CD3 mAb. Polymerized actin and tubulin, therefore, were not required for TCR-mediated signal transduction. Nocodazole, however, was found to inhibit dramatically TCR signaling, independently of its ability to depolymerize microtubules. This effect was TCR-specific, because signaling via the human muscarinic acetylcholine receptor 1 in the same cells was unaffected. A mechanism for the inhibition of TCR signaling by nocodazole was suggested by in vitro assays, which revealed that the drug inhibited the kinase activity of LCK and, to a lesser extent, FYN. The kinase activity of ZAP-70 in vitro, however, was unaffected. These results, therefore, suggested that nocodazole prevented initial phosphorylation of the TCR by LCK after stimulation, and as a result, it blocked activation of downstream signaling pathways. Immunofluorescence analyses also revealed that nocodazole and the specific SRC-family kinase inhibitor PP1 delocalized ZAP-70 from its constitutive site at the cell cortex. These effects did not require the SH2 domains of ZAP-70. The localization of ZAP-70 to the cell cortex is, therefore, regulated by the activity of SRC-family kinases, independently of their ability to phosphorylate immunoreceptor tyrosine-based activation motifs of the TCR.
Huby
R D
RD
Division of Cellular Immunology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Weiss
A
A
Ley
S C
SC
D000199:Actins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D015739:Nocodazole / Q000494:pharmacology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D014404:Tubulin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
D000199:Actins; D004791:Enzyme Inhibitors; D011948:Receptors, Antigen, T-Cell; D014404:Tubulin; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D019061:src-Family Kinases; D015739:Nocodazole