NOCODAZOLE INHIBITS SIGNAL-TRANSDUCTION BY THE T-CELL ANTIGEN RECEPTOR

The potential role of the cytoskeleton in signaling via the T cell ant igen receptor (TCR) was investigated using pharmacological agents. In Jurkat T cells, disruption of the actin-based cytoskeleton with cytoch alasin D or disruption of the microtubules with colchicine did not aff ect TCR induction of proximal signaling events triggered by CD3 mAb. P olymerized actin and tubulin, therefore, were not required for TCR-med iated signal transduction. Nocodazole, however, was found to inhibit d ramatically TCR signaling, independently of its ability to depolymeriz e microtubules. This effect was TCR-specific, because signaling via th e human muscarinic acetylcholine receptor 1 in the same cells was unaf fected, A mechanism for the inhibition of TCR signaling by nocodazole was suggested by in vitro assays, which revealed that the drug inhibit ed 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 acti vation of downstream signaling pathways. Immunofluorescence analyses a lso revealed that nocodazole and the specific SRC-family kinase inhibi tor PP1 delocalized ZAP-70 from its constitutive site at the cell cort ex. These effects did not require the SH2 domains of ZAP-70. The local ization of ZAP-70 to the cell cortex is, therefore, regulated by the a ctivity of SRC-family kinases, independently of their ability to phosp horylate immunoreceptor tyrosine-based activation motifs of the TCR.