Effect of synthetic and natural protein tyrosine kinase inhibitors on auxin efflux in zucchini (Cucurbita pepo) hypocotyls

Basipetal auxin transport along the zucchini hypocotyl requires the cell-to-cell translocation of this hormone. Several authors have demonstrated that naphthylphthalamic acid (NPA) blocks auxin efflux. It is believed that NPA binds to a regulatory protein that modulates the activity of the carrier rather than to the carrier itself. A few regula tory mechanisms have been suggested but all lack strong supporting experimental evidence. Four classes of protein tyrosine kinase inhibitors (PTKIs) have been used to investigate the NPA perception pathway. First, six synthetic tyrphostins, inhibitors of the epidermal growth factor receptor (EGF-R), were shown to displace NPA on plasmalemma fractions. The most potent compound was (3,4-dihydroxybenzylidene)thiocyanoacetamide (tyrphostin A47). The inactive counterpart of tyrphostin A47, (4-methoxybenzylidene)-malononitrile (tyrphostin Al), was unable to displace NPA. Tyrphostins did not inhibit auxin efflux but were antagonists of the inhibition of auxin efflux by NPA. Again, tyrphostin A47 was the most effective and tyrphostin Al was inactive. Second, the flavonoid genistein, also an EGF-R inhibitor, showed an effect on NPA binding and NPA antagonism similar to the one for the tyrphostins. Daidzein, the inactive counterpart of genistein, was ineffective in displacing NPA and in antagonizing its inhibition of auxin efflux. Two other PTKIs, curcumin and lavendustin A, displayed similar characteristics. Calmodulin antagonists, protein serine/threonine kinase inhibitors, and phosphatase inhibitors were inactive. These results suggest that the NPA binding protein may be related to mammalian tyrosine kinases and may regulate the auxin efflux carrier by phosphorylation.