Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a pro minent substrate for activated tyrosine kinase receptors that has been prop osed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both f or correct localization of the protein to endosomes that only partially coi ncides with early endosomal autoantigen 1 and for efficient tyrosine phosph orylation of the protein in response to epidermal growth factor stimulation . Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required fo r localization. We have used both hypertonic media and expression of a domi nant-negative form of dynamin (K44A) to inhibit endocytosis; under which co nditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pa thway to endocytosis, from which we propose that activated receptor (or ass ociated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.