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 prominent substrate for activated tyrosine kinase receptors that has been proposed 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 for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation 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 for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.
Urbé
S
S
Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, United Kingdom. urbe@liv.ac.uk
Mills
I G
IG
Stenmark
H
H
Kitamura
N
N
Clague
M J
MJ
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D056827:Endosomal Sorting Complexes Required for Transport; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D019000:Phosphotyrosine; D011956:Receptors, Cell Surface; D000077191:Wortmannin; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; C055525:phosphatidylinositol 3-phosphate