Phosphorylation of Hrs downstream of the epidermal growth factor receptor.
The hepatocyte growth factor-regulated tyrosine kinase substrate Hrs is an early endosomal protein that is thought to play a regulatory role in the trafficking of growth factor/receptor complexes through early endosomes. Stimulation of cells with epidermal growth factor (EGF) rapidly leads to phosphorylation of Hrs, raising the question whether the receptor tyrosine kinase phosphorylates Hrs directly. Here, we present evidence that a downstream kinase, rather than the active receptor kinase is responsible. We show that the nonreceptor tyrosine kinase Src is able to phosphorylate Hrs in vitro and in vivo, but that Hrs is nevertheless phosphorylated in Src-, Yes- and Fyn-negative cells. Moreover, we show that only 10-20% of Hrs is phosphorylated following EGF stimulation, and that phosphorylation occurs at multiple tyrosines located in different parts of Hrs. These results suggest that Hrs is a substrate for several kinases downstream of the EGF receptor.
Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital, Montebello, Oslo, Norway.
Kristi G
KG
Bache
Camilla
C
Raiborg
Anja
A
Mehlum
Inger Helene
IH
Madshus
Harald
H
Stenmark
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000473:pathology; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000502:physiology*; D005347:Fibroblasts / Q000201:enzymology; D016391:Genes, src; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology / Q000473:pathology; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000172:deficiency; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000201:enzymology; D019061:src-Family Kinases
D056827:Endosomal Sorting Complexes Required for Transport; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; C494957:FYN protein, human; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; C496639:Yes1 protein, mouse; D019061:src-Family Kinases; C000631346:CSK protein, human