Growth factor-induced tyrosine phosphorylation of Hrs, a novel 115-kilodalton protein with a structurally conserved putative zinc finger domain.

The activation of growth factor receptor tyrosine kinases leads to tyrosine phosphorylation of many intracellular proteins which are thought to play crucial roles in growth factor signaling pathways. We previously showed that tyrosine phosphorylation of a 115-kDa protein is rapidly induced in cells treated with hepatocyte growth factor. To clarify the structure and possible function of the 115-kDa protein (designated Hrs for hepatocyte growth factor-regulated tyrosine kinase substrate), we purified this protein from B16-F1 mouse melanoma cells by anti-phosphotyrosine immunoaffinity chromatography and determined its partial amino acid sequences. On the basis of the amino acid sequences, we molecularly cloned the cDNA for mouse Hrs. The nucleotide sequence of the cDNA revealed that Hrs is a novel 775-amino-acid protein with a putative zinc finger domain that is structurally conserved in several other proteins. This protein also contained a proline-rich region and a proline- and glutamine-rich region. The expression of Hrs mRNA was detected in all adult mouse tissues tested and also in embryos. To analyze the Hrs cDNA product, we prepared a polyclonal antibody against bacterially expressed Hrs. Using this antibody, we showed by subcellular fractionation that Hrs is localized to the cytoplasm; we also showed that that tyrosine phosphorylation of Hrs is induced in cells treated with epidermal growth factor or platelet-derived growth factor. These results suggest that Hrs plays a unique and important role in the signaling pathway of growth factors.

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Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7685-7692.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7685-7692 ◽

Cited By ~ 86

Author(s):

Sylvie Urbé ◽

Ian G. Mills ◽

Harald Stenmark ◽

Naomi Kitamura ◽

Michael J. Clague

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Membrane Trafficking ◽

Signal Transduction Pathway ◽

Dominant Negative ◽

Kinase Substrate ◽

Hepatocyte Growth ◽

Tyrosine Kinase Substrate

ABSTRACT 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.

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Tyrosine Phosphorylation of β-Catenin and Plakoglobin Enhanced by Hepatocyte Growth Factor and Epidermal Growth Factor in Human Carcinoma Cells

Cell Adhesion and Communication ◽

10.3109/15419069409097261 ◽

1994 ◽

Vol 1 (4) ◽

pp. 295-305 ◽

Cited By ~ 284

Author(s):

Sayumi Shibamoto ◽

Makio Hayakawa ◽

Kenji Takeuchi ◽

Takamitsu Hori ◽

Naoto Oku ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Hepatocyte Growth Factor ◽

Tyrosine Phosphorylation ◽

Carcinoma Cells ◽

Human Carcinoma ◽

Epidermal Growth ◽

Hepatocyte Growth

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Induction of Tyrosine Phosphorylation and Translocation of Ezrin by Hepatocyte Growth Factor/Scatter Factor (HGF/SF)

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1995.2877 ◽

1995 ◽

Vol 217 (3) ◽

pp. 1062-1069 ◽

Cited By ~ 39

Author(s):

W.G. Jiang ◽

S. Hiscox ◽

S.K. Singhrao ◽

M.C.A. Puntis ◽

T. Nakamura ◽

...

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Tyrosine Phosphorylation ◽

Scatter Factor ◽

Hepatocyte Growth

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Receptor-type Protein Tyrosine Phosphatase β (RPTP-β) Directly Dephosphorylates and Regulates Hepatocyte Growth Factor Receptor (HGFR/Met) Function

Journal of Biological Chemistry ◽

10.1074/jbc.m110.212597 ◽

2011 ◽

Vol 286 (18) ◽

pp. 15980-15988 ◽

Cited By ~ 20

Author(s):

Yiru Xu ◽

Wei Xia ◽

Dustin Baker ◽

Jin Zhou ◽

Hyuk Chol Cha ◽

...

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Phosphatase ◽

Protein Tyrosine Kinase ◽

Tyrosine Phosphatase ◽

Human Keratinocytes ◽

Primary Human Keratinocytes ◽

Protein Tyrosine ◽

Hepatocyte Growth

Protein tyrosine phosphorylation is a ubiquitous, fundamental biochemical mechanism that regulates essential eukaryotic cellular functions. The level of tyrosine phosphorylation of specific proteins is finely tuned by the dynamic balance between protein tyrosine kinase and protein tyrosine phosphatase activities. Hepatocyte growth factor receptor (also known as Met), a receptor protein tyrosine kinase, is a major regulator of proliferation, migration, and survival for many epithelial cell types. We report here that receptor-type protein tyrosine phosphatase β (RPTP-β) specifically dephosphorylates Met and thereby regulates its function. Expression of RPTP-β, but not other RPTP family members or catalytically inactive forms of RPTP-β, reduces hepatocyte growth factor (HGF)-stimulated Met tyrosine phosphorylation in HEK293 cells. Expression of RPTP-β in primary human keratinocytes reduces both basal and HGF-induced Met phosphorylation at tyrosine 1356 and inhibits downstream MEK1/2 and Erk activation. Furthermore, shRNA-mediated knockdown of endogenous RPTP-β increases basal and HGF-stimulated Met phosphorylation at tyrosine 1356 in primary human keratinocytes. Purified RPTP-β intracellular domain preferentially dephosphorylates purified Met at tyrosine 1356 in vitro. In addition, the substrate-trapping mutant of RPTP-β specifically interacts with Met in intact cells. Expression of RPTP-β in human primary keratinocytes reduces HGF induction of VEGF expression, proliferation, and motility. Taken together, the above data indicate that RPTP-β is a key regulator of Met function.

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