Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated 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.Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a prominent target for tyrosine phosphorylation following the activation of tyrosine kinase receptors (11). It was initially shown to lie downstream of the HGF (scatter factor) receptor c-met, but activation of other tyrosine kinase receptors and by cytokines such as interleukin-2 and granulocyte-macrophage colony-stimulating factor also results in phosphorylation of Hrs (1). It is localized to transferrin receptor-containing endosomes (12) and bears significant similarity (including a FYVE-finger motif and a VHS domain) to the Saccharomyces cerevisiae protein Vps27. Vps27 belongs to the class E set of Vps mutants which are defective in transport from the sorting endosome to the vacuole (2, 22). A highly related protein, Hrs-2, has been shown to interact with SNAP-25 and SNAP-23, homologous proteins which are involved in regulated exocytosis and other intracellular fusion events, respectively (3).Two proteins that bind to Hrs have been identified which have been named STAM (for signal-transducing adapter molecule) and Hrs binding protein (Hbp) (1, 31). They show 53% sequence identity, each bears an SH3 domain, and both are also tyrosine phosphorylated. In T cells, overexpression of Hrs leads to suppression of cytokine-mediated DNA synthesis, while a mutant unable to bind STAM is without effect (1). NIH 3T3 cells stably transfected with mutants of Hbp that lack the SH3 domain or the binding site for Hrs are impaired in degrading internalized platelet-derived growth factor (31). This latter result is consistent with a role for Hrs in regulating transport from early to late endosomes (or multivesicular bodies) proposed by analogy to Vps27 function in yeast.The FYVE domain is a double zinc finger domain that has been shown ...

From Tpr-Met to Met, tumorigenesis and tubes

“…Growing evidence indicates that ubiquitination of RTKs is critical for their lysosomal degradation, through their ubiquitin-dependent targeting to intralumenal vesicles of MVBs and lysosomal degradation (Urbe et al, 2000;Raiborg et al, 2002;Duan et al, 2003;Jiang et al, 2003;Yamasaki et al, 2003). RTKs, including Met, may undergo multimonoubiquitination and Lys63-linked polyubiquitination, both of which do not form a signal for proteasomal degradation (Haglund et al, 2003;Mosesson et al, 2003;Carter et al, 2004;Huang et al, 2006).…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…These coats were also shown to be enriched in EGFR, but not in TfR Sachse et al, 2002). Hrs is recruited to early endosomes by the specific interactions between its FYVE domain (a zinc finger domain, named after Fab1, YOTB/ZK632.12, Vac1 and EEA1) and PI(3)P in the limiting membrane of the endosome (Urbe et al, 2000;Raiborg et al, 2001b). Another domain in Hrs, a coiled-coil domain, is also believed to be involved in membrane microdomain-binding specificity (Raiborg et al, 2001b).…”

Possible Existence of Common Internalization Mechanisms among Arginine-rich Peptides

“…To examine if this result was also applicable to the uptake of other arginine-rich peptides, the effects of the following reagents, known as typical endocytosis inhibitors, on the internalization of the Rev-(34 -50) and Arg 8 peptides as well as the Tat-(48 -60) were examined: the microtubule-disrupting reagents colchicine (20 M) (23), taxol (20 M) (24), and nocodazole (20 M) (25); the filament-disrupting reagent cytochalasin D (5 M) (26); the inhibitor of trans-Golgi transport brefeldin A (10 M) (27); the phosphatidylinositol-3 kinase inhibitor wortmannin (50 nM) (28); and the inhibitor of the acidification of endocytic vesicles chloroquine (50 M) (29). Prior to the addition of the peptides, the HeLa cells were incubated in serum-free medium containing each of the reagents at 37°C for 30 min except in the case of the brefeldin A (10 min), and then a peptide was added to the medium.…”