Hepatocyte Growth Factor-regulated Tyrosine Kinase Research Articles

Intrinsic Tyrosine Kinase Activity is Required for Vascular Endothelial Growth Factor Receptor 2 Ubiquitination, Sorting and Degradation in Endothelial Cells

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis co-ordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.

Novel function of POSH, a JNK scaffold, as an E3 ubiquitin ligase for the Hrs stability on early endosomes

POSH (plenty of SH3s) acts as a scaffold that links activated Rac1 and downstream c-Jun N-terminal kinase (JNK) signaling modules. However, it is unknown whether it's functional domain-mediated roles including the interesting RING-finger domain or its cellular function. Here, we provide evidence that subcellular localization of POSH is regulated by a particular domain of the protein and POSH was colocalized with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) on early endosomes via interaction of Hrs with POSH's two rear SH3 domains. Moreover, the RING domain of POSH specifically regulates the stability of Hrs, but not of JNK1, via a ubiquitin-proteasomal degradation pathway. Finally, we demonstrate that JNK1 does not interact with Hrs under the conditions of POSH interacted with Hrs, but instead reduces the POSH-catalyzed ubiquitination of Hrs and their reciprocal interaction. Together, these data suggest that POSH has a distinct role as a specific E3 ubiquitin ligase for Hrs on early endosomes, and there exists a relationship between its separate activities as a scaffold and as an E3.

Hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) and guanylate kinase 1 (GUK1) are differentially expressed in GH-secreting adenomas

Pituitary tumors, adenomas in their vast majority, represent around 10-15% of the intracranial neoplasms. Pituitary carcinomas are exceedingly rare. Clinically, these neoplasms cause hormonal dysfunctions, and mass effect symptoms as headache and visual disorders in the case of macroadenomas. Pituitary tumorigenesis is still poorly understood. In order to investigate the expression of cancer-related genes in pituitary tumors, we employed a human cancer cDNA macroarray membrane with 1176 well-characterized human genes related to cancer and tumor biology. We were able to identify several differentially expressed genes, among them hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) and guanylate kinase 1 (GUK1) which were over expressed in a pool of clinically nonfunctioning pituitary adenomas, compared with a spinal cord metastasis of a nonfunctioning pituitary carcinoma. HGS and GUK1 mRNA expression were chosen to be validated by quantitative RT-qPCR, however, only GUK1 had the differential expression confirmed between the adenomas and the metastasis of a pituitary carcinoma. We have also investigated HGS and GUK1 mRNA expressions in a series of 46 pituitary adenomas (18 nonfunctioning, 12 GH-secreting, nine PRL-secreting, and seven ACTH-secreting adenomas). HGS and GUK1 were significantly over expressed in GH-secreting adenomas, compared with ACTH-secreting adenomas and nonfunctioning tumors, and with PRL-secreting adenomas, respectively. We have shown that these genes, involved in tumorigenesis in other tissues, are as well over expressed in the pituitary tumors, however, their role in the oncogenesis of these tumors need to be further investigated.

Internalization is required for proper Wingless signaling in Drosophila melanogaster

The Wnt–Wingless (Wg) pathway regulates development through precisely controlled signaling. In this study, we show that intracellular trafficking regulates Wg signaling levels. In Drosophila melanogaster cells stimulated with Wg media, dynamin or Rab5 knockdown causes reduced Super8XTOPflash activity, suggesting that internalization and endosomal transport facilitate Wg signaling. In the wing, impaired dynamin function reduces Wg transcription. However, when Wg production is unaffected, extracellular Wg levels are increased. Despite this, target gene expression is reduced, indicating that internalization is also required for efficient Wg signaling in vivo. When endosomal transport is impaired, Wg signaling is similarly reduced. Conversely, the expression of Wg targets is enhanced by increased transport to endosomes or decreased hepatocyte growth factor–regulated tyrosine kinase substrate– mediated transport from endosomes. This increased signaling correlates with greater colocalized Wg, Arrow, and Dishevelled on endosomes. As these data indicate that endosomal transport promotes Wg signaling, our findings suggest that the regulation of endocytosis is a novel mechanism through which Wg signaling levels are determined.

Endocytic trafficking of Wingless and its receptors, Arrow and DFrizzled-2, in the Drosophila wing

During animal development, Wnt/Wingless (Wg) signaling is required for the patterning of multiple tissues. While insufficient signal transduction is detrimental to normal development, ectopic activation of the pathway can be just as devastating. Thus, numerous controls exist to precisely regulate Wg signaling levels. Endocytic trafficking of pathway components has recently been proposed as one such control mechanism. Here, we characterize the vesicular trafficking of Wg and its receptors, Arrow and DFrizzled-2 (DFz2), and investigate whether trafficking is important to regulate Wg signaling during dorsoventral patterning of the larval wing. We demonstrate a role for Arrow and DFz2 in Wg internalization. Subsequently, Wg, Arrow and DFz2 are trafficked through the endocytic pathway to the lysosome, where they are degraded in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner. Surprisingly, we find that Wg signaling is not attenuated by lysosomal targeting in the wing disc. Rather, we suggest that signaling is dampened intracellularly at an earlier trafficking step. This is in contrast to patterning of the embryonic epidermis, where lysosomal targeting is required to restrict the range of Wg signaling. Thus, signal modulation by endocytic routing will depend on the tissue to be patterned and the goals during that patterning event.

Membrane-associated STAT3 and PY-STAT3 in the Cytoplasm

Signal transduction from the plasma membrane to the nucleus by STAT proteins is widely represented as exclusively a soluble cytosolic process. Using cell-fractionation methods, we observed that approximately 5% of cytoplasmic STAT3 was constitutively associated with the purified early endosome (EE) fraction in human Hep3B liver cells. By 15-30 min after interleukin-6 (IL-6) treatment, up to two-thirds of cytoplasmic Tyr-phosphorylated STAT3 can be associated with the purified early endosome fraction (Rab-5-, EEA1-, transferrin receptor-, and clathrin-positive fraction). Electron microscopy, immunofluorescence, and detergent dissection approaches confirmed the association of STAT3 and PY-STAT3 with early endosomes. STAT3 was constitutively associated with clathrin heavy chain in membrane and in the 1- to 2-MDa cytosolic complexes. The membrane association was dynamic in that, within 15 min of treatment with the vicinal-thiol cross-linker phenylarsine oxide, there was a dramatic increase in bulk STAT3 association with sedimentable membranes. The functional contribution of PY-STAT3 association with the endocytic pathway was evaluated in transient transfection assays using IL-6-inducible STAT3-reporter-luciferase constructs and selective regulators of this pathway. STAT3-transcriptional activation was inhibited by expression constructs for dominant negative dynamin K44A, epsin 2a, amphiphysin A1, and clathrin light chain but enhanced by that for the active dynamin species MxA. Taken together, these studies emphasize the contribution of the endocytic pathway to productive IL-6/STAT3 signaling.

Ubiquitylation of leptin receptor OB-Ra regulates its clathrin-mediated endocytosis

Leptin receptors are constitutively endocytosed in a ligand-independent manner. To study their endocytosis, leptin receptors OB-Ra and OB-Rb were expressed in HeLa cells. Both receptor isoforms were ubiquitylated, internalized by clathrin-mediated endocytosis and transported to Hrs-positive endosomes after their internalization. Proteasome inhibitors inhibited OB-Ra but not OB-Rb internalization from the cell surface. OB-Ra ubiquitylation occurred on lysine residues K877 and K889 in the cytoplasmic tail, the mutation of which abolished OB-Ra internalization. Fusion of an ubiquitin molecule at the C-terminus of an OB-Ra construct defective both in ubiquitylation and endocytosis restored clathrin-dependent endocytosis of the receptor. The internalization of this constitutively mono-ubiquitylated construct was no longer sensitive to proteasome inhibitors, which inhibited OB-Ra endocytosis by blocking its ubiquitylation. Fusion of an ubiquitin molecule to a transferrin receptor deleted from its own endocytosis motif restored clathrin-mediated endocytosis. We propose that mono-ubiquitin conjugates act as internalization motifs for clathrin-dependent endocytosis of leptin receptor OB-Ra.

Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity

Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin-binding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.

Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate (HRS) Interacts with PELP1 and Activates MAPK

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1) (also known as the modulator of nongenomic activity of estrogen receptor) plays a role in genomic functions of the estrogen receptor via histone interactions and in nongenomic functions via its influence on the MAPK-Src pathway. However, recent studies have shown that differential compartmentalization of PELP1 could play a crucial role in modulating the status of nongenomic signaling by using molecular mechanisms that remain poorly understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is an early endosomal protein that plays a role in regulating the trafficking of growth factor-receptor complexes through early endosomes. By using a yeast two-hybrid screen, we identified HRS as a novel PELP1-binding protein providing evidence of a physiologic interaction between HRS and PELP1. The noted HRS-PELP1 interaction was accompanied by inhibition of the basal coactivator function of PELP1 upon estrogen receptor transactivation. HRS was found to sequester PELP1 in the cytoplasm, leading to the activation of MAPK in a manner that is dependent on the epidermal growth factor receptor but independent of the estrogen receptor, Shc, and Src. In addition, stimulation of MAPK and the subsequent activation of its downstream effector pathway, Elk-1, by HRS or PELP1 were found to depend on the presence of endogenous PELP1 or HRS. Furthermore, HRS was overexpressed and correlated well with the cytoplasmic PELP1, increased MAPK, and EGFR status in breast tumors. These findings highlight a novel role of HRS in up-regulating MAPK, presumably involving interaction with PELP1.

Novel function of POSH, a JNK scaffold, as an E3 ubiquitin ligase for the Hrs stability on early endosomes

POSH ( plenty of SH3s) acts as a scaffold that links activated Rac1 and downstream c-Jun N-terminal kinase (JNK) signaling modules. However, it is unknown whether it's functional domain-mediated roles including the interesting RING-finger domain or its cellular function. Here, we provide evidence that subcellular localization of POSH is regulated by a particular domain of the protein and POSH was colocalized with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) on early endosomes via interaction of Hrs with POSH's two rear SH3 domains. Moreover, the RING domain of POSH specifically regulates the stability of Hrs, but not of JNK1, via a ubiquitin–proteasomal degradation pathway. Finally, we demonstrate that JNK1 does not interact with Hrs under the conditions of POSH interacted with Hrs, but instead reduces the POSH-catalyzed ubiquitination of Hrs and their reciprocal interaction. Together, these data suggest that POSH has a distinct role as a specific E3 ubiquitin ligase for Hrs on early endosomes, and there exists a relationship between its separate activities as a scaffold and as an E3.

HRS inhibits EGF receptor signaling in the RT4 rat schwannoma cell line

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is required for trafficking of cell surface receptors to the lysosome. Previously, we identified HRS as a protein that interacts with the neurofibromatosis 2 tumor suppressor schwannomin. In the present study, we established modified RT4 schwannoma cell lines that inducibly express HRS and constitutively express epidermal growth factor receptor (EGFR) fused to the green fluorescent protein. We demonstrated that HRS expression reduced EGFR abundance and EGF-mediated Stat3 activation. HRS expression also targeted EGFR to late endosomes. Schwannomin inhibited EGF-mediated Stat3 activation, consistent with HRS and schwannomin interacting in the same signaling pathway. Paradoxically, past studies have shown that HRS overexpression blocked EGFR trafficking to the late endosome and EGFR downregulation contrary to predictions of HRS function in HRS knockout studies. This study is the first to show that HRS can reduce the abundance of total and active EGFR and may reflect cell type-specific HRS function.

Growth factors induce differential phosphorylation profiles of the Hrs–STAM complex: a common node in signalling networks with signal-specific properties

Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule) form a heterodimeric complex that associates with endosomal membranes and is tyrosine-phosphorylated in response to a variety of growth factors including EGF (epidermal growth factor), HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). Phosphorylation of the Hrs-STAM complex requires receptor endocytosis. We show that an intact UIM (ubiquitin interaction motif) within Hrs is a conserved requirement for Hrs phosphorylation downstream of both EGF and HGF stimulations. Consistent with this, expression of a dominant-negative form of the E3 ubiquitin ligase, c-Cbl, inhibits EGF- and HGF-dependent Hrs phosphorylation. Despite this conservation, kinase inhibitor profiles using PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and SU6656 indicate that distinct non-receptor tyrosine kinases couple EGF, HGF and PDGF stimulation with the tyrosine phosphorylation of the Hrs-STAM complex. Crucially, analysis with phospho-specific antibodies indicates that these kinases generate a signal-specific, combinatorial phosphorylation profile of the Hrs-STAM complex, with the potential of diversifying tyrosine kinase receptor signalling through a common element.

CeVPS‐27 is an Endosomal Protein Required for the Molting and the Endocytic Trafficking of the Low‐Density Lipoprotein Receptor‐Related Protein 1 in Caenorhabditis elegans

Class E vacuolar protein-sorting (Vps) proteins were first described in yeast as being involved in receptor-mediated endocytosis and multivesicular body formation. Inactivation by RNA interference of the class E VPS genes of the nematode Caenorhabditis elegans revealed heterogeneous phenotypes. We have further characterized the role of the essential gene Cevps-27, ortholog of human hepatocyte growth factor-regulated tyrosine kinase substrate, during the development of C. elegans. Use of green fluorescent protein fusion constructs and antibody staining revealed that Cevps-27 localizes to endosomal membranes. It is widely expressed but enriched in epithelial cells. Cevps-27 mutants presented enlarged endosomal structures and an accumulation of autophagic vesicles as revealed by electron microscopy and the analysis of the autophagic marker LGG-1. Cevps-27 animals arrested at L2-L3 molt with an inability to degrade their old cuticle. This molting phenotype was more severe when Cevps-27 worms were grown on suboptimal concentrations of cholesterol. Furthermore, defective endocytic trafficking of the low-density lipoprotein receptor-related protein 1 (LRP-1) was also observed in Cevps-27 mutants. These results indicate that CeVPS-27 is required for endosomal and autophagic pathways in C. elegans and plays a crucial role in the control of molting through LRP-1 internalization and cholesterol traffic.

Essential role of Hrs in a recycling mechanism mediating functional resensitization of cell signaling

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is well known to terminate cell signaling by sorting activated receptors to the MVB/lysosomal pathway. Here we identify a distinct role of Hrs in promoting rapid recycling of endocytosed signaling receptors to the plasma membrane. This function of Hrs is specific for receptors that recycle in a sequence-directed manner, in contrast to default recycling by bulk membrane flow, and is distinguishable in several ways from previously identified membrane-trafficking functions of Hrs/Vps27p. In particular, Hrs function in sequence-directed recycling does not require other mammalian Class E gene products involved in MVB/lysosomal sorting, nor is receptor ubiquitination required. Mutational studies suggest that the VHS domain of Hrs plays an important role in sequence-directed recycling. Disrupting Hrs-dependent recycling prevented functional resensitization of the beta(2)-adrenergic receptor, converting the temporal profile of cell signaling by this prototypic G protein-coupled receptor from sustained to transient. These studies identify a novel function of Hrs in a cargo-specific recycling mechanism, which is critical to controlling functional activity of the largest known family of signaling receptors.

Hrs, a Mammalian Master Molecule in Vesicular Transport and Protein Sorting, Suppresses the Degradation of ESCRT Proteins Signal Transducing Adaptor Molecule 1 and 2

The degradation and sorting of cytoplasmic and cell-surface proteins are crucial steps in the control of cellular functions. We previously identified three mammalian Vps (vacuolar protein sorting) proteins, Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and signal transducing adaptor molecule (STAM) 1 and -2, which are tyrosine-phosphorylated upon cytokine/growth factor stimulation. Hrs and the STAMs each contain a ubiquitin-interacting motif and through formation of a complex are involved in the vesicle transport of early endosomes. To explore the mechanism and cellular function of this complex in mammalian cells, we established an Hrs-defective fibroblastoid cell line (hrs(-/-)); embryos with this genotype died in utero. In the hrs(-/-) cells only trace amounts of STAM1 and STAM2 were detected. Introduction of wild-type Hrs or an Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) fully restored STAM1 and STAM2 expression, whereas mutants with no STAM binding ability (Hrs-dC2, Hrs-dM) failed to express the STAMs. This regulated control of STAM expression by Hrs was independent of transcription. Interestingly, STAM1 degradation was mediated by proteasomes and was partially dependent on the ubiquitin-interacting motif of STAM1. Revertant Hrs expression in hrs(-/-) cells not only led to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also restored STAM1 levels in early endosomes and eliminated the enlarged endosome phenotype caused by the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins.

Interactions of TOM1L1 with the Multivesicular Body Sorting Machinery

Tom1L1 (Tom1-like1) and related proteins Tom1 (Target of Myb1) and Tom1L2 (Tom1-like2) constitute a new protein family characterized by the presence of a VHS (Vps27p/Hrs/Stam) domain in the N-terminal portion followed by a GAT (GGA and Tom) domain. Recently it was demonstrated that the GAT domain of both Tom1 and Tom1L1 binds ubiquitin, suggesting that these proteins might participate in the sorting of ubiquitinated proteins into multivesicular bodies (MVBs). Here we report a novel interaction between Tom1L1 and members of the MVB sorting machinery. Specifically, we found that the VHS domain of Tom1L1 interacts with Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate), whereas a PTAP motif, located between the VHS and GAT domain of Tom1L1, is responsible for binding to TSG101 (tumor susceptibility gene 101). Myc epitope-tagged Tom1L1 showed a cytosolic distribution but was recruited to endosomes following Hrs expression. In addition, Tom1L1 possesses several tyrosine motifs at the C-terminal region that mediate interactions with members of the Src family kinases and other signaling proteins such as Grb2 and p85. We showed that a fraction of Fyn kinase localizes at endosomes and that this distribution becomes more evident after epidermal growth factor internalization. Moreover, expression of a constitutive active form of Fyn also promoted the recruitment of Tom1L1 to enlarged endosomes. Taken together, we propose that Tom1L1 could act as an intermediary between signaling and degradative pathways.

Lysosomal Targeting of E-Cadherin: a Unique Mechanism for the Down-Regulation of Cell-Cell Adhesion during Epithelial to Mesenchymal Transitions

A hallmark characteristic of epithelial tumor progression as well as some processes of normal development is the loss of the epithelial phenotype and acquisition of a motile or mesenchymal phenotype. Such epithelial to mesenchymal transitions are accompanied by the loss of E-cadherin function by either transcriptional or posttranscriptional mechanisms. Here we demonstrate that, upon v-Src expression, a potent trigger of epithelial to mesenchymal transitions, E-cadherin is internalized and then shuttled to the lysosome instead of being recycled back to the lateral membrane. Thus, while E-cadherin internalization facilitates the dissolution of adherens junctions, its subsequent traffic to the lysosome serves as a means to ensure that cells do not reform their cell-cell contacts and remain motile. We also show that ubiquitin tagging of E-cadherin is essential for its sorting to the lysosome. The lysosomal targeting of E-cadherin is mediated by hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and v-Src-induced activation of the Rab5 and Rab7 GTPases. Our studies reveal that the lysosomal targeting of E-cadherin is an important posttranscriptional mechanism to deplete cellular E-cadherin during Src-induced epithelial to mesenchymal transitions.

The Inside Story on Gα s

Best known for their role in transducing extracellular signals to the intracellular pathways that mediate their effects, heterotrimeric GTP-binding proteins (G proteins) are also found on intracellular membranes, where they have been hypothesized to play a role in membrane trafficking. Zheng et al. , who previously identified RGS-PX1, which acts as a Gα s GTPase activating protein and also as a sorting nexin (SNX, a family of proteins implicated in endosomal trafficking) that affects epidermal growth factor receptor (EGFR) degradation, investigated the role of Gα s in EGFR trafficking. When overexpressed in HEK293 cells also transfected with pXER-EGFR, Gα s enhanced degradation of EGF and the EGFR (following stimulation with EGF). In contrast, Gα s knockdown with siRNA delayed EGFR degradation in Cos7 cells, as did knockdown of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Knockdown of both Gα s and Hrs led to a greater delay in EGFR degradation than knockdown of either Gα s or Hrs alone. The authors used glutathione- S -transferase pull-down assays to show that both Gα s and RGS-PX1 bound Hrs in vitro and used coimmunoprecipitation to show that Gα s , RGS-PX1, and Hrs formed a complex in vivo. Gα s transfected into Cos7 cells colocalized with Hrs or RGS-Px1 in endosomes; moreover, overexpression of either RGS-PX1 or Hrs promoted the endosomal localization of Gα s . Thus, Gα s , acting in association with RGS-PX1 and Hrs, appears to play a role in regulating EGFR trafficking and degradation. B. Zheng, C. Lavoie, T.-D. Tang, P. Ma, T. Meerloo, A. Beas, M. G. Farquhar, Regulation of epidermal growth factor receptor degradation by heterotrimeric Gαs protein. Mol. Biol. Cell 15 , 5538-5550 (2004). [Abstract] [Full Text]

Regulation of epidermal growth factor receptor degradation by heterotrimeric Galphas protein.

Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Galphas) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Galphas expression by RNA interference (RNAi) delays receptor degradation. We also show that Galphas and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Galphas coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Galphas colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Galphas on endosomes. Furthermore, knock-down of both Hrs and Galphas by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Galphas and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Galphas in EGF receptor degradation and provide mechanistic insights into the function of Galphas in endocytic sorting.

Serine 518 phosphorylation modulates merlin intramolecular association and binding to critical effectors important for NF2 growth suppression.

The neurofibromatosis 2 (NF2) tumor suppressor protein, merlin, functions as a negative growth regulator; however, the molecular mechanisms that underlie merlin regulation remain elusive. Recent studies have implicated merlin phosphorylation in regulating merlin subcellular localization and growth suppression. P21-activated kinase (PAK), a downstream target of Rac1/Cdc42, directly phosphorylates merlin at Serine 518. In this report, we show that PAK2 directly phosphorylates wild-type merlin, whereas merlin truncation mutants with impaired GST-amino-terminal domain (N-term or NTD)/GST-carboxy-terminal domain (C-term or CTD) intramolecular association exhibit impaired S518 phosphorylation. We directly demonstrate that PAK2 phosphorylation impairs merlin N-term/C-term binding in vitro and in vivo. Lastly, we show that PAK2 phosphorylation impairs the ability of merlin to bind to two interacting proteins, CD44 and hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), both critical for merlin growth suppression. These observations suggest that merlin S518 phosphorylation directly modulates merlin intramolecular and intermolecular associations important for the ability of merlin to function as a tumor suppressor.