scholarly journals Tyrosine Phosphorylation of Eps15 Is Required for Ligand-Regulated, but Not Constitutive, Endocytosis

2000 ◽  
Vol 150 (4) ◽  
pp. 905-912 ◽  
Author(s):  
Stefano Confalonieri ◽  
Anna Elisabetta Salcini ◽  
Claudia Puri ◽  
Carlo Tacchetti ◽  
Pier Paolo Di Fiore
Keyword(s):  
Tyrosine Phosphorylation ◽  
Phosphorylation Site ◽  
Growth Factor Receptor ◽  
Differential Regulation ◽  
Dominant Negative ◽  
Membrane Receptors ◽  
Kinase Substrate ◽  
Molecular Determinant ◽  
Molecular Machinery

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.

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

2000 ◽  
Vol 20 (20) ◽  
pp. 7685-7692 ◽  
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.

Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium

1999 ◽  
Vol 276 (1) ◽  
pp. C91-C101 ◽  
Author(s):  
Kurt Amsler ◽  
Scott K. Kuwada
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Basolateral Membrane ◽  
Growth Factor Receptor ◽  
Membrane Receptor ◽  
Membrane Receptors ◽  
Receptor Interaction ◽  
Signaling Proteins ◽  
Polarized Epithelial Cells

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-γ (PLC-γ) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-γ protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

scholarly journals An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor

2008 ◽  
Vol 180 (6) ◽  
pp. 1205-1218 ◽  
Author(s):  
Ingrid Roxrud ◽  
Camilla Raiborg ◽  
Nina Marie Pedersen ◽  
Espen Stang ◽  
Harald Stenmark
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Coated Pits ◽  
Kinase Substrate ◽  
Epidermal Growth

Down-regulation of activated and ubiquitinated growth factor (GF) receptors by endocytosis and subsequent lysosomal degradation ensures attenuation of GF signaling. The ubiquitin-binding adaptor protein Eps15 (epidermal growth factor receptor [EGFR] pathway substrate 15) functions in endocytosis of such receptors. Here, we identify an Eps15 isoform, Eps15b, and demonstrate its expression in human cells and conservation across vertebrate species. Although both Eps15 and Eps15b interact with the endosomal sorting protein Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) in vitro, we find that Hrs specifically binds Eps15b in vivo (whereas adaptor protein 2 preferentially interacts with Eps15). Although Eps15 mainly localizes to clathrin-coated pits at the plasma membrane, Eps15b localizes to Hrs-positive microdomains on endosomes. Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect. Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR. These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.

scholarly journals EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (8) ◽  
pp. 4294-4304 ◽  
Author(s):  
H Y Tang ◽  
A Munn ◽  
M Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Growth Factor Receptor ◽  
Cell Cortex ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Cortical Actin ◽  
Kinase Substrate ◽  
Physiological Relevance ◽  
Eh Domain

Several proteins from diverse organisms have been shown to share a region of sequence homology with the mammalian epidermal growth factor receptor tyrosine kinase substrate Eps15. Included in this new protein family, termed EH domain proteins, are two yeast proteins, Pan1p and End3p. We have shown previously that Pan1p is required for normal organization of the actin cytoskeleton and that it associates with the actin patches on the cell cortex. End3p has been shown by others to be an important factor in the process of endocytosis. End3p is also known to be required for the organization of the actin cytoskeleton. Here we report that Pan1p and End3p act as a complex in vivo. Using the pan1-4 mutant which we isolated and characterized previously, the END3 gene was identified as a suppressor of pan1-4 when overexpressed. Suppression of the pan1-4 mutation by multicopy END3 required the presence of the mutant Pan1p protein. Coimmunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other. The localization of Pan1p to the cortical actin cytoskeleton became weakened in the end3 mutant at the permissive temperature and undetectable at the restrictive temperature, suggesting that End3p may be important for proper localization of Pan1p to the cortical actin cytoskeleton. The finding that the pan1-4 mutant was defective in endocytosis as severely as the end3 mutant under nonpermissive conditions supports the notion that the association between Pan1p and End3p is of physiological relevance. Together with results of earlier reports, these results provide strong evidence suggesting that Pan1p and End3p are the components of a complex that has essential functions in both the organization of cell membrane-associated actin cytoskeleton and the process of endocytosis.

scholarly journals A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

1997 ◽  
Vol 17 (12) ◽  
pp. 7362-7374 ◽  
Author(s):  
J A Diehl ◽  
C J Sherr
Keyword(s):  
Cyclin D1 ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Cyclin Dependent Kinase ◽  
Linker Peptide ◽  
Cdk Activating Kinase

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

The serine/threonine kinase ULK1 is a target of multiple phosphorylation events

2011 ◽  
Vol 440 (2) ◽  
pp. 283-291 ◽  
Author(s):  
Markus Bach ◽  
Mark Larance ◽  
David E. James ◽  
Georg Ramm
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
Phosphorylation Site ◽  
Degradation Process ◽  
Dominant Negative ◽  
Serine Threonine Kinase ◽  
Kinase Activation ◽  
Threonine Kinase ◽  
Convergence Point ◽  
Mtor Phosphorylation

Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser555in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser774 and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr180 that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.

Transactivated EGFR mediates α1-AR-induced STAT3 activation and cardiac hypertrophy

2011 ◽  
Vol 301 (5) ◽  
pp. H1941-H1951 ◽  
Author(s):  
Yan Li ◽  
Hui Zhang ◽  
Wenqiang Liao ◽  
Yao Song ◽  
Xiaowei Ma ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Tyrosine Phosphorylation ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Growth Factor Receptor ◽  
Sprague Dawley ◽  
Inhibitory Peptide ◽  
Rat Cardiomyocytes ◽  
Hypertrophic Growth

Li Y, Zhang H, Liao W, Song Y, Ma X, Chen C, Lu Z, Li Z, Zhang Y. α1-Adrenergic receptor (α1-AR) is a crucial mediator of cardiac hypertrophy. Although numerous intracellular pathways have been implicated in α1-AR-induced hypertrophy, its precise mechanism remains elusive. We aimed to determine whether α1-AR induces cardiac hypertrophy through a novel signaling pathway-α1-AR/epidermal growth factor receptor (EGFR)/signal transducer and activator of transcription 3 (STAT3). The activation of STAT3 by α1-AR was first demonstrated by tyrosine phosphorylation, nuclear translocation, DNA binding, and transcriptional activity in neonatal Sprague-Dawley rat cardiomyocytes. Activated STAT3 showed an essential role in α1-AR-induced cardiomyocyte hypertrophic growth, as assessed by treatment with STAT3 inhibitory peptide and lentivirus-STAT3 small interfering RNA. The results were further confirmed by in vivo experiments involving intraperitoneal injection of the STAT3 inhibitor WP1066 significantly inhibiting phenylephrine-infusion-induced heart hypertrophy in male C57BL/6 mice. Furthermore, the α1-AR-activated STAT3 was associated with transactivation of EGFR because inhibition of EGFR with the selective inhibitor AG1478 prevented α1-AR-induced STAT3 tyrosine phosphorylation and its transcriptional activity, as well as cardiac hypertrophy. In summary, these results suggest that α1-AR induces the activation of STAT3, mainly through transactivation of EGFR, which plays an important role in α1-AR-induced cardiac hypertrophy.

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