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W2108697952 abstract "Transforming growth factor β (TGFβ) receptors require SARA for phosphorylation of the downstream transducing Smad proteins. SARA, a FYVE finger protein, binds to membrane lipids suggesting that activated receptors may interact with downstream signaling molecules at discrete endocytic locations. In the present study, we reveal a critical role for the early endocytic compartment in regulating Smad-dependent signaling. Not only is SARA localized on early endosomes, but also its minimal FYVE finger sequence is sufficient for early endosomal targeting. Expression of a SARA mutant protein lacking the FYVE finger inhibits downstream activin A signaling in endothelial cells. Moreover, a dominant-negative mutant of Rab5, a crucial protein for early endosome dynamics, causes phosphorylation and nuclear translocation of Smads leading to constitutive (i.e. ligand independent) transcriptional activation of a Smad-dependent promoter in endothelial cells. As inhibition of endocytosis using the K44A negative mutant of dynamin and RN-tre did not lead to activation of Smad-dependent transcription, the effects of the dominant-negative Rab5 are likely to be a consequence of altered membrane trafficking of constitutively formed TGFβ/activin type I/II receptor complexes at the level of early endosomes. The results suggest an important interconnection between early endosomal dynamics and TGFβ/activin signal transduction pathways. Transforming growth factor β (TGFβ) receptors require SARA for phosphorylation of the downstream transducing Smad proteins. SARA, a FYVE finger protein, binds to membrane lipids suggesting that activated receptors may interact with downstream signaling molecules at discrete endocytic locations. In the present study, we reveal a critical role for the early endocytic compartment in regulating Smad-dependent signaling. Not only is SARA localized on early endosomes, but also its minimal FYVE finger sequence is sufficient for early endosomal targeting. Expression of a SARA mutant protein lacking the FYVE finger inhibits downstream activin A signaling in endothelial cells. Moreover, a dominant-negative mutant of Rab5, a crucial protein for early endosome dynamics, causes phosphorylation and nuclear translocation of Smads leading to constitutive (i.e. ligand independent) transcriptional activation of a Smad-dependent promoter in endothelial cells. As inhibition of endocytosis using the K44A negative mutant of dynamin and RN-tre did not lead to activation of Smad-dependent transcription, the effects of the dominant-negative Rab5 are likely to be a consequence of altered membrane trafficking of constitutively formed TGFβ/activin type I/II receptor complexes at the level of early endosomes. The results suggest an important interconnection between early endosomal dynamics and TGFβ/activin signal transduction pathways. The transforming growth factor β (TGFβ) 1The abbreviations used are: TGFβtransforming growth factor βPI(3)Pphosphatidylinositol 3-phosphateBBCEbovine brain capillary endothelialFCSfetal calf serumSBESmad-binding elementlucluciferaseGFPgreen fluorescent proteinCMVcytomegalovirusβ-galβ-galactosidaseTRITCtetramethylrhodamine isothiocyanate 1The abbreviations used are: TGFβtransforming growth factor βPI(3)Pphosphatidylinositol 3-phosphateBBCEbovine brain capillary endothelialFCSfetal calf serumSBESmad-binding elementlucluciferaseGFPgreen fluorescent proteinCMVcytomegalovirusβ-galβ-galactosidaseTRITCtetramethylrhodamine isothiocyanate superfamily is a large group of secreted polypeptide growth factors, which include the TGFβs, the activins, and the bone morphogenetic proteins. Members of this family play critical roles during embryogenesis and in maintaining tissue homeostasis in adult life. Deregulated TGFβ family signaling has been implicated in multiple developmental disorders and in various human diseases, including cancer (1Massague J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2052) Google Scholar). Some of these disorders, such as hereditary hemorrhagic teleangiectasia and primary pulmonary hypertension, involve altered TGFβ family signaling regulating vasculogenic and angiogenic responses of endothelial cells (2Bourdeau A. Dumont D.J. Letarte M. J. Clin. Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (385) Google Scholar, 3Oh S.P. Seki T. Goss K.A. Imamura T., Yi, Y. Donahoe P.K., Li, L. Miyazono K. ten Dijke P. Kim S. Li E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2626-2631Crossref PubMed Scopus (721) Google Scholar, 4Arthur H.M. Ure J. Smith A.J. Renforth G. Wilson D.I. Torsney E. Charlton R. Parums D.V. Jowett T. Marchuk D.A. Burn J. Diamond A.G. Dev. Biol. 2000; 217: 42-53Crossref PubMed Scopus (377) Google Scholar, 5Peacock A.J. Thorax. 1999; 54: 1107-1118Crossref PubMed Scopus (82) Google Scholar, 6Galvin K.M. Donovan M.J. Lynch C.A. Meyer R.I. Paul R.J. Lorenz J.N. Fairchild-Huntress V. Dixon K.L. Dunmore J.H. Gimbrone M.A., Jr. Falb D. Huszar D. Nat. Genet. 2000; 24: 171-174Crossref PubMed Scopus (396) Google Scholar). Indeed, TGFβ1 is known to influence both endothelial cell proliferation and critical endothelial cell-pericyte interactions occurring during vessel maturation (7Beck L., Jr. D'Amore P.A. FASEB J. 1997; 11: 365-373Crossref PubMed Scopus (446) Google Scholar). We have recently shown also that activin A affects endothelial cell function leading to inhibition of angiogenesis and decreased vessel wall integrity (8Breit S. Ashman K. Wilting J. Rossler J. Hatzi E. Fotsis T. Schweigerer L. Cancer Res. 2000; 60: 4596-4601PubMed Google Scholar).The TGFβ/activin family members signal through heteromeric complexes of transmembrane type I and type II serine-threonine kinase receptors. The type II receptor kinase phosphorylates the type I receptor kinase which in turn phosphorylates the downstream transducer proteins, Smad2 and Smad3 (reviewed in Ref. 9Wrana J. Cell. 2000; 100: 189-192Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). The latter associate with Smad4 and the resulting complex translocates to the nucleus, where they control transcription of target genes. Recent data show that, in the case of the TGFβ receptor and most likely in the case of activin (10Miura S. Takeshita T. Asao H. Kimura Y. Murata K. Sasaki Y. Hanai J.-I. Beppu H. Tsukazaki T. Wrana J. Miyazono K. Sugamura K. Mol. Cell. Biol. 2001; 20: 9346-9355Crossref Scopus (148) Google Scholar), the Smad-binding protein SARA plays an important role in phosphorylation of Smad2 and Smad3 by TGFβRI and ActRIB receptors (10Miura S. Takeshita T. Asao H. Kimura Y. Murata K. Sasaki Y. Hanai J.-I. Beppu H. Tsukazaki T. Wrana J. Miyazono K. Sugamura K. Mol. Cell. Biol. 2001; 20: 9346-9355Crossref Scopus (148) Google Scholar, 11Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar). SARA recruits Smad2 and Smad3 to intracellular membranes that contain the receptor. This targeting requires a FYVE finger, which by analogy to other FYVE fingers (reviewed in Ref. 12Stenmark H. Aasland R. J. Cell Sci. 1999; 112: 4175-4183Crossref PubMed Google Scholar) has been speculated to bind specifically to phosphatidylinositol 3-phosphate (PI(3)P). Even though the subcellular localization of SARA (11Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar) is as yet uncharacterized, we hypothesized that this protein may localize to early endosomes, which are known to be enriched for PI(3)P (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar).The concept that activated receptors interact with downstream signaling molecules at discrete endocytic locations has long been suspected (reviewed in Ref. 14Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (256) Google Scholar). Furthermore, membrane trafficking plays an important role in controlling the location of signaling interactions and in regulating receptor degradation and/or recycling (reviewed in Ref. 15Leof E.B. Trends Cell Biol. 2000; 10: 343-348Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In the case of the TGFβ/activin receptors, little is known about how endocytosis and receptor trafficking influences the assembly, localization, and activation of ligand-receptor-SARA-Smad complexes. Indeed, the temporal and spatial regulation of these interactions is not fully understood. Since SARA binds to membranes via its FYVE finger providing a potential link between membrane trafficking and TGFβ/activin signaling, we have addressed the intracellular localization of SARA and investigated the requirements for its FYVE finger-membrane lipid interaction. Motivated by our finding that SARA is localized on early endosomes, we reasoned that experimental perturbation of proteins which regulate endosome function would allow us to address the contribution of the endocytic pathway in the control of TGFβ/activin signaling. Toward this purpose, we have investigated the effects on Smad-dependent signaling of constitutively active and dominant-negative mutant forms of Rab5, a small GTPase that plays a key role in endosome dynamics and receptor signaling (16Bucci C. Parton R.G. Mather I.H. Stunnenberg H. Simons K. Hoflack B. Zerial M. Cell. 1992; 70: 715-728Abstract Full Text PDF PubMed Scopus (1116) Google Scholar, 17Gorvel J.-P. Chavrier P. Zerial M. Gruenberg J. Cell. 1991; 64: 915-925Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 18Nielsen E. Severin F. Backer J.M. Hyman A.A. Zerial M. Nat. Cell Biol. 1999; 6: 376-382Crossref Scopus (392) Google Scholar, 19Barbieri M.A. Roberts R.L. Gumusboga A. Highfield H. Alvarez-Dominguez C. Wells A. Stahl P.D. J. Cell Biol. 2000; 151: 539-550Crossref PubMed Scopus (197) Google Scholar, 20Lanzetti L. Rybin V. Malabarba M.G. Christoforidis S. Scita G. Zerial M. Di Fiore P.P. Nature. 2000; 408: 374-377Crossref PubMed Scopus (233) Google Scholar).DISCUSSIONInternalization of ligand activated receptors has been considered merely as a signal attenuation mechanism (reviewed in Ref. 14Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (256) Google Scholar). However, there is increasing evidence that endocytosis of ligand-receptor complexes not only leads to signal attenuation (36Vieira A. Lamaze C. Schmid S. Science. 1996; 274: 2086-2088Crossref PubMed Scopus (820) Google Scholar), but may be also necessary to co-localize activated receptors with downstream effectors (37Daaka Y. Luttrell L.M. Ahn S. Rocca G.J.D. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Thus, the idea of signaling from the endocytic compartment is gaining momentum. In the case of the TGFβ/activin family receptors, it is unclear whether receptor internalization is required to reach the SARA-bound Smad substrate or to what extent endosome dynamics affect TGFβ family signaling.We have addressed the intracellular localization of SARA and found that SARA is present on the early endocytic compartment, suggesting that the Smad pathway signals from the early endosome. Furthermore, SARA significantly increases endosome size, suggestive of a role in endosome dynamics. We have observed by immunofluorescence that the FYVE finger of SARA is sufficient for its endosomal targeting. So far, the identified FYVE finger proteins have been shown to require additional binding partners for targeting to endosomal membranes. Thus, EEA1 and Rabenosyn 5 require the binding of an adjacent domain to endosomal Rab5GTP (29Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (905) Google Scholar, 38Nielsen E. Christoforidis S. Uttenweiler-Joseph S. Miaczynska M. Dewitte F. Wilm M. Hoflack B. Zerial M. J. Cell Biol. 2000; 151: 601-612Crossref PubMed Scopus (274) Google Scholar). Surface plasmon resonance experiments indicated that the SARA FYVE finger has a higher affinity (KD of 30 nm) than those of Hrs (KD of 38 nm) and EEA1 (KD of 45 nm) for PI(3)P. This difference in KD may partially account for the fact that the SARA FYVE finger is efficiently targeted to early endosomes, whereas the EEA1 and Hrs FYVE fingers are mainly cytosolic when expressed as such (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar, 22Gaullier J.-M. Ronning E. Gillooly D.J. Stenmark H. J. Biol. Chem. 2000; 275: 24595-24600Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Since the dimerization of FYVE domains increases their avidity for PI(3)P-containing membranes (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar, 39Sankaran V.G. Klein D.E. Sachdeva M.M. Lemmon M.A. Biochemistry. 2001; 40: 8581-8587Crossref PubMed Scopus (62) Google Scholar), it is also possible that the isolated FYVE domain of SARA has a higher propensity to dimerize than those of Hrs and EEA1. Although we cannot rule out the possibility that the surface plasmon resonance experiments underestimate the differences in ligand affinities, we favor the view that endosomal targeting of SARA may rely solely on binding to endosome-located PI(3)P (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar). For instance, we did not find any direct interactions between SARA and Rab5 by the two-hybrid system (data not shown). However, we cannot fully rule out the possibility that our minimal FYVE finger construct may contain other binding elements which participate in dimerization or endosomal targeting.In the light of our initial results showing that SARA is located on early endosomes, we sought further evidence regarding the inter-relation between endosome dynamics and signaling. We reasoned that a certain level of regulation may exist at the level of this organelle and proteins, such as Rab5, regulating early endosome function may indeed alter signaling. Moreover, Rab5 has been implicated in EGFR signaling (20Lanzetti L. Rybin V. Malabarba M.G. Christoforidis S. Scita G. Zerial M. Di Fiore P.P. Nature. 2000; 408: 374-377Crossref PubMed Scopus (233) Google Scholar) and is activated by EGF stimulation (19Barbieri M.A. Roberts R.L. Gumusboga A. Highfield H. Alvarez-Dominguez C. Wells A. Stahl P.D. J. Cell Biol. 2000; 151: 539-550Crossref PubMed Scopus (197) Google Scholar). A striking finding of the present study was that Rab5S34N, a dominant-negative Rab5 mutant, stimulated transcription of a Smad-dependent promoter, in the absence of TGFβ/activins, in serum-free conditions. This activation was associated with phosphorylation and nuclear translocation of Smad proteins. Phosphorylation of Smads by Rab5S34N was independent of indirect effects such as establishment of a TGFβ/activin autocrine loop or depolymerization of microtubules. Indeed, microtubules sequester unphosphorylated Smads, and depolymerization of the microtubular network releases active, phosphorylated Smads by an uncharacterized mechanism (33Dong C., Li, Z. Alverez R. Feng X.-H. Goldschmidt-Clermont P.J. Mol. Cell. 2000; 5: 27-34Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Because phosphorylation of Smads by Rab5S34N occurred in serum-free conditions and since it has been previously shown that the cytoplasmic domains of type II and type I TGFβ receptors interact physically and functionally with each other in a ligand-independent manner (40Feng X.H. Derynck R. J. Biol. Chem. 1996; 271: 13123-13129Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), it was reasonable to assume that Rab5S34N might be able to amplify such low-level constitutive TGFβ/activin receptor activation. Indeed, we have observed a considerable inhibition of the transcriptional activation of the Smad-dependent promoter by Rab5S34N when co-transfecting dominant-negative ALK2, ALK4, and ALK5 receptor constructs.Since Rab5S34N inhibits endocytosis, recycling, and early endosome fusion (31Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (767) Google Scholar), the data suggested a regulatory role of membrane trafficking on the intensity of signaling cascades. A negligible effect of constitutively formed TGFβ-activin receptor complexes, on Smad-dependent transcription could be grossly amplified by Rab5S34N. Such amplification was unlikely to be derived from a decreased rate of endocytosis as blocking of plasma membrane endocytosis by expression of RN-tre, a specific Rab5 GAP (20Lanzetti L. Rybin V. Malabarba M.G. Christoforidis S. Scita G. Zerial M. Di Fiore P.P. Nature. 2000; 408: 374-377Crossref PubMed Scopus (233) Google Scholar), did not augment Smad-dependent transcription. Similarly, there was no increase in Smad-dependent transcription following inhibition of clathrin-coated pit- and caveolin-dependent plasma membrane endocytosis (41Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (551) Google Scholar, 42Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (617) Google Scholar, 43Zwaagstra J.C., El- Alfy M. O'Connor-McCourt M.D. J. Biol. Chem. 2001; 276: 27237-27245Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) by the dominant-negative K44A dynamin construct (34van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (586) Google Scholar, 35Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar). These results implied that the Rab5S34N effect was rather a consequence of decreased degradative or recycling trafficking leading to accumulation of constitutively formed TGFβ-activin type I/II receptor complexes on early endosomal membranes, where SARA-Smad complexes reside. Such accumulation of TGFβ-activin type I/II receptor complexes, and increased residence time thereof, presumably accounts for the increase in signaling observed upon RabS34N expression. Likewise, the observed reduction of activin A-induced Smad promoter transcription by the constitutively active Rab5Q79L is likely to be a consequence of enhanced receptor trafficking leading to decreased residence in the early endosomal compartment. It appears that cycling of Rab5 between GTP and GDP forms may influence the length and intensity of TGFβ/activin signaling cascades by regulating TGFβ-activin type I/II receptor trafficking via the early endocytic compartment. Indeed, it has been shown that Rab5S34N reduces epidermal growth factor receptor degradation by influencing membrane trafficking (44Papini E. Satin B. Bucci C. de Bernard M. Telford J.L. Manetti R. Rappuoli R. Zerial M. Montecucco C. EMBO J. 1997; 16: 15-24Crossref PubMed Scopus (193) Google Scholar). Alternatively, Rab5 could exert its effects by directly binding to components of the TGFβ/activin pathway or affecting TGFβ/activin receptor kinase activity, for instance, by modulating receptor-associated kinases or phosphatases (45Griswold-Prenner I. Kamibayashi C. Maruoka E.M. Mumby M.C. Derynck R. Mol. Cell. Biol. 1998; 18: 6595-6604Crossref PubMed Google Scholar). Toward this end, we did not observe any direct interactions between Rab5 and SARA or Smad2/3 proteins using the yeast 2-hybrid system (data not shown).In conclusion, we have revealed a critical role of early endosomes in regulating Smad-dependent signaling. Not only is SARA localized in the early endocytic compartment but also a dominant-negative Rab5 mutant causes phosphorylation and nuclear translocation of Smads leading to transcriptional activation of a Smad-dependent promoter. Rab5S34N not only stimulated Smad-dependent transcriptional activation, but also inhibited the proliferation of endothelial cells and keratinocytes mimicking the effects of TGFβ/activins. The results suggest an interconnection between events in early endosomes with signal transduction pathways and may have important implications in understanding how cells co-ordinate their cellular functions when responding to extracellular stimuli. The transforming growth factor β (TGFβ) 1The abbreviations used are: TGFβtransforming growth factor βPI(3)Pphosphatidylinositol 3-phosphateBBCEbovine brain capillary endothelialFCSfetal calf serumSBESmad-binding elementlucluciferaseGFPgreen fluorescent proteinCMVcytomegalovirusβ-galβ-galactosidaseTRITCtetramethylrhodamine isothiocyanate 1The abbreviations used are: TGFβtransforming growth factor βPI(3)Pphosphatidylinositol 3-phosphateBBCEbovine brain capillary endothelialFCSfetal calf serumSBESmad-binding elementlucluciferaseGFPgreen fluorescent proteinCMVcytomegalovirusβ-galβ-galactosidaseTRITCtetramethylrhodamine isothiocyanate superfamily is a large group of secreted polypeptide growth factors, which include the TGFβs, the activins, and the bone morphogenetic proteins. Members of this family play critical roles during embryogenesis and in maintaining tissue homeostasis in adult life. Deregulated TGFβ family signaling has been implicated in multiple developmental disorders and in various human diseases, including cancer (1Massague J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2052) Google Scholar). Some of these disorders, such as hereditary hemorrhagic teleangiectasia and primary pulmonary hypertension, involve altered TGFβ family signaling regulating vasculogenic and angiogenic responses of endothelial cells (2Bourdeau A. Dumont D.J. Letarte M. J. Clin. Invest. 1999; 104: 1343-1351Crossref PubMed Scopus (385) Google Scholar, 3Oh S.P. Seki T. Goss K.A. Imamura T., Yi, Y. Donahoe P.K., Li, L. Miyazono K. ten Dijke P. Kim S. Li E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2626-2631Crossref PubMed Scopus (721) Google Scholar, 4Arthur H.M. Ure J. Smith A.J. Renforth G. Wilson D.I. Torsney E. Charlton R. Parums D.V. Jowett T. Marchuk D.A. Burn J. Diamond A.G. Dev. Biol. 2000; 217: 42-53Crossref PubMed Scopus (377) Google Scholar, 5Peacock A.J. Thorax. 1999; 54: 1107-1118Crossref PubMed Scopus (82) Google Scholar, 6Galvin K.M. Donovan M.J. Lynch C.A. Meyer R.I. Paul R.J. Lorenz J.N. Fairchild-Huntress V. Dixon K.L. Dunmore J.H. Gimbrone M.A., Jr. Falb D. Huszar D. Nat. Genet. 2000; 24: 171-174Crossref PubMed Scopus (396) Google Scholar). Indeed, TGFβ1 is known to influence both endothelial cell proliferation and critical endothelial cell-pericyte interactions occurring during vessel maturation (7Beck L., Jr. D'Amore P.A. FASEB J. 1997; 11: 365-373Crossref PubMed Scopus (446) Google Scholar). We have recently shown also that activin A affects endothelial cell function leading to inhibition of angiogenesis and decreased vessel wall integrity (8Breit S. Ashman K. Wilting J. Rossler J. Hatzi E. Fotsis T. Schweigerer L. Cancer Res. 2000; 60: 4596-4601PubMed Google Scholar). transforming growth factor β phosphatidylinositol 3-phosphate bovine brain capillary endothelial fetal calf serum Smad-binding element luciferase green fluorescent protein cytomegalovirus β-galactosidase tetramethylrhodamine isothiocyanate transforming growth factor β phosphatidylinositol 3-phosphate bovine brain capillary endothelial fetal calf serum Smad-binding element luciferase green fluorescent protein cytomegalovirus β-galactosidase tetramethylrhodamine isothiocyanate The TGFβ/activin family members signal through heteromeric complexes of transmembrane type I and type II serine-threonine kinase receptors. The type II receptor kinase phosphorylates the type I receptor kinase which in turn phosphorylates the downstream transducer proteins, Smad2 and Smad3 (reviewed in Ref. 9Wrana J. Cell. 2000; 100: 189-192Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). The latter associate with Smad4 and the resulting complex translocates to the nucleus, where they control transcription of target genes. Recent data show that, in the case of the TGFβ receptor and most likely in the case of activin (10Miura S. Takeshita T. Asao H. Kimura Y. Murata K. Sasaki Y. Hanai J.-I. Beppu H. Tsukazaki T. Wrana J. Miyazono K. Sugamura K. Mol. Cell. Biol. 2001; 20: 9346-9355Crossref Scopus (148) Google Scholar), the Smad-binding protein SARA plays an important role in phosphorylation of Smad2 and Smad3 by TGFβRI and ActRIB receptors (10Miura S. Takeshita T. Asao H. Kimura Y. Murata K. Sasaki Y. Hanai J.-I. Beppu H. Tsukazaki T. Wrana J. Miyazono K. Sugamura K. Mol. Cell. Biol. 2001; 20: 9346-9355Crossref Scopus (148) Google Scholar, 11Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar). SARA recruits Smad2 and Smad3 to intracellular membranes that contain the receptor. This targeting requires a FYVE finger, which by analogy to other FYVE fingers (reviewed in Ref. 12Stenmark H. Aasland R. J. Cell Sci. 1999; 112: 4175-4183Crossref PubMed Google Scholar) has been speculated to bind specifically to phosphatidylinositol 3-phosphate (PI(3)P). Even though the subcellular localization of SARA (11Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar) is as yet uncharacterized, we hypothesized that this protein may localize to early endosomes, which are known to be enriched for PI(3)P (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar). The concept that activated receptors interact with downstream signaling molecules at discrete endocytic locations has long been suspected (reviewed in Ref. 14Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (256) Google Scholar). Furthermore, membrane trafficking plays an important role in controlling the location of signaling interactions and in regulating receptor degradation and/or recycling (reviewed in Ref. 15Leof E.B. Trends Cell Biol. 2000; 10: 343-348Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In the case of the TGFβ/activin receptors, little is known about how endocytosis and receptor trafficking influences the assembly, localization, and activation of ligand-receptor-SARA-Smad complexes. Indeed, the temporal and spatial regulation of these interactions is not fully understood. Since SARA binds to membranes via its FYVE finger providing a potential link between membrane trafficking and TGFβ/activin signaling, we have addressed the intracellular localization of SARA and investigated the requirements for its FYVE finger-membrane lipid interaction. Motivated by our finding that SARA is localized on early endosomes, we reasoned that experimental perturbation of proteins which regulate endosome function would allow us to address the contribution of the endocytic pathway in the control of TGFβ/activin signaling. Toward this purpose, we have investigated the effects on Smad-dependent signaling of constitutively active and dominant-negative mutant forms of Rab5, a small GTPase that plays a key role in endosome dynamics and receptor signaling (16Bucci C. Parton R.G. Mather I.H. Stunnenberg H. Simons K. Hoflack B. Zerial M. Cell. 1992; 70: 715-728Abstract Full Text PDF PubMed Scopus (1116) Google Scholar, 17Gorvel J.-P. Chavrier P. Zerial M. Gruenberg J. Cell. 1991; 64: 915-925Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 18Nielsen E. Severin F. Backer J.M. Hyman A.A. Zerial M. Nat. Cell Biol. 1999; 6: 376-382Crossref Scopus (392) Google Scholar, 19Barbieri M.A. Roberts R.L. Gumusboga A. Highfield H. Alvarez-Dominguez C. Wells A. Stahl P.D. J. Cell Biol. 2000; 151: 539-550Crossref PubMed Scopus (197) Google Scholar, 20Lanzetti L. Rybin V. Malabarba M.G. Christoforidis S. Scita G. Zerial M. Di Fiore P.P. Nature. 2000; 408: 374-377Crossref PubMed Scopus (233) Google Scholar). DISCUSSIONInternalization of ligand activated receptors has been considered merely as a signal attenuation mechanism (reviewed in Ref. 14Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (256) Google Scholar). However, there is increasing evidence that endocytosis of ligand-receptor complexes not only leads to signal attenuation (36Vieira A. Lamaze C. Schmid S. Science. 1996; 274: 2086-2088Crossref PubMed Scopus (820) Google Scholar), but may be also necessary to co-localize activated receptors with downstream effectors (37Daaka Y. Luttrell L.M. Ahn S. Rocca G.J.D. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Thus, the idea of signaling from the endocytic compartment is gaining momentum. In the case of the TGFβ/activin family receptors, it is unclear whether receptor internalization is required to reach the SARA-bound Smad substrate or to what extent endosome dynamics affect TGFβ family signaling.We have addressed the intracellular localization of SARA and found that SARA is present on the early endocytic compartment, suggesting that the Smad pathway signals from the early endosome. Furthermore, SARA significantly increases endosome size, suggestive of a role in endosome dynamics. We have observed by immunofluorescence that the FYVE finger of SARA is sufficient for its endosomal targeting. So far, the identified FYVE finger proteins have been shown to require additional binding partners for targeting to endosomal membranes. Thus, EEA1 and Rabenosyn 5 require the binding of an adjacent domain to endosomal Rab5GTP (29Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (905) Google Scholar, 38Nielsen E. Christoforidis S. Uttenweiler-Joseph S. Miaczynska M. Dewitte F. Wilm M. Hoflack B. Zerial M. J. Cell Biol. 2000; 151: 601-612Crossref PubMed Scopus (274) Google Scholar). Surface plasmon resonance experiments indicated that the SARA FYVE finger has a higher affinity (KD of 30 nm) than those of Hrs (KD of 38 nm) and EEA1 (KD of 45 nm) for PI(3)P. This difference in KD may partially account for the fact that the SARA FYVE finger is efficiently targeted to early endosomes, whereas the EEA1 and Hrs FYVE fingers are mainly cytosolic when expressed as such (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar, 22Gaullier J.-M. Ronning E. Gillooly D.J. Stenmark H. J. Biol. Chem. 2000; 275: 24595-24600Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Since the dimerization of FYVE domains increases their avidity for PI(3)P-containing membranes (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar, 39Sankaran V.G. Klein D.E. Sachdeva M.M. Lemmon M.A. Biochemistry. 2001; 40: 8581-8587Crossref PubMed Scopus (62) Google Scholar), it is also possible that the isolated FYVE domain of SARA has a higher propensity to dimerize than those of Hrs and EEA1. Although we cannot rule out the possibility that the surface plasmon resonance experiments underestimate the differences in ligand affinities, we favor the view that endosomal targeting of SARA may rely solely on binding to endosome-located PI(3)P (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar). For instance, we did not find any direct interactions between SARA and Rab5 by the two-hybrid system (data not shown). However, we cannot fully rule out the possibility that our minimal FYVE finger construct may contain other binding elements which participate in dimerization or endosomal targeting.In the light of our initial results showing that SARA is located on early endosomes, we sought further evidence regarding the inter-relation between endosome dynamics and signaling. We reasoned that a certain level of regulation may exist at the level of this organelle and proteins, such as Rab5, regulating early endosome function may indeed alter signaling. Moreover, Rab5 has been implicated in EGFR signaling (20Lanzetti L. Rybin V. Malabarba M.G. Christoforidis S. Scita G. Zerial M. Di Fiore P.P. Nature. 2000; 408: 374-377Crossref PubMed Scopus (233) Google Scholar) and is activated by EGF stimulation (19Barbieri M.A. Roberts R.L. Gumusboga A. Highfield H. Alvarez-Dominguez C. Wells A. Stahl P.D. J. Cell Biol. 2000; 151: 539-550Crossref PubMed Scopus (197) Google Scholar). A striking finding of the present study was that Rab5S34N, a dominant-negative Rab5 mutant, stimulated transcription of a Smad-dependent promoter, in the absence of TGFβ/activins, in serum-free conditions. This activation was associated with phosphorylation and nuclear translocation of Smad proteins. Phosphorylation of Smads by Rab5S34N was independent of indirect effects such as establishment of a TGFβ/activin autocrine loop or depolymerization of microtubules. Indeed, microtubules sequester unphosphorylated Smads, and depolymerization of the microtubular network releases active, phosphorylated Smads by an uncharacterized mechanism (33Dong C., Li, Z. Alverez R. Feng X.-H. Goldschmidt-Clermont P.J. Mol. Cell. 2000; 5: 27-34Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Because phosphorylation of Smads by Rab5S34N occurred in serum-free conditions and since it has been previously shown that the cytoplasmic domains of type II and type I TGFβ receptors interact physically and functionally with each other in a ligand-independent manner (40Feng X.H. Derynck R. J. Biol. Chem. 1996; 271: 13123-13129Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), it was reasonable to assume that Rab5S34N might be able to amplify such low-level constitutive TGFβ/activin receptor activation. Indeed, we have observed a considerable inhibition of the transcriptional activation of the Smad-dependent promoter by Rab5S34N when co-transfecting dominant-negative ALK2, ALK4, and ALK5 receptor constructs.Since Rab5S34N inhibits endocytosis, recycling, and early endosome fusion (31Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (767) Google Scholar), the data suggested a regulatory role of membrane trafficking on the intensity of signaling cascades. A negligible effect of constitutively formed TGFβ-activin receptor complexes, on Smad-dependent transcription could be grossly amplified by Rab5S34N. Such amplification was unlikely to be derived from a decreased rate of endocytosis as blocking of plasma membrane endocytosis by expression of RN-tre, a specific Rab5 GAP (20Lanzetti L. Rybin V. Malabarba M.G. Christoforidis S. Scita G. Zerial M. Di Fiore P.P. Nature. 2000; 408: 374-377Crossref PubMed Scopus (233) Google Scholar), did not augment Smad-dependent transcription. Similarly, there was no increase in Smad-dependent transcription following inhibition of clathrin-coated pit- and caveolin-dependent plasma membrane endocytosis (41Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (551) Google Scholar, 42Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (617) Google Scholar, 43Zwaagstra J.C., El- Alfy M. O'Connor-McCourt M.D. J. Biol. Chem. 2001; 276: 27237-27245Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) by the dominant-negative K44A dynamin construct (34van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (586) Google Scholar, 35Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar). These results implied that the Rab5S34N effect was rather a consequence of decreased degradative or recycling trafficking leading to accumulation of constitutively formed TGFβ-activin type I/II receptor complexes on early endosomal membranes, where SARA-Smad complexes reside. Such accumulation of TGFβ-activin type I/II receptor complexes, and increased residence time thereof, presumably accounts for the increase in signaling observed upon RabS34N expression. Likewise, the observed reduction of activin A-induced Smad promoter transcription by the constitutively active Rab5Q79L is likely to be a consequence of enhanced receptor trafficking leading to decreased residence in the early endosomal compartment. It appears that cycling of Rab5 between GTP and GDP forms may influence the length and intensity of TGFβ/activin signaling cascades by regulating TGFβ-activin type I/II receptor trafficking via the early endocytic compartment. Indeed, it has been shown that Rab5S34N reduces epidermal growth factor receptor degradation by influencing membrane trafficking (44Papini E. Satin B. Bucci C. de Bernard M. Telford J.L. Manetti R. Rappuoli R. Zerial M. Montecucco C. EMBO J. 1997; 16: 15-24Crossref PubMed Scopus (193) Google Scholar). Alternatively, Rab5 could exert its effects by directly binding to components of the TGFβ/activin pathway or affecting TGFβ/activin receptor kinase activity, for instance, by modulating receptor-associated kinases or phosphatases (45Griswold-Prenner I. Kamibayashi C. Maruoka E.M. Mumby M.C. Derynck R. Mol. Cell. Biol. 1998; 18: 6595-6604Crossref PubMed Google Scholar). Toward this end, we did not observe any direct interactions between Rab5 and SARA or Smad2/3 proteins using the yeast 2-hybrid system (data not shown).In conclusion, we have revealed a critical role of early endosomes in regulating Smad-dependent signaling. Not only is SARA localized in the early endocytic compartment but also a dominant-negative Rab5 mutant causes phosphorylation and nuclear translocation of Smads leading to transcriptional activation of a Smad-dependent promoter. Rab5S34N not only stimulated Smad-dependent transcriptional activation, but also inhibited the proliferation of endothelial cells and keratinocytes mimicking the effects of TGFβ/activins. The results suggest an interconnection between events in early endosomes with signal transduction pathways and may have important implications in understanding how cells co-ordinate their cellular functions when responding to extracellular stimuli. Internalization of ligand activated receptors has been considered merely as a signal attenuation mechanism (reviewed in Ref. 14Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (256) Google Scholar). However, there is increasing evidence that endocytosis of ligand-receptor complexes not only leads to signal attenuation (36Vieira A. Lamaze C. Schmid S. Science. 1996; 274: 2086-2088Crossref PubMed Scopus (820) Google Scholar), but may be also necessary to co-localize activated receptors with downstream effectors (37Daaka Y. Luttrell L.M. Ahn S. Rocca G.J.D. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Thus, the idea of signaling from the endocytic compartment is gaining momentum. In the case of the TGFβ/activin family receptors, it is unclear whether receptor internalization is required to reach the SARA-bound Smad substrate or to what extent endosome dynamics affect TGFβ family signaling. We have addressed the intracellular localization of SARA and found that SARA is present on the early endocytic compartment, suggesting that the Smad pathway signals from the early endosome. Furthermore, SARA significantly increases endosome size, suggestive of a role in endosome dynamics. We have observed by immunofluorescence that the FYVE finger of SARA is sufficient for its endosomal targeting. So far, the identified FYVE finger proteins have been shown to require additional binding partners for targeting to endosomal membranes. Thus, EEA1 and Rabenosyn 5 require the binding of an adjacent domain to endosomal Rab5GTP (29Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (905) Google Scholar, 38Nielsen E. Christoforidis S. Uttenweiler-Joseph S. Miaczynska M. Dewitte F. Wilm M. Hoflack B. Zerial M. J. Cell Biol. 2000; 151: 601-612Crossref PubMed Scopus (274) Google Scholar). Surface plasmon resonance experiments indicated that the SARA FYVE finger has a higher affinity (KD of 30 nm) than those of Hrs (KD of 38 nm) and EEA1 (KD of 45 nm) for PI(3)P. This difference in KD may partially account for the fact that the SARA FYVE finger is efficiently targeted to early endosomes, whereas the EEA1 and Hrs FYVE fingers are mainly cytosolic when expressed as such (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar, 22Gaullier J.-M. Ronning E. Gillooly D.J. Stenmark H. J. Biol. Chem. 2000; 275: 24595-24600Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Since the dimerization of FYVE domains increases their avidity for PI(3)P-containing membranes (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar, 39Sankaran V.G. Klein D.E. Sachdeva M.M. Lemmon M.A. Biochemistry. 2001; 40: 8581-8587Crossref PubMed Scopus (62) Google Scholar), it is also possible that the isolated FYVE domain of SARA has a higher propensity to dimerize than those of Hrs and EEA1. Although we cannot rule out the possibility that the surface plasmon resonance experiments underestimate the differences in ligand affinities, we favor the view that endosomal targeting of SARA may rely solely on binding to endosome-located PI(3)P (13Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.-M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (840) Google Scholar). For instance, we did not find any direct interactions between SARA and Rab5 by the two-hybrid system (data not shown). However, we cannot fully rule out the possibility that our minimal FYVE finger construct may contain other binding elements which participate in dimerization or endosomal targeting. In the light of our initial results showing that SARA is located on early endosomes, we sought further evidence regarding the inter-relation between endosome dynamics and signaling. We reasoned that a certain level of regulation may exist at the level of this organelle and proteins, such as Rab5, regulating early endosome function may indeed alter signaling. Moreover, Rab5 has been implicated in EGFR signaling (20Lanzetti L. Rybin V. Malabarba M.G. Christoforidis S. Scita G. Zerial M. Di Fiore P.P. Nature. 2000; 408: 374-377Crossref PubMed Scopus (233) Google Scholar) and is activated by EGF stimulation (19Barbieri M.A. Roberts R.L. Gumusboga A. Highfield H. Alvarez-Dominguez C. Wells A. Stahl P.D. J. Cell Biol. 2000; 151: 539-550Crossref PubMed Scopus (197) Google Scholar). A striking finding of the present study was that Rab5S34N, a dominant-negative Rab5 mutant, stimulated transcription of a Smad-dependent promoter, in the absence of TGFβ/activins, in serum-free conditions. This activation was associated with phosphorylation and nuclear translocation of Smad proteins. Phosphorylation of Smads by Rab5S34N was independent of indirect effects such as establishment of a TGFβ/activin autocrine loop or depolymerization of microtubules. Indeed, microtubules sequester unphosphorylated Smads, and depolymerization of the microtubular network releases active, phosphorylated Smads by an uncharacterized mechanism (33Dong C., Li, Z. Alverez R. Feng X.-H. Goldschmidt-Clermont P.J. Mol. Cell. 2000; 5: 27-34Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Because phosphorylation of Smads by Rab5S34N occurred in serum-free conditions and since it has been previously shown that the cytoplasmic domains of type II and type I TGFβ receptors interact physically and functionally with each other in a ligand-independent manner (40Feng X.H. Derynck R. J. Biol. Chem. 1996; 271: 13123-13129Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), it was reasonable to assume that Rab5S34N might be able to amplify such low-level constitutive TGFβ/activin receptor activation. Indeed, we have observed a considerable inhibition of the transcriptional activation of the Smad-dependent promoter by Rab5S34N when co-transfecting dominant-negative ALK2, ALK4, and ALK5 receptor constructs. Since Rab5S34N inhibits endocytosis, recycling, and early endosome fusion (31Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (767) Google Scholar), the data suggested a regulatory role of membrane trafficking on the intensity of signaling cascades. A negligible effect of constitutively formed TGFβ-activin receptor complexes, on Smad-dependent transcription could be grossly amplified by Rab5S34N. Such amplification was unlikely to be derived from a decreased rate of endocytosis as blocking of plasma membrane endocytosis by expression of RN-tre, a specific Rab5 GAP (20Lanzetti L. Rybin V. Malabarba M.G. Christoforidis S. Scita G. Zerial M. Di Fiore P.P. Nature. 2000; 408: 374-377Crossref PubMed Scopus (233) Google Scholar), did not augment Smad-dependent transcription. Similarly, there was no increase in Smad-dependent transcription following inhibition of clathrin-coated pit- and caveolin-dependent plasma membrane endocytosis (41Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (551) Google Scholar, 42Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (617) Google Scholar, 43Zwaagstra J.C., El- Alfy M. O'Connor-McCourt M.D. J. Biol. Chem. 2001; 276: 27237-27245Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) by the dominant-negative K44A dynamin construct (34van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (586) Google Scholar, 35Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar). These results implied that the Rab5S34N effect was rather a consequence of decreased degradative or recycling trafficking leading to accumulation of constitutively formed TGFβ-activin type I/II receptor complexes on early endosomal membranes, where SARA-Smad complexes reside. Such accumulation of TGFβ-activin type I/II receptor complexes, and increased residence time thereof, presumably accounts for the increase in signaling observed upon RabS34N expression. Likewise, the observed reduction of activin A-induced Smad promoter transcription by the constitutively active Rab5Q79L is likely to be a consequence of enhanced receptor trafficking leading to decreased residence in the early endosomal compartment. It appears that cycling of Rab5 between GTP and GDP forms may influence the length and intensity of TGFβ/activin signaling cascades by regulating TGFβ-activin type I/II receptor trafficking via the early endocytic compartment. Indeed, it has been shown that Rab5S34N reduces epidermal growth factor receptor degradation by influencing membrane trafficking (44Papini E. Satin B. Bucci C. de Bernard M. Telford J.L. Manetti R. Rappuoli R. Zerial M. Montecucco C. EMBO J. 1997; 16: 15-24Crossref PubMed Scopus (193) Google Scholar). Alternatively, Rab5 could exert its effects by directly binding to components of the TGFβ/activin pathway or affecting TGFβ/activin receptor kinase activity, for instance, by modulating receptor-associated kinases or phosphatases (45Griswold-Prenner I. Kamibayashi C. Maruoka E.M. Mumby M.C. Derynck R. Mol. Cell. Biol. 1998; 18: 6595-6604Crossref PubMed Google Scholar). Toward this end, we did not observe any direct interactions between Rab5 and SARA or Smad2/3 proteins using the yeast 2-hybrid system (data not shown). In conclusion, we have revealed a critical role of early endosomes in regulating Smad-dependent signaling. Not only is SARA localized in the early endocytic compartment but also a dominant-negative Rab5 mutant causes phosphorylation and nuclear translocation of Smads leading to transcriptional activation of a Smad-dependent promoter. Rab5S34N not only stimulated Smad-dependent transcriptional activation, but also inhibited the proliferation of endothelial cells and keratinocytes mimicking the effects of TGFβ/activins. The results suggest an interconnection between events in early endosomes with signal transduction pathways and may have important implications in understanding how cells co-ordinate their cellular functions when responding to extracellular stimuli. We thank the confocal laser microscope facility of the University of Ioannina for the use of the Leica TCS-SP scanning confocal microscope. The skillful technical assistance of Lambrini Kirkou and Fanny Tahmatzoglou is gratefully acknowledged. We thank Savvas Christoforidis for critical reading of the manuscript." @default.
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W2108697952 title "Early Endosomal Regulation of Smad-dependent Signaling in Endothelial Cells" @default.
W2108697952 cites W1552824037 @default.
W2108697952 cites W1606338704 @default.
W2108697952 cites W1614056212 @default.
W2108697952 cites W1796060835 @default.
W2108697952 cites W1857386042 @default.
W2108697952 cites W1931780528 @default.
W2108697952 cites W1961999388 @default.
W2108697952 cites W1963817582 @default.
W2108697952 cites W1967965496 @default.
W2108697952 cites W1972814063 @default.
W2108697952 cites W1975244118 @default.
W2108697952 cites W1976454956 @default.
W2108697952 cites W1984536659 @default.
W2108697952 cites W1992468405 @default.
W2108697952 cites W2005574904 @default.
W2108697952 cites W2007797957 @default.
W2108697952 cites W2009319176 @default.
W2108697952 cites W2009629707 @default.
W2108697952 cites W2016637403 @default.
W2108697952 cites W2017886031 @default.
W2108697952 cites W2020475407 @default.
W2108697952 cites W2026925390 @default.
W2108697952 cites W2030662016 @default.
W2108697952 cites W2031474998 @default.
W2108697952 cites W2056399584 @default.
W2108697952 cites W2074168049 @default.
W2108697952 cites W2082379606 @default.
W2108697952 cites W2082429467 @default.
W2108697952 cites W2086043352 @default.
W2108697952 cites W2087307986 @default.
W2108697952 cites W2088013881 @default.
W2108697952 cites W2108175824 @default.
W2108697952 cites W2116422958 @default.
W2108697952 cites W2124360065 @default.
W2108697952 cites W2128002216 @default.
W2108697952 cites W2133237847 @default.
W2108697952 cites W2135175655 @default.
W2108697952 cites W2150846836 @default.
W2108697952 cites W2153689647 @default.
W2108697952 cites W2163504684 @default.
W2108697952 cites W2164615751 @default.
W2108697952 cites W2168558463 @default.
W2108697952 cites W2170437356 @default.
W2108697952 cites W4253422011 @default.
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