Early Endosomal Regulation of Smad-dependent Signaling in Endothelial Cells*

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.

[1]  P. Oh,et al.  Dynamin at the Neck of Caveolae Mediates Their Budding to Form Transport Vesicles by GTP-driven Fission from the Plasma Membrane of Endothelium , 1998, The Journal of cell biology.

[2]  R. Rappuoli,et al.  The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin , 1997, The EMBO journal.

[3]  Jeffrey L. Wrana,et al.  Hgs (Hrs), a FYVE Domain Protein, Is Involved in Smad Signaling through Cooperation with SARA , 2000, Molecular and Cellular Biology.

[4]  P. D’Amore,et al.  Vascular Development: Cellular and Molecular Regulation , 2022 .

[5]  Anthony A. Hyman,et al.  Rab5 regulates motility of early endosomes on microtubules , 1999, Nature Cell Biology.

[6]  M. Lindsay,et al.  Localization of phosphatidylinositol 3‐phosphate in yeast and mammalian cells , 2000, The EMBO journal.

[7]  D. Marchuk,et al.  Endoglin, an ancillary TGFbeta receptor, is required for extraembryonic angiogenesis and plays a key role in heart development. , 2000, Developmental biology.

[8]  G. Sutter,et al.  Non‐replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase , 1995, FEBS letters.

[9]  B. Deurs,et al.  Rab7: a key to lysosome biogenesis. , 2000, Molecular biology of the cell.

[10]  M. McNiven,et al.  Dynamin-mediated Internalization of Caveolae , 1998, The Journal of cell biology.

[11]  T. Fotsis,et al.  The N-myc oncogene in human neuroblastoma cells: down-regulation of an angiogenesis inhibitor identified as activin A. , 2000, Cancer research.

[12]  Denis Vivien,et al.  Direct binding of Smad3 and Smad4 to critical TGFβ‐inducible elements in the promoter of human plasminogen activator inhibitor‐type 1 gene , 1998, The EMBO journal.

[13]  S. Schmid,et al.  Regulation of signal transduction by endocytosis. , 2000, Current opinion in cell biology.

[14]  P. Donahoe,et al.  Activin receptor-like kinase 1 modulates transforming growth factor-beta 1 signaling in the regulation of angiogenesis. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Dumont,et al.  A murine model of hereditary hemorrhagic telangiectasia. , 1999, The Journal of clinical investigation.

[16]  M. Caron,et al.  Essential Role for G Protein-coupled Receptor Endocytosis in the Activation of Mitogen-activated Protein Kinase* , 1998, The Journal of Biological Chemistry.

[17]  J. Wrana.  Regulation of Smad Activity , 2000, Cell.

[18]  H. Stenmark,et al.  Interaction of the EEA1 FYVE Finger with Phosphatidylinositol 3-Phosphate and Early Endosomes , 2000, The Journal of Biological Chemistry.

[19]  H. Stenmark,et al.  FYVE-finger proteins--effectors of an inositol lipid. , 1999, Journal of cell science.

[20]  U. Rüther,et al.  Long-term consequences of interleukin-6 overexpression in transgenic mice. , 1992, DNA and cell biology.

[21]  R. Derynck,et al.  Receptor-associated Mad homologues synergize as effectors of the TGF-β response , 1996, Nature.

[22]  H. Stenmark,et al.  FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes. , 2001, Journal of cell science.

[23]  E. Leof.  Growth factor receptor signalling: location, location, location. , 2000, Trends in cell biology.

[24]  Xin-Hua Feng,et al.  Microtubule Binding to Smads May Regulate TGFβ Activity , 2000 .

[25]  P. L. Campbell,et al.  EEA1, an Early Endosome-Associated Protein. , 1995, The Journal of Biological Chemistry.

[26]  S. Schmid,et al.  Mutations in human dynamin block an intermediate stage in coated vesicle formation , 1993, The Journal of cell biology.

[27]  Marino Zerial,et al.  EEA1 links PI(3)K function to Rab5 regulation of endosome fusion , 1998, Nature.

[28]  R. Derynck,et al.  Physical and Functional Interactions between Type I Transforming Growth Factor β Receptors and Bα, a WD-40 Repeat Subunit of Phosphatase 2A , 1998, Molecular and Cellular Biology.

[29]  Pier Paolo Di Fiore,et al.  The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5 , 2000, Nature.

[30]  S. Schmid,et al.  Induction of mutant dynamin specifically blocks endocytic coated vesicle formation , 1994, The Journal of cell biology.

[31]  S. Narumi,et al.  E-selectin Gene Expression Is Induced Synergistically with the Coexistence of Activated Classic Protein Kinase C and Signals Elicited by Interleukin-1β but Not Tumor Necrosis Factor-α* , 1999, The Journal of Biological Chemistry.

[32]  R. Derynck,et al.  Ligand-independent Activation of Transforming Growth Factor (TGF) β Signaling Pathways by Heteromeric Cytoplasmic Domains of TGF-β Receptors* , 1996, The Journal of Biological Chemistry.

[33]  J. Massagué,et al.  TGFβ Signaling in Growth Control, Cancer, and Heritable Disorders , 2000, Cell.

[34]  M. Lemmon,et al.  High-affinity binding of a FYVE domain to phosphatidylinositol 3-phosphate requires intact phospholipid but not FYVE domain oligomerization. , 2001, Biochemistry.

[35]  A. Wells,et al.  Epidermal growth factor and membrane trafficking. EGF receptor activation of endocytosis requires Rab5a. , 2000 .

[36]  M. O’Connor-McCourt,et al.  Transforming Growth Factor (TGF)-β1 Internalization , 2001, The Journal of Biological Chemistry.

[37]  M. Zerial,et al.  Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. , 1994, The EMBO journal.

[38]  M. Zerial,et al.  Rabenosyn-5, a Novel Rab5 Effector, Is Complexed with Hvps45 and Recruited to Endosomes through a Fyve Finger Domain , 2000, The Journal of cell biology.

[39]  A. Peacock.  Primary Pulmonary Hypertension , 1999, Thorax.

[40]  Liliana Attisano,et al.  SARA, a FYVE Domain Protein that Recruits Smad2 to the TGFβ Receptor , 1998, Cell.

[41]  S. Schmid,et al.  Control of EGF Receptor Signaling by Clathrin-Mediated Endocytosis , 1996, Science.

[42]  M. Zerial,et al.  rab5 controls early endosome fusion in vitro , 1991, Cell.

[43]  Kai Simons,et al.  The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway , 1992, Cell.

[44]  M. Gimbrone,et al.  A role for Smad6 in development and homeostasis of the cardiovascular system , 2000, Nature Genetics.