Assignment of Transforming Growth Factor β1 and β3 and a Third New Ligand to the Type I Receptor ALK-1

Lux, Andreas; Attisano, Liliana; Marchuk, Douglas A.

doi:10.1074/jbc.274.15.9984

Cited by 139 publications

(121 citation statements)

References 65 publications

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“…Current data (e.g. the human diseases HHT1 and HHT2 (43,44) and their respective mouse knockout models of ALK1 (49) and endoglin (33)(34)(35)(36) as well as the observed direct interaction of endoglin and ALK1 (40)) support the hypothesis that endoglin is a component of the ALK1 signaling branch. As a first prerequisite, we have demonstrated the presence of endoglin throughout the process of transdifferentiation by Northern blot (Fig.…”

Section: Fig 9 Surface Exposition Of Endoglin In Hsc/mfbmentioning

confidence: 70%

Identification of Endoglin in Rat Hepatic Stellate Cells

Meurer

Tihaa

Lahme

et al. 2005

Journal of Biological Chemistry

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Transforming growth factor ␤ (TGF-␤) signaling is mediated by the cell surface TGF-␤ type I (ALK5), type II, and the accessory type III receptors endoglin and betaglycan. Hepatic stellate cells (HSC), the most profibrogenic cell type in the liver, express ALK5, T␤RII, and betaglycan. To monitor the expression of betaglycan in HSC, we used the commercially available antibody sc-6199 in Western blot analysis. This antibody, raised against a peptide mapping at the carboxyl terminus of the human betaglycan, is claimed to be specific for betaglycan, although it is known that the C-terminal domain is highly conserved in type III receptors. Proteins recognized in HSC by sc-6199 did not match the characteristic migration pattern of betaglycan. Moreover, the determined molecular weight (M r 160) and the observed reductant sensitivity after treatment with dithiothreitol resemble those of a closely related type III receptor, endoglin (CD105). Endoglin, a disulfide-linked homodimer, is an accessory component of the TGF-␤ receptor complex and mainly expressed on endothelial cells. The presence of endoglin in HSC of rat liver was confirmed by molecular cloning of the endoglin cDNA and immunocytochemistry. The reactivity of sc-6199 with both auxiliary TGF-␤ receptors (betaglycan and endoglin) from rats was demonstrated by Western blot and immunocytochemical analysis of cells heterologously expressing these proteins. Furthermore, Northern and Western blotting revealed that both betaglycan and endoglin genes are differentially regulated in HSC and in transdifferentiated myofibroblasts (MFB). By surface labeling and immunoprecipitation experiments, we show that endoglin is found in significant amounts exposed at the plasma membrane of HSC and MFB, which is a pivotal prerequisite for binding of and signaling in response to TGF-␤. In conclusion, we hypothesize that TGF-␤ signals in HSC and MFB are tuned by two different interconnected signaling pathways, as it was previously demonstrated for endothelial cells.The transforming growth factor family of growth factors regulates a diverse set of physiologic processes including proliferation, cellular differentiation, apoptosis, and expression of extracellular matrix genes (1-3). Signaling by the three identified mammalian TGF-␤ 1 isoforms TGF-␤1, TGF-␤2, and TGF-␤3 is initiated by binding to the high affinity cell surface receptors, the accessory TGF-␤ type III receptor (T␤RIII) (4), the type II receptor (T␤RII) (5), and type I (6) receptor (T␤RI), of which T␤RII and T␤RI possess intracellular serine/threonine kinase domains. Upon binding of TGF-␤1 or TGF-␤3 to the receptor type II, the liganded receptor dimer associates with and its constitutive active kinase transphosphorylates the type I receptor at the GS domain (7). The active type I receptor in turn phosphorylates and thereby activates the intracellular signal transducers represented by the R-Smad family members 2 and 3 (2, 7, 8).Because TGF-␤2 has a lower affinity for the type II receptor (9, 10), cells sense this ligand by eithe...

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Section: Fig 9 Surface Exposition Of Endoglin In Hsc/mfbmentioning

confidence: 70%

Identification of Endoglin in Rat Hepatic Stellate Cells

Meurer

Tihaa

Lahme

et al. 2005

Journal of Biological Chemistry

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“…We acknowledge that nonspecific binding is possible when using polyclonal antibody immunoprecipitation as a technique for sample enrichment, thus identifying false protein interactions. However, a careful review of the literature for NPM-ALK interactions revealed that the majority of the published studies utilized polyclonal antibodies in their experimental design (Fujimoto et al, 1996;Bai et al, 1998Bai et al, , 2000Hubinger et al, 1999;Lux et al, 1999;Nieborowska-Skorska et al, 2001;Slupianek et al, 2001;Bonvini et al, 2002;Miyake et al, 2002;Raetz et al, 2002;Zamo et al, 2002;Zhang et al, 2002;Amin et al, 2003;Baba et al, 2003;Khoury et al, 2003;Ruchatz et al, 2003). In this regard, we have chosen to categorize proteins identified using both monoclonal and polyclonal antibodies.…”

Section: Discussionmentioning

confidence: 99%

Identification of NPM-ALK interacting proteins by tandem mass spectrometry

et al. 2004

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Constitutive overexpression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a key oncogenic event in anaplastic large-cell lymphomas with the characteristic chromosomal aberration t(2;5)(p23;q35). Proteins that interact with ALK tyrosine kinase play important roles in mediating downstream cellular signals, and are potential targets for novel therapies. Using a functional proteomic approach, we determined the identity of proteins that interact with the ALK tyrosine kinase by co-immunoprecipitation with anti-ALK antibody, followed by electrospray ionization and tandem mass spectrometry (MS/ MS). A total of 46 proteins were identified as unique to the ALK immunocomplex using monoclonal and polyclonal antibodies, while 11 proteins were identified in the NPM immunocomplex. Previously reported proteins in the ALK signal pathway were identified including PI3-K, Jak2, Jak3, Stat3, Grb2, IRS, and PLCc1. More importantly, many proteins previously not recognized to be associated with NPM-ALK, but with potential NPM-ALK interacting protein domains, were identified. These include adaptor molecules (SOCS, Rho-GTPase activating protein, RAB35), kinases (MEK kinase 1 and 4, PKC, MLCK, cyclin G-associated kinase, EphA1, JNK kinase, MAP kinase 1), phosphatases (meprin, PTPK, protein phosphatase 2 subunit), and heat shock proteins (Hsp60 precursor). Proteins identified by MS were confirmed by Western blotting and reciprocal immunoprecipitation. This study demonstrates the utility of antibody immunoprecipitation and subsequent peptide identification by tandem mass spectrometry for the elucidation of ALKbinding proteins, and its potential signal transduction pathways.

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“…It has been shown that Smad5 differs from the other BMP-regulated Smads (Smad1 and Smad8) in its DNAbinding abilities and more resembles the TGF-b-specific Smad3 in those aspects (Li et al, 2001). The alternative TGF-b type I receptor, ALK1, can transduce TGF-b signals through Smad1, Smad5 and Smad8 in ECs (Roelen et al, 1997;Lux et al, 1999;Oh et al, 2000). An intriguing possibility would be that the accessory receptor endoglin could play a role in this regard.…”

Section: Gastrulation / Embryoid Body Formationmentioning

confidence: 99%

“…The activated TGF-b receptor complex phosphorylates the R-Smads Smad2 and Smad3, which also act as mediators of the activin signaling pathway . In addition to signaling via ALK5, it has been shown that TGF-b can signal through another type I receptor (ALK1) in ECs, which phosphorylates a different set of R-Smads: Smad1, Smad5 and Smad8 (Roelen et al, 1997;Lux et al, 1999;Oh et al, 2000). These Smads are the intracellular mediators of the BMP signaling pathway.…”

Section: R-smadsmentioning

confidence: 99%