Early Endosomal Regulation of Smad-dependent Signaling in Endothelial Cells

Abstract

Transforming growth factor ␤ (TGF␤) 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 Smaddependent transcription, the effects of the dominantnegative Rab5 are likely to be a consequence of altered membrane trafficking of constitutively formed TGF␤/ activin type I/II receptor complexes at the level of early endosomes. The results suggest an important interconnection between early endosomal dynamics and TGF␤/ activin signal transduction pathways.The transforming growth factor ␤ (TGF␤) 1 superfamily is a large group of secreted polypeptide growth factors, which include the TGF␤s, 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 TGF␤ family signaling has been implicated in multiple developmental disorders and in various human diseases, including cancer (1). Some of these disorders, such as hereditary hemorrhagic teleangiectasia and primary pulmonary hypertension, involve altered TGF␤ family signaling regulating vasculogenic and angiogenic responses of endothelial cells (2-6). Indeed, TGF␤1 is known to influence both endothelial cell proliferation and critical endothelial cell-pericyte interactions occurring during vessel maturation (7). We have recently shown also that activin A affects endothelial cell function leading to inhibition of angiogenesis and decreased vessel wall integrity (8).The TGF␤/activin family members signal through heteromeric complexes of transmembrane type I and type II serinethreonine 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. 9). 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 TGF␤ receptor and most likely in the case of activin (10), the Smad-binding protein SARA plays an important role in phosphorylation of Smad2 and Smad3 by TG...

Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes

“…Early endosomes have been recognized as an important signaling center for TGF-β/Smad signaling [6,8,36]. In cells co-expressing EGFP-Smad3, caveolin-1-mRFP and Rab5Q79L, and immunostained for EEA1, we observed the accumulation of EGFP-Smad3 in the enlarged caveolin-1-positive early endosomes ( Figure 5A).…”

Endocytic regulation of TGF-β signaling

“…As Rab5 S34N inhibits clathrinmediated recycling and early endosome fusion [81], it was suggested that the stimulatory effect of Rab5 S34N on Smad-dependent signaling could be a consequence of decreased degradative or recycling trafficking of TGF-b/ activin receptors, which results in their accumulation in early endosomes, where other TGF-b/activin signalingpromoting factors SARA, endofin and Hrs reside [77] (Figure 2). However, this hypothesis remains to be tested as RN-tre, a specific Rab5 GTPase-activating protein that facilitates GTP hydrolysis, does not increase the expres-npg sion of the Smad-responsive reporter [77]. Nevertheless, these data strongly indicate that early endosomes are a signaling organelle for the TGF-b/Smad pathway.…”

“…It was shown that Rab5 S34N, a GDP-bound dominant-negative Rab5 mutant, can stimulate the expression of a Smad3-responsive reporter in a ligand-independent manner, whereas the GTP-bound Rab5 Q79L mutant attenuates the transcriptional activity induced by activin [77]. As Rab5 S34N inhibits clathrinmediated recycling and early endosome fusion [81], it was suggested that the stimulatory effect of Rab5 S34N on Smad-dependent signaling could be a consequence of decreased degradative or recycling trafficking of TGF-b/ activin receptors, which results in their accumulation in early endosomes, where other TGF-b/activin signalingpromoting factors SARA, endofin and Hrs reside [77] (Figure 2). However, this hypothesis remains to be tested as RN-tre, a specific Rab5 GTPase-activating protein that facilitates GTP hydrolysis, does not increase the expres-npg sion of the Smad-responsive reporter [77].…”

“…The FYVE domain is a zinc-binding motif known to bind phosphatidylinositol 3-phosphate, a lipid enriched in early endosomes, and can specifically recruit the FYVEdomain-containing proteins to early endosomes [76]. The FYVE domain is essential for the stimulatory effect of these proteins on TGF-b signaling [71,73,75,77], indicating that their localization in early endosomes is important to fulfill their functions. It was shown that SARA functions as a scaffold protein by specifically interacting with Smad2/3 and TGF-b receptors to facilitate Smad2/3 phosphorylation by the TGF-b receptor [71].…”

hARIP2 is a Putative Growth-promoting Factor Involved in Human Colon Tumorigenesis

“…Upon activation, the type I receptor will bind to and phosphorylates a subset of cytoplasmic Smad proteins. These phosphorylated Smadswill be translocated to the nucleus which controls the transcription of target genes (Panopoulou et al, 2002).…”