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...

Fotsis T. VEGF autoregulates its proliferative and migratory ERK1/2 and p38 cascades by enhancing the expression of DUSP1 and DUSP5 phosphatases in endothelial cells. Am J Physiol Cell Physiol 297: C1477-C1489, 2009. First published September 9, 2009 doi:10.1152/ajpcell.00058.2009.-Vascular endothelial growth factor (VEGF) is a key angiogenic factor that regulates proliferation and migration of endothelial cells via phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) and p38, respectively. Here, we demonstrate that VEGF strongly induces the transcription of two dual-specificity phosphatase (DUSP) genes DUSP1 and DUSP5 in endothelial cells. Using fluorescence microscopy, fluorescence lifetime imaging (FLIM), and fluorescence cross-correlation spectroscopy (FCCS), we found that DUSP1/mitogen-activated protein kinases phosphatase-1 (MKP-1) was localized in both the nucleus and cytoplasm of endothelial cells, where it existed in complex with p38 (effective dissociation constant, K D eff , values of 294 and 197 nM, respectively), whereas DUSP5 was localized in the nucleus of endothelial cells in complex with ERK1/2 (K D eff 345 nM). VEGF administration affected differentially the K D eff values of the DUSP1/p38 and DUSP5/ERK1/2 complexes. Gain-of-function and lack-of-function approaches revealed that DUSP1/MKP-1 dephosphorylates primarily VEGF-phosphorylated p38, thereby inhibiting endothelial cell migration, whereas DUSP5 dephosphorylates VEGF-phosphorylated ERK1/2 inhibiting proliferation of endothelial cells. Moreover, DUSP5 exhibited considerable nuclear anchoring activity on ERK1/2 in the nucleus, thereby diminishing ERK1/2 export to the cytoplasm decreasing its further availability for activation. vascular endothelial growth factors; extracellular signal regulated kinase-1/2; p38; dual-specificity phosphatase 1/mitogen-activated protein kinase phosphatase-1; dual-specificity phosphatase-5 VASCULAR ENDOTHELIAL GROWTH FACTORS (VEGFs) are the most important regulators of vessel morphogenesis. Not only do they participate in the regulation of both vasculogenesis and angiogenesis, but they also are among the most important molecules involved in the pathogenesis of angiogenic diseases such as diabetic retinopathy and cancer (6). VEGFs are secreted dimeric glycoproteins of ϳ40 kDa, and in mammals the VEGF family consists of five members: VEFG-A, B, C, D and placental growth factor (PLGF) (6). Moreover, alternative splicing of several of the VEGF family members gives rise to isoforms with different biological activities. The VEGF ligands bind in an overlapping pattern to three receptor tyrosine kinases known as VEGF receptor-1, -2, and -3 (VEGFR1-3), as well as to coreceptors such as heparan sulfate proteoglycans and neuropilins (22). VEGFR2 promotes migration, proliferation, and differentiation of endothelial cells (ECs) being critical for the regulation of angiogenic sprouting (1). Indeed, during sprouting, tip cells need to acquire an invasive and motile phenotype, whereas ECs in the stalk exhibi...

The tumor suppressor function of activin A, together with our findings that activin A is an inhibitor of angiogenesis, which is down-regulated by the N-MYC oncogene, prompted us to investigate in more detail its role in the malignant transformation process of neuroblastomas. Indeed, neuroblastoma cells with restored activin A expression exhibited a diminished proliferation rate and formed smaller xenograft tumors with reduced vascularity, whereas lung metastasis rate remained unchanged. In agreement with the decreased vascularity of the xenograft tumors, activin A inhibited several crucial angiogenic responses of cultured endothelial cells, such as proteolytic activity, migration, and proliferation. Endothelial cell proliferation, activin A, or its constitutively active activin receptor-like kinase 4 receptor (ALK4T206D), increased the expression of CDKN1A (p21), CDKN2B (p15), and CDKN1B (p27) CDK inhibitors and down-regulated the expression of vascular endothelial growth factor receptor-2, the receptor of a key angiogenic factor in cancer. The constitutively active forms of SMAD2 and SMAD3 were both capable of inhibiting endothelial cell proliferation, whereas the dominant-negative forms of SMAD3 and SMAD4 released the inhibitory effect of activin A on endothelial cell proliferation by only 20%. Thus, the effects of activin A on endothelial cell proliferation seem to be conveyed via the ALK4/SMAD2-SMAD3 pathways, however, non-SMAD cascades may also contribute. These results provide novel information regarding the role of activin A in the malignant transformation process of neuroblastomas and the molecular mechanisms involved in regulating angiogenesis thereof. (Cancer Res 2005; 65(5): 1877-86)

Summary SARA, an early endosomal protein, plays a key role in TGFb signalling, as it presents SMAD2 and SMAD3 for phosphorylation by the activated TGFb receptors. Here, we show that ERBIN is a new SARA-interacting protein that can be recruited by SARA to early endosomes. ERBIN was recently shown to bind and segregate phosphorylated SMAD2 and SMAD3 (SMAD2/3) in the cytoplasm, thereby inhibiting SMAD2/3-dependent transcription. SARA binds to ERBIN using a new domain, which we have called the ERBID (ERBIN-binding domain), whereas ERBIN binds to SARA using a domain (amino acids 1208-1265) that also interacts with SMAD2 and SMAD3, which we have called the SSID (SARA-and SMAD-interacting domain). We additionally show that SARA competes with SMAD2/3 for binding to ERBIN. In agreement, overexpression of SARA or the ERBID peptide reverses the inhibitory effect of ERBIN on SMAD2/3-dependent transcription. Taken together, these data suggest that the response of cells to TGFb and activin A can be influenced by the relative concentrations of SARA, ERBIN and SMAD2/3.

Ligand/receptor complexes formed at the plasma membrane are internalised via various endocytic pathways that influence the ultimate signalling output by regulating the complex's selection of interaction partners along the trafficking route. We report that in differentiated cells Activin A/receptor complexes are internalised via Clathrin-mediated endocytosis (CME) and macropinocytosis (MP), whereas in human embryonic stem cells (hESCs) internalisation occurred via CME. We further show that hESCs are devoid of MP, which becomes functional upon differentiation towards endothelial cells through mesoderm mediators. Our results reveal, for the first time, that MP is an internalisation route of Activin A in differentiated cells, which is not active in hESCs and is induced as cells differentiate.