Serhiy Souchelnytskyi

TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4.

Smad family members are newly identified essential intracellular signalling components of the transforming growth factor‐β (TGF‐β) superfamily. Smad2 and Smad3 are structurally highly similar and mediate TGF‐β signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF‐β or activin stimulation. Here we show that Smad2 and Smad3 interacted with the kinase‐deficient TGF‐β type I receptor (TβR)‐I after it was phosphorylated by TβR‐II kinase. TGF‐β1 induced phosphorylation of Smad2 and Smad3 in Mv1Lu mink lung epithelial cells. Smad4 was found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF‐β1 stimulation. Similar results were obtained using HSC4 cells, which are also growth‐inhibited by TGF‐β. Smads 2 and 3 interacted with Smad4 after TβR activation in transfected COS cells. In addition, we observed TβR‐activation‐dependent interaction between Smad2 and Smad3. Smads 2, 3 and 4 accumulated in the nucleus upon TGF‐β1 treatment in Mv1Lu cells, and showed a synergistic effect in a transcriptional reporter assay using the TGF‐β‐inducible plasminogen activator inhibitor‐1 promoter. Dominant‐negative Smad3 inhibited the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF‐β induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF‐β signal transduction.

The L45 loop in type I receptors for TGF-β family members is a critical determinant in specifying Smad isoform activation

Transforming growth factor‐β (TGF‐β) and bone morphogenetic proteins (BMPs) signal via distinct type I and type II receptors and Smad proteins. A nine amino acid sequence between kinase subdomains IV and V in type I receptors, termed the L45 loop, has been shown to be important in conferring signalling specificity. We examined the responses of a mutant TGF‐β type I receptor (TβR‐I) and a mutant BMPR‐IB, in which the L45 regions of these two receptors were exchanged. Swapping the four amino acid residues that are different in BMPR‐IB for those in TβR‐I, and vice versa, switched their type I receptor‐restricted Smad activation and specificity in transcriptional responses. These studies identify the L45 loop regions in type I receptors as critical determinants in specifying Smad isoform activation.

Phosphorylation of Ser465 and Ser467 in the C Terminus of Smad2 Mediates Interaction with Smad4 and Is Required for Transforming Growth Factor-β Signaling

Members of the Smad family of intracellular signal transducers are essential for transforming growth factor-β (TGF-β) to exert its multifunctional effects. After activation of TGF-β receptors, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. Thereafter, these activated Smad complexes translocate to the nucleus, where they may direct transcriptional responses. Here we report that TGF-β mediates phosphorylation of Smad2 at two serine residues in the C terminus,i.e. Ser465 and Ser467, which are phosphorylated in an obligate order; phosphorylation of Ser465 requires that Ser467 be phosphorylated. Transfection of Smad2 with mutation of Ser465 and/or Ser467 to alanine residues into Mv1Lu cells resulted in dominant-negative inhibition of TGF-β signaling. These Smad2 mutants were found to stably interact with an activated TGF-β receptor complex, in contrast to wild-type Smad2, which interacts only transiently. Mutation of Ser465 and Ser467 in Smad2 abrogated complex formation of this mutant with Smad4 and blocked the nuclear accumulation not only of Smad2, but also of Smad4. Thus, heteromeric complex formation of Smad2 with Smad4 is required for nuclear translocation of Smad4. Moreover, peptides from the C terminus of Smad2 containing phosphorylated Ser465 and Ser467 were found to bind Smad4 in vitro, whereas the corresponding unphosphorylated peptides were less effective. Thus, phosphorylated Ser465 and Ser467 in Smad2 may provide a recognition site for interaction with Smad4, and phosphorylation of these sites is a key event in Smad2 activation.

IDENTIFICATION OF SMAD2, A HUMAN MAD-RELATED PROTEIN IN THE TRANSFORMING GROWTH FACTOR BETA SIGNALING PATHWAY

Transforming growth factor-β (TGF-β) superfamily members are multifunctional cytokines that exert their effects via heteromeric complexes of two distinct serine and threonine kinase receptors. Drosophila mothers against decapentaplegic and related genes in Caenorhabditis elegans, Xenopus, and mammals were shown to function downstream in the intracellular signaling pathways of TGF-β superfamily members. Here we report the cloning of a Mad-related protein, termed Sma- and Mad-related protein 2 (Smad2). TGF-β stimulated the phosphorylation and nuclear translocation of Smad2 in nontransfected Mv1Lu cells. In addition, we demonstrated that TGF-β and activin mediated phosphorylation of Smad2 after its overexpression with appropriate type I and II receptors in COS cells. Smad2 and Smad1 were found to be broadly expressed in human tissues. Smad2 is closely linked to DPC4 on chromosome 18q21.1, a region often deleted in human cancers. Cells that lack Smad2 may escape from TGF-β-mediated growth inhibition and promote cancer progression.

Physical and Functional Interaction of Murine andXenopus Smad7 with Bone Morphogenetic Protein Receptors and Transforming Growth Factor-β Receptors

Members of the transforming growth factor-β (TGF-β) family transmit signals from membrane to nucleus via intracellular proteins known as Smads. A subclass of Smad proteins has recently been identified that antagonize, rather than transduce, TGF-β family signals. Smad7, for example, binds to and inhibits signaling downstream of TGF-β receptors. Here we report that the C-terminal MAD homology domain of murine Smad7 (mSmad7) is sufficient for both of these activities. In addition, we show that mSmad7 interacts with activated bone morphogenetic protein (BMP) type I receptors (BMPR-Is), inhibits BMPR-I-mediated Smad phosphorylation, and phenocopies the effect of known BMP antagonists when overexpressed in ventral cells of Xenopus embryos. Xenopus Smad7 (XSmad7, previously termed Smad8) and mSmad7 are nearly identical within their bioactive C-domain, but have quite distinct N-domains. We found that XSmad7, similar to mSmad7, interacted with BMP and TGF-β type I receptors and inhibited receptor-mediated phosphorylation of downstream signal-transducing Smads. However, XSmad7 is a less efficient inhibitor of TβR-I-mediated responses in mammalian cells than is mSmad7. Furthermore, overexpression of XSmad7 in Xenopus embryos produces patterning defects that are not observed following overexpression of mSmad7, suggesting that mSmad7 and XSmad7 may preferentially target distinct signaling pathways. Our results are consistent with the possibility that the C-domain of antagonistic Smads is an effector domain whereas the N-domain may confer specificity for distinct signaling pathways.

Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site

Src-family tyrosine kinases are regulatory proteins that play a pivotal role in the disorganization of cadherin-dependent cell–cell contacts. We previously showed that Src was associated with vascular endothelial (VE)-cadherin and that tyrosine phosphorylation level of VE-cadherin was dramatically increased in angiogenic tissues as compared to quiescent tissues. Here, we examined whether VE-cadherin was a direct substrate for Src in vascular endothelial growth factor (VEGF)-induced VE-cadherin phosphorylation, and we identified the target tyrosine sites. Co-transfections of Chinese hamster ovary cells (CHO) cells with VE-cadherin and constitutively active Src (Y530F) resulted in a robust tyrosine phosphorylation of VE-cadherin that was not detected with kinase-dead Src (K298M). In an in vitro Src assay, the VE-cadherin cytoplasmic domain is directly phosphorylated by purified Src as well as the tyrosine residue 685 (Tyr)685-containing peptide RPSLY685AQVQ. VE-cadherin peptide mapping from human umbilical vein endothelial cells stimulated by VEGF and VE-cadherin-CHO cells transfected with active Src revealed that Y685 was the unique phosphorylated site. The presence of PhosphoY685 was confirmed by its ability to bind to C-terminal Src kinase-SH2 domain in a pull-down assay. Finally, we found that in a VEGF-induced wound-healing assay, cadherin adhesive activity was impaired by Src kinase inhibitors. These data identify that VEGF-induced-VE-cadherin tyrosine phosphorylation is mediated by Src on Y685, a process that appears to be critical for VEGF-induced endothelial cell migration.

Functional proteomics of transforming growth factor‐β1‐stimulated Mv1Lu epithelial cells: Rad51 as a target of TGFβ1‐dependent regulation of DNA repair

Transforming growth factor‐β (TGFβ) conveys regulatory signals through multiple intracellular pathways, subsequently affecting various cellular functions. To identify new targets for TGFβ, we studied the changes in the proteome of Mv1Lu lung epithelial cells in response to TGFβ1 treatment. Thirty‐eight non‐abundant protein spots, affected by TGFβ1, were selected, and proteins were identified by peptide mass‐fingerprinting (PMF). Among them, proteins involved in regulation of immune response, apoptosis, regulation of TGFβ signalling, metabolism and DNA repair were identified. Twenty‐eight of the 38 proteins are new targets for TGFβ1, thus suggesting novel ways of integration of TGFβ signalling in intracellular regulatory processes. We show that TGFβ1‐dependent decrease in expression of one of the new targets, Rad51, correlates with a decrease in DNA repair efficiency. This was evaluated by formation of nuclear Rad51‐containing DNA repair complexes in response to DNA damage, by single cell gel electrophoresis and by cell survival assay. The TGFβ1‐dependent inhibition of DNA repair was reversed by ectopic overexpression of Rad51. Therefore, TGFβ can promote DNA instability through down‐regulation of Rad51 and inhibition of DNA repair.

Phosphorylation of Ser165 in TGF-beta type I receptor modulates TGF-beta1-induced cellular responses.

Transforming growth factor-beta (TGF-beta) signals via an oligomeric complex of two serine/threonine kinase receptors denoted TGF-beta type I receptor (TbetaR-I) and type II receptor (TbetaR-II). We investigated the in vivo phosphorylation sites in TbetaR-I and TbetaR-II after complex formation. Phosphorylation of TbetaR-II was observed at residues in the C-terminus (Ser549 and Ser551) and at residues in the juxtamembrane domain (Ser223, Ser226 and Ser227). TGF-beta1 induced in vivo phosphorylation of serine and threonine residues in the juxtamembrane domain of TbetaR-I in a region rich in glycine, serine and threonine residues (GS domain; Thr185, Thr186, Ser187, Ser189 and Ser191), and more N-terminal of this region (Ser165). Phosphorylation in the GS domain has been shown previously to be involved in activation of the TbetaR-I kinase. We show here that phosphorylation of TbetaR-I at Ser165 is involved in modulation of TGF-beta1 signaling. Mutations of Ser165 in TbetaR-I led to an increase in TGF-beta1-mediated growth inhibition and extracellular matrix formation, but, in contrast, to decreased TGF-beta1-induced apoptosis. A transcriptional activation signal was not affected. Mutations of Ser165 changed the phosphorylation pattern of TbetaR-I. These observations suggest that TGF-beta receptor signaling specificity is modulated by phosphorylation of Ser165 of TbetaR-I.

Expression of Smad proteins in human colorectal cancer

Escape from transforming growth factor‐β (TGF‐β)–induced inhibition of proliferation has been observed in many tumor cells and may contribute to loss of growth control. Smad proteins have been identified as major components in the intracellular signaling of TGF‐β family members. In this study, we examined the expression of receptor‐activated, common‐mediator and inhibitory Smads by immunohistochemistry in human colorectal cancers. We found increased expression of receptor‐activated Smads in a fraction of the tumor cells, while no immunostaining for Smad2, Smad3 or Smad5 and only occasional staining for Smad1/8 was found in epithelial mucosa of normal colon. No or only weak staining for receptor‐activated Smads, common‐mediator Smad4 and inhibitory Smads was observed in the tumor stroma. Common‐mediator Smad4 and inhibitory Smads were detected in cells of both tumor and normal tissues. We observed a distinct pattern of Smad4 immunostaining of epithelial cells along colon crypts, with high expression in zones of terminal differentiation. Our data show selective up‐regulation of receptor‐activated Smad proteins in human colorectal cancers and suggest involvement of Smad4 in differentiation and apoptosis of surface epithelial cells of normal crypts. Int. J. Cancer 82:197–202, 1999.

Intracellular signaling of osteogenic protein-1 through Smad5 activation

Smad proteins play pivotal roles in the intracellular signaling of the multifunctional transforming growth factor‐β (TGF‐β) family members downstream of serine/threonine kinase type I and type II receptors. Smad2 and Smad3 are specific mediators of TGF‐β and activin, while Smad1 and Smad5 are involved in bone morphogenetic protein‐2 (BMP‐2) and BMP‐4 signaling. Here we report that osteogenic protein‐1 (OP‐1), also termed BMP‐7, binds predominantly to BMPR‐IB in the rat osteoprogenitor‐like cell line, ROB‐C26. Smad1, Smad5, and Smad8, but not Smad2 and Smad3, were found to stably interact with the kinase‐deficient BMPR‐IB after it was phosphorylated by the BMPR‐II kinase. In ROB‐C26 cells, which express Smad2, Smad3, Smad4, and Smad5, OP‐1 was found to stimulate the phosphorylation of Smad5. Whereas transfection of wild‐type Smad5 enhanced the OP‐1‐induced response, transfection of wild‐type Smad2 had no effect on OP‐1 signaling. A Smad5‐2SA mutant, in which the two most carboxy‐terminal serine residues were mutated to alanine residues, was found to act as a dominant negative inhibitor of OP‐1‐induced responses upon its transfection into various cell types, including ROB‐C26 cells, in contrast to ectopic expression of a Smad2‐2SA mutant which was without effect. Smad5, therefore, is a key component in the intracellular signaling of OP‐1. J. Cell. Physiol. 177:355–363, 1998.