y g by addition of nonrecombinant pCDNA3, as necessary. Results represent three t o six different transfected cultures, with the experiments performed on at least two different days. For dark conditions, plates of NIH 313 cells were wrapped in foil immediately after transfection until the time of harvesting (48 hours), except for a single brief medium change performed under dim red light (Kodak Safelight, Wratten Series 1A) 24 hours before harvesting. 18. E. A. Griffin Jr., D. Staknis, C. J. Weitz, unpublished data. (17) and light conditions were processed in parallel and incubated together. For light conditions, cells were illuminated (Fiber-Lite High Intensity Illuminator, Series 180; set at 10 relative illumination units) immediately after transfection until the time of harvesting. Plates were positioned 15 t o 30 cm from the tips of the twin fiberoptic guides. Transfected cells under dark

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The receptor-regulated Smad proteins are essential intracellular mediators of signal transduction by the transforming growth factor-beta (TGF-beta) superfamily of growth factors and are also important as regulators of gene transcription. Here we describe a new role for TGF-beta-regulated Smad2 and Smad3 as components of a ubiquitin ligase complex. We show that in the presence of TGF-beta signalling, Smad2 interacts through its proline-rich PPXY motif with the tryptophan-rich WW domains of Smurf2, a recently identified E3 ubiquitin ligases. TGF-beta also induces the association of Smurf2 with the transcriptional co-repressor SnoN and we show that Smad2 can function to mediate this interaction. This allows Smurf2 HECT domain to target SnoN for ubiquitin-mediated degradation by the proteasome. Thus, stimulation by TGF-beta can induce the assembly of a Smad2-Smurf2 ubiquitin ligase complex that functions to target substrates for degradation.

Smad proteins are critical intracellular mediators of the transforming growth factor-␤, bone morphogenic proteins (BMPs), and activin signaling. Upon ligand binding, the receptor-associated R-Smads are phosphorylated by the active type I receptor serine/threonine kinases. The phosphorylated R-Smads then form heteromeric complexes with Smad4, translocate into the nucleus, and interact with various transcription factors to regulate the expression of downstream genes. Interaction of Smad proteins with cellular partners in the cytoplasm and nucleus is a critical mechanism by which the activities and expression of the Smad proteins are modulated. Here we report a novel step of regulation of the R-Smad function at the inner nuclear membrane through a physical interaction between the integral inner nuclear membrane protein MAN1 and R-Smads. MAN1, through the RNA recognition motif, associates with R-Smads but not Smad4 at the inner nuclear membrane in a ligand-independent manner. Overexpression of MAN1 results in inhibition of R-Smad phosphorylation, heterodimerization with Smad4 and nuclear translocation, and repression of transcriptional activation of the TGF␤, BMP2, and activin-responsive promoters. This repression of TGF␤, BMP2, and activin signaling is dependent on the MAN1-Smad interaction because a point mutation that disrupts this interaction abolishes the transcriptional repression by MAN1. Thus, MAN1 represents a new class of R-Smad regulators and defines a previously unrecognized regulatory step at the nuclear periphery.The transforming growth factor-␤ (TGF␤) 1 superfamily of cytokines, including TGF␤s, BMPs, and activins, plays important roles in the regulation of various aspects of mammalian embryogenesis and carcinogenesis. The signals initiated by these cytokines are transduced by their receptors and the downstream Smad proteins (1-5). Upon ligand binding, activin or TGF␤ receptor serine/threonine kinase phosphorylates and activates the type I receptor kinase, which then phosphorylates the downstream Smad proteins. The Smad proteins are critical mediators of TGF␤ superfamily signaling. Upon phosphorylation by the activated type I receptor kinases, the receptorassociated R-Smads (Smad2 and Smad3 for TGF␤ and activin; Smad1, Smad5, and Smad8 for BMPs) oligomerize with the common mediator Smad4, translocate into the nucleus where they interact with various transcription factors, bind to DNA, and regulate transcription of downstream genes.The expression and activity of the Smad proteins can be modulated by interaction with various cellular proteins at the plasma membrane or in the cytoplasm and nucleus (2, 6, 7). For example, Smad proteins can interact with various transcriptional co-activators on promoter DNAs to regulate the activation of TGF␤, activin, or BMP target genes (2, 6). The activity or intracellular localization of the Smads can be modulated through binding to adaptors molecules such as SARA (8), Hgs (9, 10), chaperones (11), microtubules (12), or various co-repressors such as Ski, SnoN, SNIP,. O...

Transforming growth factor ␤ (TGF␤) activates transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene through a major TGF␤-responsive region (؊740 and ؊647) in the PAI-1 promoter. This process requires the Smad family of signaling molecules. Upon phosphorylation by the TGF␤ receptors, Smad2 and Smad3 homoligomerize and heteroligomerize with Smad4, translocate to the nucleus and activate transcription of TGF␤ responsive genes. Smad3 and Smad4 have been shown to bind to various sites in the PAI-1 promoter. To determine the number of Smad-binding sites within the 94-base pair major TGF␤-responsive region and the mechanism of Smad-mediated transactivation, we systematically mapped the Smad-binding sites and show that Smad4 and Smad3 bind cooperatively to two adjacent DNA elements in this region. Both elements were required for TGF␤-induced, Smad3-and Smad4-dependent activation of PAI-1 transcription. Contrary to previous reports, transactivation of the PAI-1 promoter was mediated by the amino-but not carboxyl-terminal domains of the Smads. Furthermore, oligomerization of Smad3 markedly enhanced its binding to the two binding sites. Finally, a Smad4 mutation identified in a human pancreatic carcinoma that inactivates Smad4 signaling abolished Smad4 DNA binding activity, hence preventing transactivation of TGF␤-responsive genes. These results underscore the importance of the Smad4 DNA binding activity in controlling cell growth and carcinogenesis. Transforming growth factor-␤ (TGF␤)1 is a multipotent cytokine that elicits many biological functions including inhibition of the growth of cells of epithelial, endothelial, and lymphoid origins, production of extracellular matrix components, and regulation of differentiation of many cell types (1). These activities are mediated by the cell surface types I and II TGF␤ receptors, T␤RI and T␤RII, which are receptor serine/threonine kinases (2-4). In the absence of ligand, while the T␤RI kinase is inactive, the T␤RII kinase is constitutively active and the receptor is autophosphorylated (2, 5). Binding of TGF␤1 to T␤RII results in the formation of a heteromeric complex containing T␤RI and T␤RII, followed by transphosphorylation of T␤RI by the T␤RII kinase (6). Phosphorylation of T␤RI by T␤RII is thought to activate the T␤RI kinase activity, allowing it to phosphorylate and activate downstream Smad2 (7-10) and Smad3 proteins (11, 12).The Smad family proteins are critical components of the TGF␤ signaling pathway. Depending on their mechanisms of action, the Smads can be divided into three classes: pathwayrestricted Smads, common-mediator Smads, and inhibitory Smads (13). All Smad proteins share considerable homology in their primary sequence and most contain two highly conserved Mad homology domains: MH1 in the amino-terminal half and MH2 in the carboxyl-terminal half separated by a diverse proline-rich linker. Upon stimulation by TGF␤1, the pathway restricted Smads, Smad2, and Smad3, interact with the TGF␤ receptor complex, and become phosphorylated on three ser...

Ste5 is essential for pheromone response and binds components of a mitogen-activated protein kinase (MAPK) cascade: Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK). Pheromone stimulation releases G␤␥ (Ste4-Ste18), which recruits Ste5 and Ste20 (p21-activated kinase) to the plasma membrane, activating the MAPK cascade. A RING-H2 domain in Ste5 (residues 177-229) negatively regulates Ste5 function and mediates its interaction with G␤␥. Ste5(C177A C180A), carrying a mutated RING-H2 domain, cannot complement a ste5⌬ mutation, yet supports mating even in ste4⌬ ste5⌬ cells when artificially dimerized by fusion to glutathione S-transferase (GST). In contrast, wild-type Ste5 fused to GST permits mating of ste5⌬ cells, but does not allow mating of ste4⌬ ste5⌬ cells. This differential behavior provided the basis of a genetic selection for STE5 gain-of-function mutations. MATa ste4⌬ ste5⌬ cells expressing Ste5-GST were mutagenized chemically and plasmids conferring the capacity to mate were selected. Three independent single-substitution mutations were isolated. These constitutive STE5 alleles induce cell cycle arrest, transcriptional activation, and morphological changes normally triggered by pheromone, even when G␤␥ is absent. The first, Ste5(C226Y), alters the seventh conserved position in the RING-H2 motif, confirming that perturbation of this domain constitutively activates Ste5 function. The second, Ste5(P44L), lies upstream of a basic segment, whereas the third, Ste5(S770K), is situated within an acidic segment in a region that contacts Ste7. None of the mutations increased the affinity of Ste5 for Ste11, Ste7, or Fus3. However, the positions of these novel-activating mutations suggested that, in normal Ste5, the N terminus may interact with the C terminus. Indeed, in vitro, GST-Ste5(1-518) was able to associate specifically with radiolabeled Ste5(520-917). Furthermore, both the P44L and S770K mutations enhanced binding of full-length Ste5 to GST-Ste5(1-518), whereas they did not affect Ste5 dimerization. Thus, binding of G␤␥ to the RING-H2 domain may induce a conformational change that promotes association of the N-and C-terminal ends of Ste5, stimulating activation of the MAPK cascade by optimizing orientation of the bound kinases and/or by increasing their accessibility to Ste20-dependent phosphorylation (or both). In accord with this model, the novel Ste5 mutants copurified with Ste7 and Fus3 in their activated state and their activation required Ste20. INTRODUCTIONThe pheromone response pathway of the yeast Saccharomyces cerevisiae has provided a system for elucidating mechanisms that convert an extracellular signal into both a morphological response and a change in the pattern of gene expression (reviewed in Bardwell et al., 1994;Leberer et al., 1997a). Mating of haploid cells (MATa and MAT␣) requires the action of peptide pheromones: MATa cells secrete a-factor, and MAT␣ cells secrete ␣-factor. The cell surface receptors for these peptides (Ste2 in MATa cells binds ␣-factor, and Ste3 in MAT␣ cells binds a-factor) a...

Smad proteins mediate transforming growth factor-β (TGF-β) signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGF-β signaling that functions to maintain the repressed state of TGF-β target genes in the absence of ligand. On TGF-β stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGF-β target genes. We show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase-promoting complex (APC) and the UbcH5 family of ubiquitin-conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box (D box)-dependent manner. In addition to the D box, efficient ubiquitination and degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGF-β signaling. Our studies elucidate an important mechanism and pathway for the degradation of SnoN and more importantly, reveal a novel role of the APC in the regulation of TGF-β signaling.

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