REVIEWSTIBS 25 –FEBRUARY 2000640968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: S0968-0004(99)01519-4TRANSFORMING GROWTH FACTOR-b(TGF-b) family members, which includeTGF-bs, activins and bone morpho-genetic proteins (BMPs), are secretedsignaling molecules that regulate aplethora of cellular responses, such asproliferation, differentiation, migrationand apoptosis. TGF-b family membershave critical roles during embryogenesisand in maintaining tissue homeostasisduring adult life. Deregulated TGF-b fam-ily signaling has been implicated in mul-tiple developmental disorders and in var-ious human diseases, including cancer,fibrosis and auto-immune diseases1.TGF-b family members transducetheir signals across the plasma mem-brane by inducing the formation of het-eromeric complexes of specific type Iand type II serine/threonine kinase re-ceptors (Fig. 1). The type I receptor isphosphorylated and activated by thetype II receptor and has been shown todetermine signaling specificity withinthe receptor complex. Activated type Ireceptors initiate intracellular signalingthrough activation of specific Smad pro-teins that relay signals into the nucleuswhere they, together with other pro-teins, direct transcriptional responses1.Thus, reminiscent of many other tran-scription factors, Smad activity is regu-lated by translocation from the cyto-plasm to the nucleus. In this review, wewill focus on the function of vertebrateSmads as nuclear effectors of TGF-b sig-naling and on how other signaling inputsaffect the TGF-b/Smad pathway.Smad family: R-Smads, Co-Smads and I-SmadsThe TGF-b/Smad pathway has beenconserved throughout evolution. Smad-related genes were first discoveredthrough genetic screens in Drosophilaand Caenorhabditis elegans. The nameSmad is a fusion of two gene names,Drosophila mothers against dpp (Mad )and C. elegans Sma, the gene products ofwhich were found to perform criticalroles downstream of a BMP homolog,Dpp, and Daf-4 serine/threonine kinasereceptor, respectively1. Nine distinct ver-tebrate Smad family members have nowbeen identified, and Smads can be classi-fied into three groups1. Receptor-regu-lated Smads (R-Smads) are phosphory-lated differentially by activated type Ireceptor kinases on two serine residuesin a SS[V/M]S motif at their extreme C termini (Figs 1,2) upon which they formheteromeric complexes with common-partner Smads (Co-Smads)1. One mam-malian Smad4 is known, and two distinctCo-Smads (Smad4a and Smad4b) havebeen identified in Xenopus2,3. Searchesfor other mammalian Co-Smads are ac-tively being pursued in several laborato-ries. The inhibitory Smads (I-Smads;Smad6 and Smad7) act in opposition tosignal-transducing R- and Co-Smads,forming stable associations with acti-vated type I receptors and thus prevent-ing the phosphorylation of R-Smads(Fig. 3). Smad6 has also been shown tocompete with Smad4 for heteromericcomplex formation for activated Smad1(Ref. 1).R-Smads and Co-Smads share two do-mains of high sequence similarity attheir N and C termini (Fig. 2)1. TheseMad-homology domains (MH1 and MH2)are separated by a proline-rich linker re-gion of variable length and have distinctfunctions (Fig. 2 and Table 1). The MH2domain is important for homo- and het-eromeric complex formation and fortranscriptional activation and repres-sion1. The L3 loops in the MH2 domainsmediate interaction with type I recep-tors4, and the H1 a helix in the MH2 do-main is important for the interaction ofALK-1 with Smad15. The MH1 domains ofSmad3 and Smad4 have intrinsic DNA-binding activities and also associatewith other transcription factors.Furthermore, MH1 and MH2 domains in-teract with and repress the activity ofeach other (Table 1 and see below)1. I-Smads have conserved MH2 domainsthat are sufficient for type I receptor in-teraction and inhibitory activity. The N-terminal regions of I-Smads might directsignaling specificity to I-Smads.Activation of R- and Co-SmadsThe first intracellular step in theTGF-b/Smad pathway, the recruitmentof Smad2 and Smad3 to the TGF-b recep-tor complex, is controlled by a mem-brane-associated FYVE-domain-contain-ing protein, termed Smad anchor foractivation (SARA)6. SARA presentsSmad2 and Smad3 to the activated type Ireceptor by binding cooperatively toboth non-phosphorylated Smads andthe TGF-b receptor complex. SARA doesnot interact with Smad1, Smad4 or I-Smads. Ectopic expression of SARAmutants that lack the phospholipid-binding FYVE domain interfere quitestrongly with TGF-b signaling6.R-Smads interact directly with acti-vated type I receptors1; the L45 loopSignaling inputs converge onnuclear effectors in TGF-bsignalingPeter ten Dijke, Kohei Miyazono and Carl-Henrik HeldinRecent studies have consolidated the pivotal role of Smads as intracellulareffectors of TGF-b family members. Upon binding to their specific type Iand type II serine/threonine kinase receptors, each family member acti-vates a particular subset of Smad proteins. Activated, receptor-regulatedSmads form hetero-oligomeric complexes with common-partner Smadsthat translocate into the nucleus, where they control the expression of tar-get genes in a cell-type-specific manner. Smads appear to function not onlyas nuclear effectors for TGF-b family members, but as signal integratorswithin an extensive intracellular network.P. ten Dijke and C-H. Heldin are at theLudwig Institute for Cancer Research, Box595, S-751 24 Uppsala, Sweden; andK. Miyazono is at the Dept of Biochemistry,The Cancer Institute of Japanese Foundationfor Cancer Research (JFCR), 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan.P. ten Dijke is currently at the Division ofCellular Biochemistry, The NetherlandsCancer Institute, Plesmanlaan 121, 1066 CXAmsterdam, The Netherlands.Email: [email protected] 25 –FEBRUARY 200065that protrudes from the ki-nase domain and controlstype I receptor signalingspecificity was found to be acritical determinant in speci-fying Smad isoform interac-tion7. Upon phosphorylationof Smad2 and Smad3 by acti-vated type I receptors, R-Smads and SARA dissociatefrom the TGF-b receptor com-plex. Phosphorylation of R-Smads relieves the auto-inhibitory MH1–MH2 interac-tion and allow R-Smads toform complexes with Smad4through their MH2 domains,an interaction that is mutuallyexclusive with binding toSARA6. The released SARA iscapable of recruiting othernonactivated Smad2 or Smad3for receptor presentation.SARA expression and sub-cellular location thus