TGFbeta signaling: receptors, transducers, and Mad proteins.

The primary structure of Mad proteins does not contain any motif that would reveal their function. Mads are proteins of ∼450 amino acids with highly conserved N-terminal and C-terminal domains and a variable proline-rich intervening region (Figure 3Figure 3). Intriguingly, all the described null mutations in Mad, sma2, and sma3 are missense or nonsense mutations that fall within a highly conserved, short portion of the C-domain (16xSavage, C, Das, P, Finelli, A, Townsend, S.R, Sun, C.-Y, Baird, S.E, and Padgett, R.W. Proc. Natl. Acad. Sci. USA. 1996; 93: 790–794Crossref | PubMed | Scopus (352)See all References, 17xSekelsky, J.J, Newfeld, S.J, Raftery, L.A, Chartoff, E.H, and Gelbart, W.M. Genetics. 1995; 139: 1347–1358PubMedSee all References). Mutations in this hot spot have also surfaced in DPC4 (deleted in pancreatic carcinoma), a human Mad identified as a candidate tumor suppressor in human chromosome 18q21.1 that is mutated or deleted in half of all pancreatic malignancies (Hahn et al. 1996xHahn, S.A, Schutte, M, Hoque, A.T.M.S, Moskaluk, C.A, da Costa, L.T, Rozenblum, E, Weinstein, C.L, Fischer, A, Yeo, C.J, Hruban, R.H, and Kern, S.E. Science. 1996; 271: 350–353Crossref | PubMedSee all ReferencesHahn et al. 1996). TGFβ, activins, and BMPs can cause cell cycle arrest, terminal differentiation, or apoptosis in different cell types, and DPC4 might be essential for some of these responses.Figure 3Domain Structure of Mad Proteins and Similarity among Family MembersThe structures are aligned based on a highly conserved N-terminal motif (LVKKLKK). The level of identity refers to the combined N- and C-domains and excludes other regions.View Large Image | View Hi-Res Image | Download PowerPoint SlideSmad1, also called Madr1 (the Mad nomenclature needs unification and revision), undergoes two striking modifications in response to BMP2 or BMP4: serine phosphorylation (Hoodless et al. 1996xHoodless, P.A, Haerry, T, Abdollah, S, Stapleton, M, O'Connor, M.B, Attisano, L, and Wrana, J.L. Cell. 1996; 85: 489–500Abstract | Full Text | Full Text PDF | PubMed | Scopus (566)See all ReferencesHoodless et al. 1996) and accumulation in the nucleus (7xHoodless, P.A, Haerry, T, Abdollah, S, Stapleton, M, O'Connor, M.B, Attisano, L, and Wrana, J.L. Cell. 1996; 85: 489–500Abstract | Full Text | Full Text PDF | PubMed | Scopus (566)See all References, 10xLiu, F, Hata, A, Baker, J, Doody, J, Carcamo, J, Harland, R, and Massague, J. Nature. 1996; 381: 620–623Crossref | PubMed | Scopus (512)See all References). Both events are rapidly induced by BMP2 or BMP4 and require BMP receptors. TGFβ induces phosphorylation of products immunologically related to Madr1/Smad1, indicating the generality of this phenomenon (8xLechleider, R.J, de Caestecker, M.P, Dehejia, A, Polymeropoulos, M.H, and Roberts, A.B. J. Biol. Chem., in press. 1996; See all References, 22xYingling, J.M, Das, P, Savage, C, Zhang, C, Padgett, R.W, and Wang, X.-F. Proc. Natl. Acad. Sci. USA, in press. 1996; See all References). Madr1/Smad1 phosphorylation is prevented by a missense mutation in a hot spot glycine (Hoodless et al. 1996xHoodless, P.A, Haerry, T, Abdollah, S, Stapleton, M, O'Connor, M.B, Attisano, L, and Wrana, J.L. Cell. 1996; 85: 489–500Abstract | Full Text | Full Text PDF | PubMed | Scopus (566)See all ReferencesHoodless et al. 1996). It is presently unclear whether phosphorylation is catalyzed by the receptor kinase or an intermediate kinase or is secondary to entry into the nucleus. The subcellular localization of hot spot mutants may help clarify this question.A correlation between nuclear localization and Mad function has been observed using Xenopus embryo assay systems (Baker and Harland 1996xBaker, J and Harland, R.M. Genes Dev., in press. 1996; See all ReferencesBaker and Harland 1996). In the secondary axis generated by injected lacZ-tagged Madr2 mRNA, the protein is exclusively nuclear in cells of the anterior portion of the axis and is cytoplasmic in other cells. Coinjection with activin mRNA enhances the nuclear localization of lacZ–Madr2. Based on these results, it has been suggested that when Madr2 is in the correct embryonic context to respond to mesoderm-inducing signals, it sensitizes the cells to these signals, accumulates in the nucleus, and induces dorso–anterior fate (Baker and Harland 1996xBaker, J and Harland, R.M. Genes Dev., in press. 1996; See all ReferencesBaker and Harland 1996). Interestingly, the C-domains of Madr2 and Smad1 mimic the activity of their full-length counterparts in mesoderm induction assays (2xBaker, J and Harland, R.M. Genes Dev., in press. 1996; See all References, 10xLiu, F, Hata, A, Baker, J, Doody, J, Carcamo, J, Harland, R, and Massague, J. Nature. 1996; 381: 620–623Crossref | PubMed | Scopus (512)See all References). Furthermore, a lacZ-tagged Madr2 C-domain becomes localized in the nucleus of all cells expressing it (Baker and Harland 1996xBaker, J and Harland, R.M. Genes Dev., in press. 1996; See all ReferencesBaker and Harland 1996). The C-domain might be an active domain, whereas the N-domain might act as an inhibitor of nuclear translocation, a direct repressor of the C-domain, or both.What is it that Mads do once they enter the nucleus? It has been observed that the C-domains of Smad1 and DPC4 display transcriptional activity when bound to DNA via a GAL4 DNA-binding domain (Liu et al. 1996xLiu, F, Hata, A, Baker, J, Doody, J, Carcamo, J, Harland, R, and Massague, J. Nature. 1996; 381: 620–623Crossref | PubMed | Scopus (512)See all ReferencesLiu et al. 1996). This activity is eliminated by a hot spot nonsense mutation found in various Mad and DPC4 alleles. A full-length Smad1 fusion product is inactive in this assay, but can be specifically activated in response to BMP2 or BMP4 in combination with BMP receptors. Thus, as in the nuclear localization assays, an activity of the C-domain appears to be repressed by the N-domain and this repression eliminated by incoming BMP signals. These observations raise the possibility that the function of Mad proteins might have something to do with the ability to transactivate. Before this can be established, however, it will be necessary to demonstrate that native Mad proteins can physically interact with specific response elements in physiological target genes and that they can activate transcription of these genes in response to receptor signals. Once activated, Mads might interact with other transcriptional regulators, possibly including homologs of schnurri, a Drosophila transcription factor genetically implicated in DPP signaling (1xArora, K, Dai, H, Kazuko, S.G, Jamal, J, O'Connor, M.B, Letsou, A, and Warrior, R. Cell. 1995; 81: 781–790Abstract | Full Text PDF | PubMed | Scopus (182)See all References, 5xGrieder, N.C, Nellen, D, Burke, R, Basler, K, and Affolter, M. Cell. 1995; 81: 791–800Abstract | Full Text PDF | PubMed | Scopus (163)See all References).In sum, TGFβ signaling continues to unfold as a classic process to bring to the nucleus instructions dictated from the cell surface and shaped in the cytoplasm. The advent of Mad proteins gives us an additional handle on the components that mediate the diverse and remarkable effects of the TGFβ family.