Transforming growth factor-beta TGF-beta is the prototype for a family of extracellular proteins that affect cell proliferation and tissue differentiation. TGF-beta-related factors, including BMP-2/4, Dpp and activin, act through two types of serine/threonine kinase receptors which can form a heteromeric complex. However, the mechanism of signal transduction by these receptors is largely unknown. In Drosophila, Mad is required for signalling by Dpp. We have isolated complementary DNAs for four human Mad homologues, one of which, hMAD-4, is identical to DPC-4, a candidate tumour suppressor. hMAD-3 and -4 synergized to induce strong ligand-independent TGF-beta-like responses. When truncated at their carboxy termini, hMAD-3 and -4 act as dominant-negative inhibitors of the normal TGF-beta response. The activity of hMAD-3 and -4 was regulated by the TGF-beta receptors, and hMAD-3 but not hMAD-4 was phosphorylated and associated with the ligand-bound receptor complex. These results define hMAD-3 and -4 as effectors of the TGF-beta response and demonstrate a function for DPCA-4/hMAD-4 as a tumour suppressor.

Smads regulate transcription of defined genes in response to TGF-β receptor activation, although the mechanisms of Smad-mediated transcription are not well understood. We demonstrate that the TGF-β-inducible Smad3 uses the tumor suppressor Smad4/DPC4 and CBP/p300 as transcriptional coactivators, which associate with Smad3 in response to TGF-β. The association of CBP with Smad3 was localized to the carboxyl terminus of Smad3, which is required for transcriptional activation, and a defined segment in CBP. Furthermore, CBP/p300 stimulated both TGF-β- and Smad-induced transcription in a Smad4/DPC4-dependent fashion. Smad3 transactivation and TGF-β-induced transcription were inhibited by expressing E1A, which interferes with CBP functions. The coactivator functions and physical interactions of Smad4 and CBP/p300 with Smad3 allow a model for the induction of gene expression in response to TGF-β.

Homologs of Drosophila Mad function as downstream mediators of the receptors for transforming growth factor ␤ (TGF-␤)-related factors. Two homologs, the receptor-associated Smad3 and the tumor suppressor Smad4/DPC4, synergize to induce ligand-independent TGF-␤ activities and are essential mediators of the natural TGF-␤ response. We now show that Smad3 and Smad4 associate in homomeric and heteromeric interactions, as assessed by yeast two-hybrid and coimmunoprecipitation analyses. Heteromeric interactions are mediated through the conserved C-terminal domains of Smad3 and Smad4. In Smad3, the homomeric interaction is mediated by the same domain. In contrast, the homomeric association of Smad4 requires both the N-terminal domain and the C-terminal domain, which by itself does not homomerize. Mutations that have been associated with impaired Mad activity in Drosophila or decreased tumor suppressor activity of Smad4/ DPC4 in pancreas cancer, including a short C-terminal truncation and two point mutations in the conserved C-terminal domains, impair the ability of Smad3 and Smad4 to undergo homo-and heteromeric associations. Analyses of the biological activity of Smad3 and Smad4 and their mutants show that full signaling activity correlates with their ability to undergo efficient homo-and heteromeric interactions. Mutations that interfere with these interactions result in decreased signaling activity. Finally, we evaluated the ability of Smad3 or Smad4 to induce transcriptional activation in yeast. These results correlate the ability of individual Smads to homomerize with transcriptional activation and additionally with their biological activity in mammalian cells.Transforming growth factor ␤ (TGF-␤)-related factors play important regulatory roles in cell differentiation and proliferation and in tissue morphogenesis in animals ranging from flies and nematodes to mammals (12,16). Among the many members of the TGF-␤ superfamily, which include activins and bone morphogenetic proteins (BMPs) (12, 16), TGF-␤1 is considered the prototype factor to characterize the various cellular responses and the mechanism of receptor signaling. The growth-inhibitory effect of TGF-␤ in a variety of cell types, such as epithelial cells, and its ability to induce gene expression that leads to increased extracellular matrix deposition have been best characterized (5). Similarly to BMP-2/4 and activin, TGF-␤ exerts its activities through two types of cell surface serine/threonine kinase receptors, type II and type I receptors. The type II and type I receptors have the ability to form a heterotetrameric complex, consisting of two type II and two type I receptors, and this complex is thought to represent the fully functional receptor complex that mediates TGF-␤ signaling following ligand binding. In this tetrameric complex, the constitutively active type II receptors phosphorylate the type I receptor cytoplasmic domains, and this phosphorylation is required for activation of the signaling pathways leading to the TGF-␤ response (4,18,22).The down...

PC12-E2 cells, a stable variant subcloned from native cell populations, produce neurites in a rapid, transcription-independent manner upon exposure to nerve growth factor (NGF) or basic fibroblast growth factor (bFGF). They also give a similar morphological response to interleukin-6 (IL-6), which is, however, transcription-dependent and with a slower onset, a phenomenon basically not observed in native PC12 cells. The response profile of PC12-E2 cells to NGF and bFGF is similar to that observed for native PC12 cells pre-exposed (primed) to NGF, and such cells also respond to IL-6 in a fashion indistinguishable from PC12-E2 cells. Mechanistically, NGF and bFGF induce a sustained phosphorylation and activation of ERK1 and ERK2 in both cells, while IL-6 produces only a transient and weak tyrosine phosphorylation. However, it does stimulate a prolonged and biphasic tyrosine phosphorylation and nuclear translocation of Stat3 (signal transducers and activators of transcription 3; at least 24 h) and, to a lesser extent, Stat1. Gel shift and supershift analyses confirm that IL-6 predominantly activates Stat3 (and some Stat1) and stimulates sis-inducible element binding activity. Other members of the same cytokine subfamily, including ciliary neurotrophic factor and leukemia inhibitory factor, also cause a transient initial phase of tyrosine phosphorylation and activation of Stat1 and Stat3 (up to 1 h) but fail to stimulate a second phase of response and do not produce significant neurites. These results suggest that sustained signaling of either STAT or ERK pathways in PC12-E2 cells leads to induction of neuronal differentiation. However, only the latter is effective in native PC12 cells as the activation of Stat3 and Stat1 in native PC12 cells by IL-6 fails to induce neuronal differentiation. Thus, the response of PC12-E2 cells to IL-6 suggests the constitutive expression of a required factor(s) for differentiation, that is induced in native PC12 cells by NGF or bFGF (possibly by ERK activation), but not by IL-6 via Janus kinase/STAT activation. This factor(s), which has a sufficient half-life to allow primed cells to remain responsive to IL-6 for several days, is necessary but not sufficient for differentiation (as measured by neurite proliferation) to occur.

LIM domains contain about 50 amino acids and have a cysteine-rich sequence: CX 2 CX 16 -23 CX 2 HX 2 CX 2 CX 2 CX 16 -23 -CX 2 C (1). They bind two atoms of Zn 2ϩ with coordination being S 4 , S 3 N 1 , S 3 O 1 , or S 2 N 1 O 1 (2). The NMR structure of the carboxyl-terminal LIM domain of chicken cysteine-rich protein (CRP) 1 revealed that the two Zn 2ϩ binding modules are located at the ends of a hydrophobic core composed of antiparallel ␤ sheets (3). LIM domains were initially recognized in the primary sequences of the homeodomain proteins Lin 11 (4), Isl-1 (5), and Mec-3 (6) and have subsequently been identified in a variety of homeodomain proteins (7,8), in cytoskeleton-associated proteins (9, 10), in LIM domain-only proteins (11-13), in protein kinases (14), and in proteins of undefined function (8).Most LIM proteins contain more than one LIM domain. The sequence of an individual LIM domain is, in general, more closely related to the same LIM domain in analogous proteins from other species than to other LIM domains within the same protein (8). Although the NMR structure of LIM2 of CRP resembles the DNA binding domain of the GATA-1 transcription factor (3), most available evidence indicates that LIM domains function in protein⅐protein rather than protein⅐DNA interactions.Two structural targets for LIM domains have been identified. Using gel overlay techniques, Schmeichel and Beckerle (15) found that the LIM domains of zyxin interacted with the LIM-only protein CRP. Specificity was evident from the observation that LIM1 but not LIM2 or LIM3 of zyxin-bound CRP. Feurstein, et al. (16) also found evidence for LIM⅐LIM interactions involving CRP but did not observe specificity for the LIM domain. The carboxyl-terminal LIM domain of the cytoplasmic protein Enigma was found to specifically interact with exon 16 of the insulin receptor (InsR) (17). Mutations in exon 16 that disrupted the major endocytic code and ligand-induced endocytosis of InsR (18) also disrupted interaction with Enigma. The endocytic code of InsR, like that of many receptors (19), consists of 4 -6 amino acids that form a tyrosine-containing tight turn (20). A generalized tight-turn motif, which functioned in endocytosis of mutant EGFR (21), and which contained two copies of an Asn-Asn-Ala-Tyr-Phe motif interacted with a wider range of LIM domains, suggesting that specific Tyr-based tight turns would provide interaction targets for specific LIM domains. There is functional evidence for LIM domain interactions with a variety of transcription factors (22-24), suggesting that additional target specificities exist.We recently found that in addition to InsR, Enigma specifically interacted with the receptor tyrosine kinase Ret (25). Given that Enigma, which contains three LIM domains at its carboxyl terminus, 2 was found to interact with two receptor tyrosine kinases, determining the molecular basis of this recognition became important. We found that LIM2 of Enigma