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Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases.

TGF-β is the prototypic member of a large family of structurally related cytokines including the TGF-βs, activins, and bone morphogenetic proteins (BMPs), which regulate cell fate and extracellular matrix deposition through the transcriptional regulation of diverse gene targets. These ligands initiate intracellular signals by associating with two classes of interacting transmembrane receptor serine-threonine kinases (Derynck and Feng 1997; Massague 1998). Smad proteins, which serve as direct targets of phosphorylation by the activated type I receptors, have been demonstrated to be downstream mediators of specific TGF-β/BMP-signaling pathways from the receptors to the nucleus. Three classes of Smads have been identified and are referred to as the receptor-activated Smads, co-Smads, and inhibitory Smads. Receptor-activated Smads (R-Smads), Smad2, and Smad3 are specific mediators of TGF-β and activin signaling pathways, whereas Smad1, Smad5, and Smad8 are involved in BMP responses (Massague 1998). Smad4 is a co-Smad and is not regulated by phosphorylation, but acts as a common and essential mediator of TGF-β, activin, and BMP-signaling responses (Candia et al. 1997; de Caestecker et al. 1997). A third class of Smads includes Smad6 and Smad7, and act as an inhibitor of this pathway by binding to the activated receptors or to R-Smads, thereby sequestering the transcriptionally active complex from the promoter. Like many transcription factors, the R-Smad proteins interact with the paralogous transcriptional coactivators CBP and p300 through their MH2 domains (Feng et al. 1998; Janknecht et al. 1998; Pouponnot et al. 1998; Topper et al. 1998). Although these two proteins were characterized by different means, they are now considered to be orthologous and to act as a crucial scaffold to bring together transcription factors and basal factors in the transcriptional initiation complex. They are essential coactivators for a wide variety of transcriptional factors including nuclear hormone receptors and NF-κB (Mannervik et al. 1999). Besides possessing intrinsic histone acetyltransferase (HAT) activity, these proteins are also able to interact with other HATs such as p/CAF and steroid receptor coactivators (SRCs) (Torchia et al. 1998). Although recent publications have concentrated on the HAT activity of CBP/p300, these proteins also contain three highly conserved cysteine-histidine-rich domains (C/H1, C/H2, and C/H3). Among these, the C/H1 domain was shown to interact with the Smad activation domain (SAD) of Smad4, which is necessary for transcriptional activating activity of the Smads (de Caestecker et al. 2000), whereas the C/H3 domain is the locus of interaction with R-Smads (Feng et al. 1998; Janknecht et al. 1998; Nishihara et al. 1998; Shen et al. 1999). Many recent investigations have focused on identification of proteins that interact with Smad proteins to modulate both their trafficking and stability in the cytoplasm and their transcriptional activating activity in the nucleus. Cytoplasmic interactors include Smurf1, which was found to modulate the BMP signal-transduction pathway by regulating the level of Smad1 and Smad5 in the cell (Zhu et al. 1999), and SARA, which interacts directly with Smad2 and Smad3 and functions to recruit Smad2 to the TGF-β receptor (Tsukazaki et al. 1998). An increasing number of nuclear activators including the AP-1 complex, vitamin D receptor, and TFE3 transcriptional factor (Hua et al. 1998; Zhang et al. 1998; Yanagi et al. 1999; Yanagisawa et al. 1999) have been shown to interact specifically with Smad proteins. In addition, a number of transcriptional inhibitors have been shown to interact with Smad proteins and to repress their transcriptional activating activity. These include the oncoproteins Evi-1 (Kurokawa et al. 1998), Ski and SnoN, (Luo et al. 1999), TGIF, which recruits the histone deacetylase HDAC (Wotton et al. 1999), and the δEF1/Zfh-1 family member SIP1 (Verschueren et al. 1999). In this study, we characterize a novel nuclear protein, termed SNIP1 for Smad nuclear interacting protein 1, which also serves to suppress the TGF-β-signaling pathway. We demonstrate that, whereas this inhibitor can interact with the MH2 domain of Smad4 in a TGF-β dependent manner, its principal mechanism of action appears to be through inhibition of transcription by binding to CBP/p300 and interfering with the ability of these coactivators to interact with Smad4.

To define DNA regulatory elements that mediate the response of the keratin 1 (K1) gene to Ca(2+)-induced differentiation, regions spanning the 5'- and 3'-flanking sequences, coding regions, and introns from the human K1 gene were cloned into vectors containing the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into cultured mouse keratinocytes. A 4.3-kilobase (kb) region located 3' to the K1 gene stimulated CAT activity in response to increasing Ca2+ concentrations from 0.05 mM (basal cells) to 1.2 mM (differentiated cells). The 4.3-kb fragment was also active in human epidermal cells but inactive in NIH 3T3 cells and primary mouse fibroblasts. Deletion analysis localized the activity to the terminal 1682 base pairs (bp) of the flanking sequence which retained Ca2+ sensitivity in epidermal cells but was not active in mesenchymal cells. Removal of a 207-base pair element created an enhancer which was active in both epidermal and mesenchymal cells but was still Ca(2+)-inducible. Further deletions identified two elements which functioned synergistically to give maximal Ca(2+)-sensitive activity. Stably transfected epidermal cell lines expressed CAT under the direction of these elements when grafted onto nude mice to reconstitute an intact epidermis. Previously reported keratin regulatory motifs were not contained in the 1682-bp fragment, but an AP-1 site was identified in one of the synergistic subunits.

The intracellular signaling events of the Bone Morphogenetic Proteins (BMPs) involve the R-Smad family members Smad1, Smad5, Smad8 and the Co-Smad, Smad4. Smads are currently considered to be DNA-binding transcriptional modulators and shown to recruit the master transcriptional co-activator CBP/p300 for transcriptional activation. SNIP1 is a recently discovered novel repressor of CBP/p300. Currently, the detailed molecular mechanisms that allow R-Smads and Co-Smad to co-operatively modulate transcription events are not fully understood. Here we report a novel physical and functional link between Smad1 and the 26S proteasome that contributes to Smad1- and Smad4-mediated transcriptional regulation. Smad1 forms a complex with a proteasome β subunit HsN3 and the ornithine decarboxylase antizyme (Az). The interaction is enhanced upon BMP type I receptor activation and occur prior to the incorporation of HsN3 into the mature 20S proteasome. Furthermore, BMPs trigger the translocation of Smad1, HsN3 and Az into the nucleus, where the novel CBP/p300 repressor protein SNIP1 is further recruited to Smad1/HsN3/Az complex and degraded in a Smad1-, Smad4- and Az-dependent fashion. The degradation of the CBP/p300 repressor SNIP1 is likely an essential step for Smad1-, Smad4-mediated transcriptional activation, since increased SNIP1 expression inhibits BMP-induced gene responses. Our studies thus add two additional important functional partners of Smad1 into the signaling web of BMPs and also suggest a novel mechanism for Smad1 and Smad4 to co-modulate transcription via regulating proteasomal degradation of CBP/p300 repressor SNIP1.