In order to investigate potential roles for Mam in Notch signaling, we searched sequence databases for related proteins. A human cDNA sequence that shows significant similarity to DMam (30
) was identified (Fig. a). This cDNA, KIAA0200, was isolated during a project to clone long cDNAs (19
). No function for this sequence has been reported. The arrangements of basic and acidic amino acid clusters in the protein and DMam are well conserved, implying that their higher-order structures are related (Fig. b). Based on our observations reported below, we have tentatively named KIAA0200 hMam-1.
FIG. 1 Primary structure of Mam proteins. (a) Primary sequences of DMam (GenBank accession number X54251) and hMam-1 (D83785). Sequences were aligned using the ClustalW algorithm. Identical amino acids are in shaded boxes. Similar amino acids are in open boxes. (more ...)
To identify the product of a full-length cDNA of hMam-1, we attached an HA tag to the portion of the coding region corresponding to the N terminus and cloned it into a mammalian expression vector. When this vector was transfected to 293T cells, a single protein with an apparent molecular mass of 140 kDa was detected by Western blotting analysis with an anti-HA antibody (Fig. a). Immunocytochemistry with the same antibody showed that this antigen is in the nuclei of the transfected cells (Fig. b), as previously reported for DMam (3
FIG. 2 Identification and nuclear localization of the hMam-1 protein. (a) Identification of the hMam-1 protein. 293T cells were transfected with an expression vector for hMam-1 N-HA or the empty control vector (pEF-BOS). Whole-cell extracts prepared from these (more ...)
If hMam-1 is a homolog or ortholog of DMam, its expression should have effects on the mammalian Notch signaling pathway. Ligand binding to Notch on cell surfaces induces cleavage of the receptor in or near its transmembrane domain (site 3), releasing the IC domain of the receptor from the membrane (15
). The Notch1-mediated activation of the HES-1
promoter has been shown to require this cleavage (28
), and this can be mimicked experimentally by expression of the IC domain of Notch (12
). We examined whether hMam-1 acts synergistically with Notch1IC in this system by cotransfecting the expression vector of hMam-1 with that of the IC domain of Notch1 into NIH 3T3 cells. As shown in Fig. a, the HES-5
promoter was activated by the expression of Notch1IC alone but not by the expression of hMam-1 alone. Coexpression of Notch1IC and hMam-1 augmented the activation of the HES-5
promoter. More-modest effects were observed using the HES-1
promoter (data not shown).
FIG. 3 Effects of full-length and truncated forms of hMam-1 on transactivation of the HES-5 promoter. NIH 3T3 cells were transfected with expression vectors for Notch and Mam or their empty counterparts as controls. The transfection also contains the pHES-5 (more ...)
The mRNA of hMam-1 is expressed ubiquitously in human tissues (19
) and many cell lines (Y. Konuma, K. Harigaya, and M. Kitagawa, unpublished data). Thus, we assumed that the murine version of the Mam-1 protein is expressed in NIH 3T3 cells and contributes to the activation of the HES
promoters observed in the absence of the transfection of hMam-1. In Drosophila
, it has been shown that overexpression of C-terminally truncated versions of DMam disrupts Notch pathway function (8
). We made similar truncations of hMam-1 and examined the effects of their expression on the transactivation of the HES
promoters in the transactivation assay system. Coexpression of the shortest truncated form of hMam-1 reduced the Notch1IC-induced activation of HES-5
(Fig. b) and HES-1
(data not shown). Additionally, expression of Notch2IC in combination with full-length and truncated hMam-1 elicited similar effects on the activation of HES
promoters (data not shown). These data suggest that hMam-1 may act like DMam does in the insect system, as a positive regulator of Notch signaling and HES
promoter activity. They suggest further that hMam-1 may act in concert with the IC domain of Notch.
The Notch IC domain has been shown to translocate to the nucleus and associate with the sequence-specific DNA binding protein CSL (9
). Therefore, we tested for physical interaction of the hMam-1 protein with this complex. For this purpose, we employed an EMSA using an oligonucleotide probe derived from the HES-1
promoter. This sequence is essential for activation by the Notch1IC domain (12
). As shown in Fig. a, lane 2, an extract from cells transfected with a major form of mammalian CSL proteins (RBP-J, also known as CBF-1) (7
) exhibited two specific bands. Cotransfection of Notch1IC with RBP-J induced another complex that migrates more slowly than the bands mentioned above (Fig. a, lane 3). Interestingly, coexpression of hMam-1 (either tagged or not) with these two proteins abolished all the complexes and induced two novel bands that migrate more slowly (Fig. a, lane 4, and b, lane 8). The complexes from the extract of RBP-J-transfected cells were supershifted by incubation with an antibody against the RBP-J protein (Fig. b, lanes 1 to 3), verifying involvement of RBP-J (34
). In the extract cotransfected with RBP-J and Notch, two faster-migrating bands that show apparent similarity in their mobility to the RBP-J complex could be supershifted by the anti-RBP-J antibody but not by the anti-Notch1 antibody (Fig. b, lanes 4 to 7). The more slowly migrating complex could be supershifted by the anti-Notch1 antibody and diminished or cleared by the anti-RBP-J antibody, indicating its identity as the Notch1IC–RBP-J complex. The amount of the complex supershifted by the anti-Notch1 antibody (Fig. b, lane 6) is relatively large compared to the Notch1IC–RBP-J band. This phenomenon is reproducible and might be due to stabilization of the complex by binding to the antibody. Cotransfection of Notch1IC with RBP-J also makes the slower-migrating form of the RBP-J-specific complexes stronger (Fig. a, lane 3, and b, lane 4). This might be due to the induction of modification of RBP-J by the presence of Notch1IC. In the extract transfected with RBP-J, Notch1IC, and the tagged hMam-1, the anti-Notch1 antibody shifted the faster band (Fig. b, lane 10). The appearance of this supershift by the anti-Notch1 antibody is very different from that of the Notch1IC–RBP-J band by the same antibody (compare lanes 6 and 10 of Fig. b), indicating that the faster band that lies just above the Notch1IC–RBP-J band is indeed distinct from it. The anti-HA antibody did produce a minor shift (Fig. b, lane 11); however, there is no direct evidence of the involvement of RBP-J in these complexes, as available antibody against RBP-J does not react with them (Fig. b, lane 9). Stereospecific inhibition might inhibit access of the antibodies to the antigen in this particular context. We did observe that transfection of hMam-1 and Notch1IC without RBP-J greatly reduced the quantities of these complexes (Fig. a, lane 8) and that RBP-J is coimmunoprecipitated with hMam-1 and Notch1IC (Fig. e; also see below), which is strong evidence that RBP-J is present. Expression of Notch2IC with hMam-1 and RBP-J induced slow-migrating complexes that look similar to those found in the case of Notch1IC expression (data not shown). Expression of hMam-1 without Notch1IC does not significantly alter the DNA binding activity of RBP-J or endogenous RBP-J-like factor (Fig. a, lanes 1, 2, 5, and 6).
FIG. 4 Formation of the Mam-Notch-CSL complex on its target promoter elements. (a) Extracts of transfected 293T cells exhibit activities that bind to the RBP-J element of the HES-1 promoter. Cells were transfected with the expression vectors for the indicated (more ...)
To map the domain(s) of hMam-1 required for these physical associations, we examined the effects of C-terminal truncations of hMam-1 on the DNA binding complexes involving RBP-J and Notch1IC. The truncations include those exhibiting dominant negative effects on the transcription activation assay. As shown in lanes 3 to 6 of Fig. c, all the truncations up to amino acid 103 greatly diminished the complexes involving RBP-J only. Furthermore, each of these truncations induced more slowly migrating complexes whose mobility correlates with the molecular mass of each truncation (Fig. d). These results support the idea that both of the slowly migrating complexes contain the hMam-1 protein. The faster-migrating complex involving shorter truncations of hMam-1 migrates more rapidly than the Notch1IC–RBP-J complex (Fig. a and b). This could be due to a change in complex stoichiometry or composition. Furthermore, approximately 100 amino acids from the N terminus are sufficient to alter the mobility of the DNA binding complexes, and a deletion construct lacking amino acids 26 to 100 exhibits virtually no activity on the complexes (Fig. c, lane 7). Thus, the N-terminal region of hMam-1 is necessary and sufficient to mediate the physical association.
Coimmunoprecipitation assays revealed that hMam-1 associates with Notch1IC and RBP-J even in the absence of the binding site of DNA (Fig. e, lane 3). This assay also revealed that hMam-1 associates with Notch1IC only in the presence of RBP-J (Fig. e, lanes 3 and 6). In EMSA, expression of hMam-1 without Notch1IC does not significantly alter the DNA binding activity of RBP-J (Fig. a, lanes 2 and 6), indicating that hMam-1 associates with RBP-J only in the presence of Notch1IC. These results suggest that hMam-1 associates with the complex of the two proteins but not with the single protein species. The coimmunoprecipitation assays further reveal that expression of hMam-1 enhances the physical association of Notch1IC and RBP-J (12
) (Fig. e, lanes 2 and 3). The two shortest truncations could also be coimmunoprecipitated with Notch1 with RBP-J, enhancing the association of these two proteins in varying degrees (Fig. e, lanes 4 and 5). More carboxyl portions of the hMam-1 protein presumably contain a domain(s) necessary for transcriptional activation, as overexpression of the N-terminal region hampers the transactivation induced by Notch signaling (Fig. b).
There is substantial genetic evidence implicating DMam as a positive effector in Notch signaling (1
). In contrast, no genetic information is yet available for hMam-1, and additionally, hMam-1 has diverged significantly from the DMam sequence outside the charged amino acid clusters (Fig. a). Therefore, additional evidence that linked the functions of these two proteins was sought. We investigated whether DMam can form a complex with DNotch and Su(H) (Drosophila
CSL) proteins in Drosophila
S2 cells endogenously express DMam. S2 cells were cotransfected with either Myc epitope-tagged Su(H) [Su(H)-Myc] and DNotch1IC or Su(H)-Myc, DNotchIC, and DMam. Coimmunoprecipitations showed that endogenous DMam (Fig. a, lane 2) or transfected DMam (Fig. a, lane 3) exists in a complex with Su(H) and DNotchIC. These data also revealed that DNotchIC is required for DMam's association with the complex (Fig. a, lanes 1 and 4). As reported earlier (5
), cotransfection of a Notch IC domain and Su(H) activates expression of an E
) reporter (Fig. b). Consistent with the hMam-1 data, we observed that cotransfection of DMam augments the activation levels of the E
) reporter (Fig. b).
FIG. 5 DMam function in cell culture. (a) Physical association of DMam with DNotchIC and Su(H). S2 cell extracts were immunoprecipitated with an anti-c-Myc antibody. The precipitates were resolved on an SDS gel, transferred to a membrane, and stained sequentially (more ...)