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From the Department of Pathology, Kochi Medical School, Kochi, Japan
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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The ubiquitination of the intracellular region of receptors and their ligands commonly plays a pivotal role in ligand-dependent receptor activation. Of particular note, it is well recognized that ubiquitination of the Notch receptor is important for proteolytic cleavage, endocytosis, and signal transduction.5 Interestingly, endocytosis of the Notch ligand is also essential for ligand-dependent Notch activation.6,7
Recently, a skeletrophin-related molecule, DIP-1 and the (identical) mib molecule, which together with skeletrophin may constitute a new subfamily of ubiquitin ligase, was found to be involved in the endocytosis of Delta, a Notch ligand.6 Jin and colleagues8 identified a novel murine E3 ubiquitin ligase, designated DIP-1 (DAPK-interacting protein), which targets a death-associated protein kinase (DAPK). DIP-1 promotes tumor necrosis factor-induced apoptosis. Itoh and colleagues6 isolated a novel ubiquitin ligase, designated mib, which was thought to be responsible for the phenotype of the zebrafish mind bomb mutant. Interestingly, mib ubiquitin ligase mediated endocytosis of Delta by ubiquitination, thereby facilitating transendocytosis of the Notch extracellular domain, resulting in the transportation of the Notch intracellular domain to the nucleus, where it activates various genes. Further studies eventually showed that DIP-1 and mib were in fact the same molecule. Because DIP has been used for the nomenclature for the distinct molecule, mDia-interacting protein,9 before Jin and colleagues8 reporting their identified molecule, we provisionally use the name DIP-1/mib for this RING ubiquitin ligase.
The molecular structure of skeletrophin is closely related to that of human DIP-1/mib protein. Both skeletrophin and DIP-1/mib have, from the N- to C-terminus, a Mib/Herc2 region, a ZZ zinc finger domain, ankyrin repeat domains, and RING finger domains (see schematic representation of molecular structures of DIP-1/mib and skeletrophin in Figure 2A
). The significant identity between human skeletrophin and human DIP-1/mib, 36.0% by deduced amino acid sequence, clearly indicates that skeletrophin and DIP-1/mib are in a new E3 ubiquitin ligase subfamily. However, further experimental evidence is necessary to conclude whether skeletrophin is also a ubiquitin ligase, because several molecules containing the RING motif are known to lack ubiquitin ligase activity. To understand the biological and potent pathogenetic properties of skeletrophin, it is essential to identify its substrates and/or target molecules.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Expression of the glutathione S-transferase (GST) fusion RING domain of skeletrophin, the intracellular region of Jagged-1, and -2, and Delta-1, -3, and -4, was driven by isopropyl-1-thio-ß-D-galactopyranoside-inducible promoter elements as previously described.11
Briefly, human skeletrophin (amino acids 824 to 998) cDNA encoding two RING-HC domains was amplified from human brain cDNAs (BD Biosciences Clontech, Palo Alto, CA) by polymerase chain reaction (PCR) with a sense (5'-CGACTCGAGGGTCGCGTGCTCAAGGCCC-3') primer (in which the XhoI site is underlined), and an anti-sense (5'-TCACACGAAGATCTGGATGCGG-3') primer. The amplified RING finger domain was first cloned into pTarget-T vector (Promega, Madison, WI), where the NotI cut site was present at the 3'-region, followed by digestion with XhoI and NotI, and subcloned in-frame into the pGEX-5X-1 vector (Amersham Biosciences Co., Piscataway, NJ). The construct was verified by sequencing and designated skRING. The amino acid substitution, C969S, was introduced into skRING by PCR site-directed mutagenesis by using a 5'-GGGCGCAATGCGCCGTGGCACTCTGGAAC-3' primer, verified by sequencing, and designated skRING-C969S. Subsequently, the amino acid substitution, C891S, was introduced into skRING or skRING-C969S by PCR site-directed mutagenesis with 5'-CACACACGGTGCGGTGCTGGCTAGGCGAG-3', verified by sequencing, and designated skRING-C891S and skRING-C891S/C969S, respectively. Notably, recombinant proteins generated by using skRING-C891S or skRING-C969S contained one intact RING domain and one mutated domain, C891S and C969S, respectively, whereas both of the two RING domains of the skRING-C891S/C969S recombinant protein were mutated (Figure 1)
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), subcloned into pGEX-5X-1, and verified by sequencing. All GST-fused recombinant proteins were expressed in log phase Escherichia coli BL21-CodonPlus-RP (Stratagene, La Jolla, CA). Sufficient expression and solubilization of GST-fused recombinant proteins were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by staining with Coomassie Brilliant Blue.
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In Vitro Ubiquitination Assays
In vitro ubiquitination assays were performed according to the methods of Lorick and colleagues.12 Recombinant FLAG-tagged ubiquitin and recombinant ubiquitin carrier proteins UbcH5b and UbcH7 were purchased from Sigma-Aldrich Inc. (Saint Louis, MO). Recombinant murine ubiquitin-activating enzyme E1 was purchased from Calbiochem (La Jolla, CA). Using the UbcH5b and UbcH7 as E2s, the autoubiquitination assay was performed as follows: GST-fused RING proteins were bound to glutathione Sepharose 4B (GT) beads (Amersham Biosciences Co.), washed with wash buffer (50 mmol/L Tris-HCl, pH 7.4, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 2 µg/ml aprotinin), and mixed with a reaction mixture (50 µl) containing 50 mmol/L Tris-HCl, pH 7.4, 5 mmol/L MgCl2, 4 mmol/L ATP, 0.5 mmol/L dithiothreitol, 15 µg of FLAG-ubiquitin, and 200 ng E1 in the presence or absence of 200 ng of E2 at 30°C for 60 minutes. The beads were washed with wash buffer. Proteins bound to beads were subjected to SDS-PAGE under reducing conditions and detected by Western immunoblotting using an antibody to the FLAG tag (Sigma-Aldrich Inc.) or GST (Amersham Biosciences).
GST Pull-Down Assays
Cell extracts including the recombinant full-length skeletrophin protein and the GST-fused intracellular region of Delta-1, -3, -4, and Jagged-1 and -2 were mixed in a binding buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.5% Nonidet P-40, 0.5% bovine serum albumin, 0.5 mmol/L phenylmethyl sulfonyl fluoride, and 2 µg/ml aprotinin) and incubated for 6 hours at 4°C. Then, the GST-fused molecules were pulled-down by GT beads, washed four times with binding buffer containing 300 mmol/L KCl, suspended in SDS sample buffer, and analyzed by SDS-PAGE followed by Western immunoblotting with anti-skeletrophin antibody or anti-GST antibody.
Ubiquitination of the Intracellular Region of Jagged-2 by Skeletrophin
In vitro ubiquitination assays were performed by incubating FLAG-tagged ubiquitin, E1, UbcH5b, recombinant full-length skeletrophin, or C969S-skeletrophin, with the GST-fused intracellular region of Jagged-2. Briefly, GST-fused Jagged-2 proteins were bound to GT-Sepharose beads, washed with wash buffer (50 mmol/L Tris-HCl, pH 7.4, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 2 µg/ml aprotinin), and mixed with a reaction mixture (50 µl) containing 50 mmol/L Tris-HCl, pH 7.4, 5 mmol/L MgCl2, 4 mmol/L ATP, 0.5 mmol/L dithiothreitol, 15 µg of FLAG-ubiquitin, 200 ng E1, 200 ng UbcH5b in the presence of recombinant full-length skeletrophin or C969S-skeletrophin at 30°C for 60 minutes. Proteins bound to GT beads were analyzed by Western immunoblotting with anti-GST and anti-FLAG antibodies.
Immunohistochemical Staining
Details of the immunohistochemical staining procedures including the preparation of affinity-purified rabbit antibody to skeletrophin have been described previously.1 In the present study, we newly generated a monoclonal antibody to intracellular region of Jagged-2. Briefly, the recombinant GST-fused intracellular region of Jagged-2, described above, was digested with bovine factor Xa (Seikagaku Co. Ltd., Osaka, Japan) to separate Jagged-2 from GST tag. Factor Xa was subsequently adsorbed with Xa Removal Resin (Qiagen GmbH, Hilden, Germany). BALB/c mice were immunized intraperitoneally with the recombinant intracellular region of Jagged-2. Monoclonal antibody was generated according to the modified method of Koehler and Milstein13 and Takeuchi and colleagues.14 One of the established hybridoma clones secreted an antibody, which could immunostain the intracellular domain of Jagged-2 in the routinely processed paraffin-embedded tissue sections. Western immunoblotting was also performed to evaluate the specificity of the antibody. To identify the Jagged-2 band in the Western immunoblotting, we also used commercially available rabbit antibody to Jagged-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Extracted protein mixtures from human thymus were purchased from BD Biosciences Clontech.
In the present study, we examined archival tissue specimens comprising a spectrum of various intramedullar multiple myeloma and nonneoplasmic plasma cells (summarized in Table 2
). When we examined multiple myeloma specimens, we focused on exploring the initial bone marrow biopsy specimens of patients with untreated intramedullary multiple myeloma. To demonstrate skeletrophin expression in the brain, which exhibited comparatively high levels of mRNA expression in our previous study,1
we stained a small part of the forebrain surgically resected from patients with hemorrhage due to amyloid angiopathy. Normocellular nonneoplasmic bone marrow tissues were obtained from patients with various cancers to evaluate the metastasis to bone marrow, and were used in the present study. All specimens were fixed in 10% buffered formalin, and paraffin-embedded. Deparaffinized sections were placed in plastic Coplin jars (Asone, Tokyo, Japan) filled with preheated citrate buffer (pH 6.0) and microwave-irradiated for 5 minutes at 650 W. Next, the slides were incubated for 30 minutes in fetal bovine serum. The tissues were then immunostained with affinity-purified rabbit anti-human skeletrophin peptide, monoclonal antibody to intracellular region of Jagged-2, or control antibodies using a streptavidin-biotin complex peroxidase kit (DAKO LSAB kit; Dakopatts, Kyoto, Japan). The procedures were performed according to the manufacturer’s protocol. In several experiments, anti-skeletrophin antibody or anti-Jagged-2 antibody was preadsorbed with the immunized skeletrophin peptide or recombinant intracellular domain of Jagged-2.
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Cell Culture, Reverse Transcriptase (RT)-PCR, Transfection, and Co-Culture Assay
A murine bone marrow-derived stromal cell line, ST-2,15 was obtained from the Riken BioSource Center (Tsukuba, Japan). A murine myeloma cell line P3U1 was maintained in our laboratory. A human myeloma cell line, RPMI8226, was obtained from the Japan Heath Science Research Resources Bank (Osaka, Japan). Another human myeloma cell line, U266, was a kind gift from Dr. Ikezoe Takayuki (KMS, Department of Hematology and Respiratory Medicine, Kochi University, Kochi, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Grand Island, NY) containing 10% fetal bovine serum and 50 µg/ml of gentamicin (Life Technologies, Inc.).
The construction and the method for obtaining the stable transfection of a pCI-neo expression vector (Promega) containing a human wild-type skeletrophin has been previously reported.1 A pCI-neo vector containing a RING finger mutant, C969S, was constructed by substitution of an Eco47III-Not1 part from skRING-C969S. P3U1 clones, which expressed human full-length or C969S-mutated skeletrophin, were established after the limiting dilution. Culture wells of six-well plates were seeded with 1 x 104 ST-2 cells 16 hours before co-culture assay. Then, 1 x 103 P3U1 cells were co-cultured with ST-2 cells with or without an insert for 14 hours. After incubation, P3U1 cells were removed by shaking the plates and aspiration when P3U1 was allowed to be in direct contact with ST-2, without an insert.
RT-PCR analysis was performed as previously described.16
Briefly, total cellular RNA was prepared from cell lysates using RNA-zol B (Biotex Laboratory, Houston, TX). In several experiments, serial fivefold dilutions were made from master lysates and used as a set in RT-PCR. The cDNA synthesis from total RNA and subsequent PCR were performed using an RNA LA (long and accurate) PCR kit (Takara Co. Ltd., Ohtsu, Japan). The procedure was performed according to the manufacturer’s instructions. The primer sets used for RT-PCR are summarized in Table 2
. The PCR-amplified product was electrophoretically separated on a 2% agarose gel. To assure reproducibility of the results, the RT-PCR amplification was repeated by using different clones at least three times.
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Results |
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Ubiquitination requires sequential reactions of three enzymes. First, ubiquitin is activated by E1 through the formation of a thioester bond in the presence of ATP. Subsequently, ubiquitin is transferred to E2 via the thioester bond. Finally, E2 transfers ubiquitin, generally with the help of ubiquitin ligase E3, to an 
-amino group of an internal lysine residue of the substrate protein. Ubiquitin ligase is sometimes itself ubiquitinated and therefore in vitro demonstration of substrate-independent autoubiquitination is considered to be a good indicator of ligase activity.
As shown in Figure 1B
, the recombinant GST-fused RING motif of skeletrophin protein promoted autoubiquitination in a UbcH5b-dependent manner. The high molecular mass of FLAG-tagged proteins, which indicates ubiquitination, was observed when ubiquitin, E1, UbcH5b, and the GST-fused RING domain of skeletrophin were incubated. In the absence of UbcH5b, these ladders were not observed. UbcH7 could not confer sufficient ubiquitination. These results demonstrate that skeletrophin has a ubiquitin ligase activity similar to that observed in DIP-1/mib.6,8
Alternation of RING motif, C969S, eliminated autoubiquitination activity, whereas C891S mutation did not affect the activity (Figure 1, C and D)
. These results indicated that skeletrophin is a ubiquitin ligase, the activity of which depends on a RING domain of C-terminus. In addition, we also examined autoubiquitination of GST-fused full-length skeletrophin or RING-mutated, C969S, skeletrophin. Autoubiquitination of the former, but not the latter, was observed (data not shown).
Skeletrophin Bound and Ubiquitinated the Intracellular Region of Jagged-2
Itoh and colleagues6
demonstrated that DIP-1/mib, the molecular structure of which is close to skeletrophin (Figure 2A)
, targets the intracellular domain of Delta, a ligand of Notch. Therefore, we speculated that skeletrophin ubiquitin ligase might also target the intracellular domain of Notch ligands. To determine whether skeletrophin can bind to human Notch ligands, we performed GST pull-down assays using recombinant Delta-1, -3, and -4, and Jagged-1 and -2.
As demonstrated in Figure 2B
, full-length skeletrophin molecules bound to the recombinant GST-fused intracellular domain of Jagged-2, but not to the other GST-fused intracellular domains of Notch ligands tested. Subsequently, we explored whether the intracellular domain of Jagged-2 was ubiquitinized by skeletrophin. The result is shown in Figure 2C
. The band corresponding to the ubiquitinated intracellular domain of Jagged-2, which migrated slower than that of the intact one, was observed by Western immunoblotting using a specific antibody to GST and the FLAG tag. These observations indicate that skeletrophin is a ubiquitin ligase that acts toward the intracellular domain of Jagged-2.
Aberrant Expression of Skeletrophin in Multiple Myeloma Cells
An increasing number of articles state that Jagged-1 and -2 are overexpressed in multiple myeloma and that ligand-dependent Notch activation in bone marrow stromal cells has a pivotal role in the pathogenesis of multiple myeloma, possibly by accelerating paracrine interleukin (IL)-6 secretion.10,17
Because recombinant skeletrophin was able to bind and mediate the ubiquitination of the intracellular domain of Jagged-2, we were interested in the expression of skeletrophin in multiple myeloma cells. The results of immunohistochemical staining are summarized in Tables 3 and 4
and representative results are shown in Figure 3
. First, we demonstrate the staining pattern in neural cells to show the protein expression in skeletrophin mRNA-rich tissues (Figure 3A)
.1
As demonstrated in Figure 3B
, no significant staining was observed in normocellular nonneoplastic bone marrow. This result is compatible with our preliminary findings by dot-blot analysis, in which we did not detect skeletrophin mRNA in nonneoplastic bone marrow tissues (data not shown). Compared to neural cells, skeletrophin was expressed weakly or not at all in most nonneoplasmic plasma cells in various chronically inflamed tissues. A few dispersed plasma cells were weakly stained in the cytoplasm (Figure 3C)
. In contrast, strong staining was observed in malignant plasma cells in 23 of 40 specimens. In skeletrophin-positive cases, skeletrophin was localized in the entire cytoplasm (Figure 3D)
. We could not find any significant relationship between skeletrophin expression and the morphology of the infiltrating myeloma cell type (the presence of cleaved and/or large nuclei, nucleated or not), growth pattern (interstitial or nodular), the percentage of plasma cells in the bone marrow, or the serum level of lactate dehydrogenase. However, skeletrophin expression was more commonly observed in multiple myeloma specimens from patients with osteolytic bone lesions (Table 4
, Figure 3D
). Preadsorption of antibody with immunized peptide diminished the staining (Figure 3E)
. We also demonstrated the staining of the specimens from patients without osteolytic bone lesions (Figure 3F)
. These data obtained by immunohistochemical staining revealed that skeletrophin was significantly overexpressed in a considerable number of malignant myeloma specimens, especially skeletrophin from patients with an osteolytic region.
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Next, we investigated the correlation of skeletrophin and Jagged-2 expression in malignant plasma cells. To perform this study, we newly generated a monoclonal antibody to explore the Jagged-2 expression in routinely processed archival tissues. Western immunoblotting verified the specificity of this monoclonal antibody (Figure 4
, left). Most myeloma cells expressed Jagged-2, as recently reported by Houde and colleagues.10
Malignant plasma cells in 37 of 40 multiple myeloma specimens examined in this study appeared to express Jagged-2. Of particular interest, Jagged-2 tended to be localized in the cell surface membrane in skeletrophin-negative myeloma cases (2 of 16 specimens; Figure 4A
), whereas Jagged-2 was often found in both surface, perisurface cytoplasm, and/or entire cytoplasm in skeletrophin-positive cases (14 of 21; Figure 4, B and C
). In five skeletrophin-positive myeloma specimens, we observed strong Jagged-2 expression in the entire cytoplasm (Figure 4C)
. The result is summarized in Table 5
and representative staining is shown in Figure 4
, right.
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Finally, we investigated whether skeletrophin expression in myeloma cells could activate ligand-dependent Notch receptor activation in bone marrow stromal cells. To perform this experiment, we first examined the skeletrophin expression in several cultured myeloma cells. As shown in Figure 5A
, both RPMI8226 and U266 appeared to express abundant skeletrophin mRNA by RT-PCR. We also examined Jagged-2 expression and found that both RPMI8226 and U266 expressed Jagged-2 mRNA (Houde and colleagues10
had already reported the Jagged-2 expression in RPMI8226 and U266). Interestingly, a murine myeloma cell line, P3U1, did express Jagged-2, but did not express the detectable skeletrophin by RT-PCR (Figure 5A)
. A murine bone marrow-derived stromal cell line, ST-2, expressed Notch1, Notch2, and Notch3 as previously described by Yamada and colleagues,18
but did not express Jagged-1 or -2 (Figure 5B)
. Lack of Jagged-2 expression in ST-2 stromal cells is also consistent with the report from Nefedova and colleagues,19
who demonstrated that Jagged-2 was not detectable in bone marrow stromal cells from healthy donors.
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, induced Hes-1 gene expression in ST-2 stromal cells, by direct cell-cell contact (Figure 5D)
. By contrast, RING-mutated skeletrophin, indicated as C969S-sk, did not enhance Hes-1 expression. Notably, P3U1, in which exogenous skeletrophin or RING-mutated skeletrophin was overexpressed, expressed Jagged-2 as well as original P3U1 cells (Figure 5C)
. Figure 5
shows the representative results, which were obtained by using three independent clones. Hes-1 has been shown to be a target molecule of the activated Notch receptor.20
Therefore, we think that skeletrophin expression in myeloma cells could enhance ligand-dependent Notch activation in bone marrow stromal cells through direct cell-cell contact. |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In the present study, we first characterized skeletrophin as a novel ubiquitin ligase, which targeted the intracellular region of Jagged-2. Subsequently, we demonstrated that skeletrophin was overexpressed in malignant plasma cells in many multiple myeloma specimens. Premyeloma or myeloma cells may adhere to stromal cells, and then skeletrophin may mediate ligand-dependent Notch activation by ubiquitination, followed by endocytosis of the intracellular domain of Jagged-2. In line with this speculation, significant cytoplasmic localization of Jagged-2 was observed in skeletrophin-positive myeloma specimens. However, we also found that Jagged-2 was localized in the cytoplasm in two skeletrophin-negative specimens. In seven skeletrophin-positive cases, Jagged-2 was only found in the cell surface membrane. These data indicate that molecular events other than skeletrophin overexpression may be involved in Jagged-2 localization in malignant plasma cells.
Next, exogenously expressed skeletrophin in P3U1 cells induced a Notch downstream effector gene, Hes-1, through direct cell-cell contact. Very interestingly, Houde and colleagues10
reported that Jagged-2 was overexpressed in multiple myeloma cells and induced stromal cells to secrete IL-6. Skeletrophin may therefore be involved in the endocytosis of Jagged-2 to facilitate ligand-dependent Notch signal activation to increase IL-6 production from stromal cells. The finding that skeletrophin expression was related to the presence of osteolytic lesions in patients may also be related to IL-6 secretion, because patients with multiple myeloma with high IL-6 levels have a higher incidence of osteolytic bone lesions.24
However, further study, including the expression pattern of the Notch receptor in the stromal cells of bone marrow, is necessary to verify our working hypothesis, which is summarized in Figure 6A
.
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Recently, Ikeuchi and Sisodia25
revealed that Delta-1 and Jagged-2 are subject to presenilin-dependent, intramembranous 
-secretase processing. They also demonstrated that nuclear transport of intracellular region of Delta-1 mediated transcriptional stimulation. Therefore, Delta-1 and Jagged-2 can play dual roles as activators of Notch receptor signaling and as receptors that mediate nuclear signaling events via -secretase processing in the intracellular region. The intracellular region of Jagged-2 contains the putative nuclear localization signals, RKRR and KRRK. Similarly, the Delta-1 intracellular region also contains PDRKRPE and RKRP putative nuclear localization signals. From this perspective, it is particularly interesting that Jagged-2 was found in the nuclei of a few malignant plasma cells in skeletrophin-positive myeloma specimens (Figure 5C)
. Matsuda and colleagues26
recently performed a large-scale identification and characterization of the human genes that activate the nuclear factor (NF)-
B pathway. One of the molecules that strongly activated the NF-B pathway is a splicing form of skeletrophin. This finding may indicate that skeletrophin is involved in the pathogenesis of multiple myeloma, not only by activating the Notch-Jagged-2 interaction in myeloma cells and stromal cells, but also directly by suppressing apoptosis by activating the NF-B pathway in malignant plasma cells. We speculate that skeletrophin may also facilitate the production of the soluble intracellular region of Jagged-2, which might activate the transcriptional events, including the induction of a molecule, which could activate the NF-B pathway (Figure 6B)
.
Skeletrophin and DIP-1/mib ubiquitin ligase may play a complementary role in Notch-ligand interaction by binding to each Notch-ligand target, Jagged-2 and Delta, respectively. They may also be involved in the generation of soluble cytoplasmic protein by facilitating the presenilin-dependent -secretase of their substrates. Further extensive studies will be required to reveal the relationship between the biological properties of skeletrophin and DIP-1/mib ubiquitin ligase, especially with respect to their ligands and the role in presenilin-dependent -secretase cleavage of Jagged-2 and Delta-1. However, it is likely that skeletrophin and DIP-1/mib, which have very similar molecular structures (see Figure 2A
and the Introduction), may constitute a unique tribe of the RING-HC-dependent ubiquitin ligase family.
Lastly, skeletrophin did not down-regulate the expression of its substrate, Jagged-2, in myeloma cells. This indicated that the effect of skeletrophin on the Jagged-2 molecule is incessant in many malignant plasma cells. Therefore, therapeutic approaches intended to disturb skeletrophin function may have a potent therapeutic effect in patients with multiple myeloma. In the present study, we focused on specimens from untreated patients; however, we also did a preliminary exploration of skeletrophin expression in three patients, whose bone marrow tissues at initial diagnosis were not stained with anti-skeletrophin. With time, malignant plasma cells became resistant to various chemotherapy agents. Notably, skeletrophin expression was increasingly observed in these progressing malignant plasma cells. Further study to unravel the relationship between skeletrophin and resistance to chemotherapy is ongoing.
In conclusion, skeletrophin appears to be a novel ubiquitin ligase targeted toward the intracellular region of Jagged-2, which is aberrantly overexpressed in multiple myeloma cells. A precise understanding of the molecular mechanism that is involved in Notch and its ligand activation is likely to be beneficial to the development of specific therapeutic approaches for malignant myeloma.27,28
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Acknowledgements |
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Footnotes |
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Supported by the Ministry of Education of Japan (grants KAKEN12670165, 13670177), the Medical Research Fund of the Kochi Medical School, and the Vice-Chancellor of the Kochi Medical School (project grant for overseas study).
Accepted for publication February 25, 2005.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-secretase" cleavage. J Biol Chem 2003, 278:7751-7754This article has been cited by other articles:
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  M. Carrasco-Rando and M. Ruiz-Gomez Mind bomb 2, a founder myoblast-specific protein, regulates myoblast fusion and muscle stability Development, March 1, 2008; 135(5): 849 - 857. [Abstract] [Full Text] [PDF]
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 H. T. Nguyen, F. Voza, N. Ezzeddine, and M. Frasch Drosophila mind bomb2 is required for maintaining muscle integrity and survival J. Cell Biol., October 22, 2007; 179(2): 219 - 227. [Abstract] [Full Text] [PDF]
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 K. G. Leong and A. Karsan Recent insights into the role of Notch signaling in tumorigenesis Blood, March 15, 2006; 107(6): 2223 - 2233. [Abstract] [Full Text] [PDF]
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C. Pitsouli and C. Delidakis The interplay between DSL proteins and ubiquitin ligases in Notch signaling Development, September 15, 2005; 132(18): 4041 - 4050. [Abstract] [Full Text] [PDF]
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