Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IR<sup>A</sup> Signal in Malignant Mesothelioma
EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using...
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MDPI AG
2023-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/8/7380 |
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| author | Pierluigi Scalia Carmen Merali Carlos Barrero Antonio Suma Vincenzo Carnevale Salim Merali Stephen J. Williams |
| author_facet | Pierluigi Scalia Carmen Merali Carlos Barrero Antonio Suma Vincenzo Carnevale Salim Merali Stephen J. Williams |
| author_sort | Pierluigi Scalia |
| collection | DOAJ |
| description | EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein–protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as “DTX3c”), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway. |
| first_indexed | 2024-03-11T04:56:31Z |
| format | Article |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T04:56:31Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-cf203344c2c04db588f9902480733f6f2023-11-17T19:39:35ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-04-01248738010.3390/ijms24087380Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IR<sup>A</sup> Signal in Malignant MesotheliomaPierluigi Scalia0Carmen Merali1Carlos Barrero2Antonio Suma3Vincenzo Carnevale4Salim Merali5Stephen J. Williams6The ISOPROG-Somatolink EPFP Research Network, Philadelphia, PA 19102, USA and 93100 Caltanissetta, ItalyProteomics and Metabolomics Facility, Moulder Center for Drug Discovery Research, School of Pharmacy, Temple University, Philadelphia, PA 19140, USAProteomics and Metabolomics Facility, Moulder Center for Drug Discovery Research, School of Pharmacy, Temple University, Philadelphia, PA 19140, USAInstitute of Computational Molecular Science, College of Science and Technology, Temple University, Philadelphia, PA 19122, USAInstitute of Computational Molecular Science, College of Science and Technology, Temple University, Philadelphia, PA 19122, USAProteomics and Metabolomics Facility, Moulder Center for Drug Discovery Research, School of Pharmacy, Temple University, Philadelphia, PA 19140, USAThe ISOPROG-Somatolink EPFP Research Network, Philadelphia, PA 19102, USA and 93100 Caltanissetta, ItalyEphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein–protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as “DTX3c”), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.https://www.mdpi.com/1422-0067/24/8/7380IGF2insulin-like growth factor-IIIR-Ainsulin receptor isoform-AEphB4eph receptor B4 |
| spellingShingle | Pierluigi Scalia Carmen Merali Carlos Barrero Antonio Suma Vincenzo Carnevale Salim Merali Stephen J. Williams Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IR<sup>A</sup> Signal in Malignant Mesothelioma International Journal of Molecular Sciences IGF2 insulin-like growth factor-II IR-A insulin receptor isoform-A EphB4 eph receptor B4 |
| title | Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IR<sup>A</sup> Signal in Malignant Mesothelioma |
| title_full | Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IR<sup>A</sup> Signal in Malignant Mesothelioma |
| title_fullStr | Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IR<sup>A</sup> Signal in Malignant Mesothelioma |
| title_full_unstemmed | Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IR<sup>A</sup> Signal in Malignant Mesothelioma |
| title_short | Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IR<sup>A</sup> Signal in Malignant Mesothelioma |
| title_sort | novel isoform dtx3c associates with ube2n uba1 and cdc48 p97 as part of the ephb4 degradation complex regulated by the autocrine igf ii ir sup a sup signal in malignant mesothelioma |
| topic | IGF2 insulin-like growth factor-II IR-A insulin receptor isoform-A EphB4 eph receptor B4 |
| url | https://www.mdpi.com/1422-0067/24/8/7380 |
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