CMIP interacts with WT1 and targets it on the proteasome degradation pathway

ABSTRACT Insulin receptor substrate 1 (IRS-1) plays an important role in the insulin signaling cascade. In vitro and in vivo studies from many investigators have suggested that lowering of IRS-1 cellular levels may be a mechanism of disordered insulin action (so-called insulin resistance). We previously reported that the protein levels of IRS-1 were selectively regulated by a proteasome degradation pathway in CHO/IR/IRS-1 cells and 3T3-L1 adipocytes during prolonged insulin exposure, whereas IRS-2 was unaffected. We have now studied the signaling events that are involved in activation of the IRS-1 proteasome degradation pathway. Additionally, we have addressed structural elements in IRS-1 versus IRS-2 that are required for its specific proteasome degradation. Using ts20 cells, which express a temperature-sensitive mutant of ubiquitin-activating enzyme E1, ubiquitination of IRS-1 was shown to be a prerequisite for insulin-induced IRS-1 proteasome degradation. Using IRS-1/IRS-2 chimeric proteins, the N-terminal region of IRS-1 including the PH and PTB domains was identified as essential for targeting IRS-1 to the ubiquitin-proteasome degradation pathway. Activation of phosphatidylinositol 3-kinase is necessary but not sufficient for activating and sustaining the IRS-1 ubiquitin-proteasome degradation pathway. In contrast, activation of mTOR is not required for IRS-1 degradation in CHO/IR cells. Thus, our data provide insight into the molecular mechanism of insulin-induced activation of the IRS-1 ubiquitin-proteasome degradation pathway.

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Increased Expression of Proteasome-Related Genes In Patients with Primary Myelofibrosis

Blood ◽

10.1182/blood.v116.21.4117.4117 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4117-4117

Author(s):

Vibe Skov ◽

Thomas Stauffer Larsen ◽

Mads Thomassen ◽

Caroline Riley ◽

Morten Krogh Jensen ◽

...

Keyword(s):

Gene Expression ◽

Network Analysis ◽

Whole Blood ◽

Proteasome Inhibitor ◽

Single Gene ◽

Primary Myelofibrosis ◽

Degradation Pathway ◽

Gene Analysis ◽

Proteasome Degradation ◽

Nf Kappab

Abstract Abstract 4117 Introduction: The proteasome is an ubiquituous enzyme complex that plays a critical role in the degradation of many proteins involved in cell cycle regulation, apoptosis, and angiogenesis. Since these pathways and functions are often deregulated in cancer cells, inhibition of the proteasome is an attractive potential anticancer therapy. Bortezomib (Velcade, formerly PS-341) is an extremely potent and selective proteasome inhibitor that shows strong activity against many solid and hematologic tumor types. Moreover, bortezomib, mainly by inhibition of the NF-kappaB pathway, has a chemosensitizing effect when administered together with other antitumoral drugs. Bortezomib is a well-established treatment in multiple myeloma and studies are focusing in the potential benefit of bortezomib in other haematological malignancies, including malignant lymphomas. Since the NF-kappaB pathway is considered to be implicated in the abnormal release of cytokines in primary myelofibrosis (PMF), the proteasome inhibitor bortezomib might be a potential therapy. In a murine model, bortezomib has been demonstrated to inhibit thrombopoietin (TPO)-induced NF-kappaB activation in megakaryocytes and to reduce myeloproliferation induced by high TPO levels. Accordingly, from in vitro studies it was concluded that bortezomib might be a promising therapy for future treatment of PMF patients. Surprisingly, however, these encouraging results have not been achieved in clinical trials testing bortezomib in patients with myelofibrosis. We have performed gene expression profiling of patients with PMF and in patients with other chronic myeloproliferative neoplasms (CMPNs) in order to describe aberrant genes in the proteasome pathway in PMF. Materials and methods: The HG-U133 Plus 2.0 microarray from Affymetrix was used to profile expression of 38500 genes in whole blood from 70 patients with CMPNs, including 9 patients with PMF and 61 patients with other CMPNs. All patients were diagnosed according to the WHO criteria of a CMPN (ET=19, PV=41, PMF=9). The patients were diagnosed and followed in two institutions. Most patients were studied on cytoreductive therapy, which for the large majority included hydroxyurea. Total RNA was purified from whole blood and amplified to biotin-labeled aRNA and hybridized to microarray chips. Differences in gene expression between the two groups were calculated for each gene in the dataset by using Welch two sample t test, and the Benjamini Hochberg method was applied to control for multiple hypothesis testing (false discovery rate (FDR) < 0.05). Data were integrated with biological pathways and networks using Gene Microarray Pathway Profiler (GenMAPP 2.1) and Cytoscape 2.6.3, respectively. Hypothesis driven discovery was used to find significantly differentially expressed genes and pathways associated with PMF. Results: Single gene analysis demonstrated significantly elevated expression of seventeen proteasomal subunit genes in patients with PMF (PSMA1, PSMA2, PSMA6, PSMA7, PSMB4, PSMB5, PSMB6, PSMB7, PSMC2, PSMC3, PSMD10, PSMD14, PSMD4, PSMD8, PSMD9, PSMG1, and PSMG3 (FDR < 0.05). Only one gene, PSMB4, was significantly downregulated (FDR < 0.05). Global pathway analysis showed a significant upregulation of the proteasome degradation pathway (adjusted P < 0.03), and the network analysis revealed a significant subnetwork only composed of upregulated genes (CDC25A, CDC6, CDT1, GMNN, ORC1L, PSMA6, PSMA7, PSMB5, PSMB6, PSMB7, PSMC3, PSMD5, PSMD8, PSMD9, PSMD14) of which 10 were proteasomal genes (Z=2.6). Conclusion: In this study, we have for the first time described the gene signature of the proteasome in peripheral blood cells from patients with myelofibrosis and patients with ET and PV. Using single gene analysis, global pathway and network analysis, we found significant upregulation of the proteasomal transcriptome in patients with PMF as compared to patients with ET and PV as a group. This study has added new important information of the genes involved in the upregulation of the proteasome degradation pathway in these patients. Disclosures: No relevant conflicts of interest to declare.

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