| 1. |
- Akhoondi, Shahab, et al.
(författare)
-
FBXW7/hCDC4 is a general tumor suppressor in human cancer
- 2007
-
Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 67:19, s. 9006-9012
-
Tidskriftsartikel (refereegranskat)abstract
- The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skpl-Cdc53/ Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin El, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of ∼ 6%. The highest mutation frequencies were found in tumors of the bile duct (cholangio-carcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational "hotspots," which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer.
|
|
| 2. |
- Dagnell, Markus
(författare)
-
Redox-regulation of PTPs : mechanisms and impact on PDGFR signaling
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein tyrosine phosphatases (PTPs) are reversibly oxidized upon activation of platelet-derived growth factor receptor beta (PDGFβR). Dys-regulation of the PDGFβR signaling pathway is associated with several diseases, including cancers and cardiovascular disease, and is thus a known driver of disease progression. Ligand dependent PDGFβR phosphorylation stimulates cell proliferation and migration. The aim of this thesis was to elucidate redox-regulatory mechanisms of protein tyrosine phosphatases impacting on PDGFβR signaling. In Paper I, we analysed effects of mitochondria-derived ROS on PTP oxidation in models of hypoxia and hypoxia/re-oxygenation (H/R) in vitro and in vivo. We found an increase in PTP oxidation of multiple PTPs, including SHP-2, PTP1B and DEP-1, after exposure of NIH3T3 fibroblasts to H/R. An increase in total PTP oxidation and SHP-2 was also seen in rat cardiomyoblasts after H/R. Furthermore, H/R induced a delay of PDGFR dephosphorylation and also an antioxidant sensitive activation of downstream effectors ERK1/2. In addition, H/R enhanced PDGF-dependent cytoskeletal re-arrangements, which could be abolished by antioxidant treatment. Finally, we found an increase in total PTP oxidation and SHP2 oxidation in tissue extracts from an ex-vivo model of rat heart ischemia-reperfusion. In paper II, we studied expression and activity of PDGFβR pathway components in human pulmonary artery smooth muscle cells (hPASMC) subjected to hypoxia. We show that hypoxia- induced HIF-1α in hPASMC, both in vivo and in vitro, negatively regulate expression of PDGFβR associated PTPs, including PTP1B, DEP-1, TC-PTP and SHP2. The negatively regulation of these PDGFβR-associated PTPs occurred together with an enhanced PDGF receptor activation and an increase in both proliferation and migration of hPASMC. In paper III, we found that p66Shc dependent mitochondrial derived ROS contribute to inactivation of the PDGFβR associated PTPs PTP1B and SHP-2 upon ligand stimulation. In addition, deletion of p66Shc reduced downstream intracellular signaling after PDGF-BB stimulation. Furthermore, p66Shc KO cells displayed a decrease in migratory response to PDGF-BB treatment. In the final study paper IV, we studied the reactivation of oxidized PTPs and its impact on PDGFβR signaling. We showed that cells lacking expression of thioredoxin reductase 1 (TrxR1) displayed an increase in oxidation of PTP1B but not of SHP-2. Furthermore, in vivo oxidized PTP1B was re-activated by addition of Trx system components to cell lysates, whereas SHP-2 was not re-activated. Oxidized recombinant PTP1B was also re-activated by treatment with Trx system components while SHP-2 remained largely unaffected. Intriguingly, the Trx related protein TRP14 also reactivated PTP1B but not SHP-2. Furthermore, PDGFβR phosphorylation and signaling was enhanced in Txnrd1-/- fibroblasts leading to an enhanced proliferative response after PDGF-BB stimulation.
|
|
| 3. |
- Weibrecht, Irene, et al.
(författare)
-
Oxidation sensitivity of the catalytic cysteine of the protein-tyrosine phosphatases SHP-1 and SHP-2
- 2007
-
Ingår i: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849 .- 1873-4596. ; 43:1, s. 100-110
-
Tidskriftsartikel (refereegranskat)abstract
- Reversible oxidation of the catalytic cysteine of protein-tyrosine phosphatases (PTPs) has emerged as a putative mechanism of activity regulation by physiological cell stimulation with growth factors, and by cell treatments with adverse agents such as UV irradiation. We compared SHP-1 and SHP-2, two structurally related cytoplasmic protein-tyrosine phosphatases with different cellular functions and cell-specific expression patterns, for their intrinsic susceptibility to oxidation by H(2)O(2). The extent of oxidation was monitored by detecting the modification of the PTP catalytic cysteine by three different methods, including a modified in-gel PTP assay, alkylation with a biotinylated iodoacetic acid derivative, and an antibody against oxidized PTPs. Dose-response curves for oxidation of the catalytic domains of SHP-1 and SHP-2 were similar. SHP-1 and -2 require relatively high H(2)O(2) concentrations for oxidation (half-maximal oxidation at 0.1-0.5 mM). For SHP-1, the SH2 domains had a significant protective function with respect to oxidation. In EOL-1 cells, SHP oxidation by exogenous H(2)O(2) in general and SHP-2 oxidation in particular was strongly diminished compared to HEK293 cells, at least partially related to a generally lower oxidant sensitivity of the EOL-1 cells. The data suggest that the differential cell functions of SHP-1 and SHP-2 are not related to differences in oxidation sensitivity. The modulating effects of SH2 domains for oxidation of these PTPs are in support of an enhanced oxidation susceptibility of activated SHPs.
|
|
