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SCF(β-TRCP) suppresses angiogenesis and thyroid cancer cell migration by promoting ubiquitination and destruction of VEGF receptor 2.

Shaik S, Nucera C, Inuzuka H, Gao D, Garnaas M, Frechette G, Harris L, Wan L, Fukushima H, Husain A, Nose V, Fadda G, Sadow PM, Goessling W, North T, Lawler J, Wei W - J. Exp. Med. (2012)

Bottom Line: Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC.We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells.These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology and Angiogenesis, Department of Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
The incidence of human papillary thyroid cancer (PTC) is increasing and an aggressive subtype of this disease is resistant to treatment with vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. VEGFR2 promotes angiogenesis by triggering endothelial cell proliferation and migration. However, the molecular mechanisms governing VEGFR2 stability in vivo remain unknown. Additionally, whether VEGFR2 influences PTC cell migration is not clear. We show that the ubiquitin E3 ligase SCF(β-TRCP) promotes ubiquitination and destruction of VEGFR2 in a casein kinase I (CKI)-dependent manner. β-TRCP knockdown or CKI inhibition causes accumulation of VEGFR2, resulting in increased activity of signaling pathways downstream of VEGFR2. β-TRCP-depleted endothelial cells exhibit enhanced migration and angiogenesis in vitro. Furthermore, β-TRCP knockdown increased angiogenesis and vessel branching in zebrafish. Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC. We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells. These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

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Phosphorylation of VEGFR2 by CKI at multiple sites triggers its ubiquitination and degradation by SCFβ-TRCP. (A) Illustration of VEGFR2 protein and various VEGFR2 mutants generated for this study. There are three DSG degrons present in the C-terminal tail of VEGFR2 that can be identified by β-TRCP1. AA represents two alanines substituted from two serines in DSG1, DSG2, and DSG3 degrons (Fig. S1, A–C). AAA represents three alanines substituted from three threonines in the DDTD degron (Fig. S1 D). (B) Immunoblot analysis of 293T cells transfected with the indicated HA-VEGFR2 and Flag–β-TRCP1 plasmids in the presence or absence of Myc-CKIδ. Where indicated, cells were treated with the proteasome inhibitor MG132. Data shown is representative of two independent experiments. (C) Purified CKIδ protein was incubated with 5 µg of the indicated glutathione S-transferase (GST)–VEGFR2 fusion proteins in the presence of γ-[32P]ATP. The protein kinase reaction products were resolved by SDS-PAGE, and phosphorylation was detected by autoradiography. Top: autoradiogram of phosphorylated GST-VEGFR2; bottom panel: staining of GST-VEGFR2 to demonstrate equal loading. Data shown is representative of two independent experiments. Black lines indicate that intervening lanes were spliced out. (D) Autoradiograms show a recovery of 35S-labeled β-TRCP1 protein bound to the indicated GST-VEGFR2 fusion proteins (GST protein as a negative control) incubated with CKIδ before pulldown assays. Top: autoradiogram of β-TRCP1 bound with GST-VEGFR2; bottom: staining of GST-VEGFR2 to demonstrate equal loading. Data shown is representative of two independent experiments. Black lines indicate that intervening lanes were spliced out. (E) Immunoblot analysis of 293T cells transfected with the indicated HA-VEGFR2 and Flag–β-TRCP1 plasmids in the presence or absence of Myc-CKIδ. Data shown is representative of two independent experiments. (F) Immunoblot analysis of WCL from 293 cells transfected with indicated constructs. Where indicated, cells were treated with 100 ng/ml VEGF-A for 2 h before harvesting. Data shown is representative of two independent experiments. (G) Affinity-purified SCFβ-TRCP complexes were incubated with purified recombinant GST-VEGFR2 proteins, purified E1 and E2, and ubiquitin as indicated at 30°C for 45 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the anti-VEGFR2 antibody. Data shown is representative of two independent experiments.
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fig3: Phosphorylation of VEGFR2 by CKI at multiple sites triggers its ubiquitination and degradation by SCFβ-TRCP. (A) Illustration of VEGFR2 protein and various VEGFR2 mutants generated for this study. There are three DSG degrons present in the C-terminal tail of VEGFR2 that can be identified by β-TRCP1. AA represents two alanines substituted from two serines in DSG1, DSG2, and DSG3 degrons (Fig. S1, A–C). AAA represents three alanines substituted from three threonines in the DDTD degron (Fig. S1 D). (B) Immunoblot analysis of 293T cells transfected with the indicated HA-VEGFR2 and Flag–β-TRCP1 plasmids in the presence or absence of Myc-CKIδ. Where indicated, cells were treated with the proteasome inhibitor MG132. Data shown is representative of two independent experiments. (C) Purified CKIδ protein was incubated with 5 µg of the indicated glutathione S-transferase (GST)–VEGFR2 fusion proteins in the presence of γ-[32P]ATP. The protein kinase reaction products were resolved by SDS-PAGE, and phosphorylation was detected by autoradiography. Top: autoradiogram of phosphorylated GST-VEGFR2; bottom panel: staining of GST-VEGFR2 to demonstrate equal loading. Data shown is representative of two independent experiments. Black lines indicate that intervening lanes were spliced out. (D) Autoradiograms show a recovery of 35S-labeled β-TRCP1 protein bound to the indicated GST-VEGFR2 fusion proteins (GST protein as a negative control) incubated with CKIδ before pulldown assays. Top: autoradiogram of β-TRCP1 bound with GST-VEGFR2; bottom: staining of GST-VEGFR2 to demonstrate equal loading. Data shown is representative of two independent experiments. Black lines indicate that intervening lanes were spliced out. (E) Immunoblot analysis of 293T cells transfected with the indicated HA-VEGFR2 and Flag–β-TRCP1 plasmids in the presence or absence of Myc-CKIδ. Data shown is representative of two independent experiments. (F) Immunoblot analysis of WCL from 293 cells transfected with indicated constructs. Where indicated, cells were treated with 100 ng/ml VEGF-A for 2 h before harvesting. Data shown is representative of two independent experiments. (G) Affinity-purified SCFβ-TRCP complexes were incubated with purified recombinant GST-VEGFR2 proteins, purified E1 and E2, and ubiquitin as indicated at 30°C for 45 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the anti-VEGFR2 antibody. Data shown is representative of two independent experiments.

Mentions: Multi-subunit Cullin–Ring complexes comprise the largest known class of E3 ubiquitin ligases (Petroski and Deshaies, 2005). Cullins directly interact with Roc1, a Ring finger protein, and the Cullin–Roc1 complex comprises the core module of a series of E3 ubiquitin ligases. Thus, we started our investigation by examining whether a specific Cullin–Ring complex interacts with VEGFR2. We found that Cullin 1, but not other members of the Cullin family that we examined, specifically interacts with VEGFR2 (Fig. 1, A and B). This suggests that the SCF complex, which contains Cullin 1, might be involved in the regulation of VEGFR2 stability. In keeping with this notion, depletion of endogenous Cullin 1, but not Cullin 4A, resulted in the up-regulation of VEGFR2 expression in human microvascular endothelial cells (HMVECs; Fig. 1 C). Next, we sought to explore which F-box protein, when complexed with Cullin 1, is responsible for VEGFR2 degradation. β-TRCP, one of the well characterized F-box proteins, binds to its substrates by recognizing a specific DSG(XX)S phosphodegron motif, within which the two serine residues are phosphorylated (Frescas and Pagano, 2008). We noticed that the cytoplasmic tail of VEGFR2 contains three DSG(XX)S motifs that could potentially be recognized by β-TRCP and are conserved among different species (see Fig. 3 A). This prompted us to examine whether β-TRCP interacts with VEGFR2 in vitro. Using coimmunoprecipitation, we found that both β-TRCP1 and β-TRCP2 interact with VEGFR2 (Fig. 1 D). The interaction between VEGFR2 and β-TRCP1 was abolished when the C-terminal WD40 repeat motif of β-TRCP1, which has been shown to mediate the interaction with most of its substrates (Wu et al., 2003), was mutated (Fig.1 D). Furthermore, we detected an interaction between endogenous VEGFR2 and endogenous β-TRCP1 in HMVECs (Fig. 1 E) and further demonstrated that phosphatase treatment abolished the interaction between VEGFR2 and β-TRCP1 (Fig. 1 F). In support of an important role for β-TRCP in regulating VEGFR2 abundance, depletion of either β-TRCP1 or β-TRCP2 led to up-regulation of VEGFR2 levels (Fig. 1 G). More importantly, depletion of β-TRCP did not affect significantly VEGFR2 mRNA levels (see Fig. 4 D), suggesting that the observed increase in VEGFR2 abundance was mainly through posttranscriptional mechanisms. In support of this notion, using cycloheximide, we demonstrated that the half-life of VEGFR2 was markedly extended after depletion of β-TRCP1 (Fig. 1, H and I).


SCF(β-TRCP) suppresses angiogenesis and thyroid cancer cell migration by promoting ubiquitination and destruction of VEGF receptor 2.

Shaik S, Nucera C, Inuzuka H, Gao D, Garnaas M, Frechette G, Harris L, Wan L, Fukushima H, Husain A, Nose V, Fadda G, Sadow PM, Goessling W, North T, Lawler J, Wei W - J. Exp. Med. (2012)

Phosphorylation of VEGFR2 by CKI at multiple sites triggers its ubiquitination and degradation by SCFβ-TRCP. (A) Illustration of VEGFR2 protein and various VEGFR2 mutants generated for this study. There are three DSG degrons present in the C-terminal tail of VEGFR2 that can be identified by β-TRCP1. AA represents two alanines substituted from two serines in DSG1, DSG2, and DSG3 degrons (Fig. S1, A–C). AAA represents three alanines substituted from three threonines in the DDTD degron (Fig. S1 D). (B) Immunoblot analysis of 293T cells transfected with the indicated HA-VEGFR2 and Flag–β-TRCP1 plasmids in the presence or absence of Myc-CKIδ. Where indicated, cells were treated with the proteasome inhibitor MG132. Data shown is representative of two independent experiments. (C) Purified CKIδ protein was incubated with 5 µg of the indicated glutathione S-transferase (GST)–VEGFR2 fusion proteins in the presence of γ-[32P]ATP. The protein kinase reaction products were resolved by SDS-PAGE, and phosphorylation was detected by autoradiography. Top: autoradiogram of phosphorylated GST-VEGFR2; bottom panel: staining of GST-VEGFR2 to demonstrate equal loading. Data shown is representative of two independent experiments. Black lines indicate that intervening lanes were spliced out. (D) Autoradiograms show a recovery of 35S-labeled β-TRCP1 protein bound to the indicated GST-VEGFR2 fusion proteins (GST protein as a negative control) incubated with CKIδ before pulldown assays. Top: autoradiogram of β-TRCP1 bound with GST-VEGFR2; bottom: staining of GST-VEGFR2 to demonstrate equal loading. Data shown is representative of two independent experiments. Black lines indicate that intervening lanes were spliced out. (E) Immunoblot analysis of 293T cells transfected with the indicated HA-VEGFR2 and Flag–β-TRCP1 plasmids in the presence or absence of Myc-CKIδ. Data shown is representative of two independent experiments. (F) Immunoblot analysis of WCL from 293 cells transfected with indicated constructs. Where indicated, cells were treated with 100 ng/ml VEGF-A for 2 h before harvesting. Data shown is representative of two independent experiments. (G) Affinity-purified SCFβ-TRCP complexes were incubated with purified recombinant GST-VEGFR2 proteins, purified E1 and E2, and ubiquitin as indicated at 30°C for 45 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the anti-VEGFR2 antibody. Data shown is representative of two independent experiments.
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Show All Figures
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fig3: Phosphorylation of VEGFR2 by CKI at multiple sites triggers its ubiquitination and degradation by SCFβ-TRCP. (A) Illustration of VEGFR2 protein and various VEGFR2 mutants generated for this study. There are three DSG degrons present in the C-terminal tail of VEGFR2 that can be identified by β-TRCP1. AA represents two alanines substituted from two serines in DSG1, DSG2, and DSG3 degrons (Fig. S1, A–C). AAA represents three alanines substituted from three threonines in the DDTD degron (Fig. S1 D). (B) Immunoblot analysis of 293T cells transfected with the indicated HA-VEGFR2 and Flag–β-TRCP1 plasmids in the presence or absence of Myc-CKIδ. Where indicated, cells were treated with the proteasome inhibitor MG132. Data shown is representative of two independent experiments. (C) Purified CKIδ protein was incubated with 5 µg of the indicated glutathione S-transferase (GST)–VEGFR2 fusion proteins in the presence of γ-[32P]ATP. The protein kinase reaction products were resolved by SDS-PAGE, and phosphorylation was detected by autoradiography. Top: autoradiogram of phosphorylated GST-VEGFR2; bottom panel: staining of GST-VEGFR2 to demonstrate equal loading. Data shown is representative of two independent experiments. Black lines indicate that intervening lanes were spliced out. (D) Autoradiograms show a recovery of 35S-labeled β-TRCP1 protein bound to the indicated GST-VEGFR2 fusion proteins (GST protein as a negative control) incubated with CKIδ before pulldown assays. Top: autoradiogram of β-TRCP1 bound with GST-VEGFR2; bottom: staining of GST-VEGFR2 to demonstrate equal loading. Data shown is representative of two independent experiments. Black lines indicate that intervening lanes were spliced out. (E) Immunoblot analysis of 293T cells transfected with the indicated HA-VEGFR2 and Flag–β-TRCP1 plasmids in the presence or absence of Myc-CKIδ. Data shown is representative of two independent experiments. (F) Immunoblot analysis of WCL from 293 cells transfected with indicated constructs. Where indicated, cells were treated with 100 ng/ml VEGF-A for 2 h before harvesting. Data shown is representative of two independent experiments. (G) Affinity-purified SCFβ-TRCP complexes were incubated with purified recombinant GST-VEGFR2 proteins, purified E1 and E2, and ubiquitin as indicated at 30°C for 45 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the anti-VEGFR2 antibody. Data shown is representative of two independent experiments.
Mentions: Multi-subunit Cullin–Ring complexes comprise the largest known class of E3 ubiquitin ligases (Petroski and Deshaies, 2005). Cullins directly interact with Roc1, a Ring finger protein, and the Cullin–Roc1 complex comprises the core module of a series of E3 ubiquitin ligases. Thus, we started our investigation by examining whether a specific Cullin–Ring complex interacts with VEGFR2. We found that Cullin 1, but not other members of the Cullin family that we examined, specifically interacts with VEGFR2 (Fig. 1, A and B). This suggests that the SCF complex, which contains Cullin 1, might be involved in the regulation of VEGFR2 stability. In keeping with this notion, depletion of endogenous Cullin 1, but not Cullin 4A, resulted in the up-regulation of VEGFR2 expression in human microvascular endothelial cells (HMVECs; Fig. 1 C). Next, we sought to explore which F-box protein, when complexed with Cullin 1, is responsible for VEGFR2 degradation. β-TRCP, one of the well characterized F-box proteins, binds to its substrates by recognizing a specific DSG(XX)S phosphodegron motif, within which the two serine residues are phosphorylated (Frescas and Pagano, 2008). We noticed that the cytoplasmic tail of VEGFR2 contains three DSG(XX)S motifs that could potentially be recognized by β-TRCP and are conserved among different species (see Fig. 3 A). This prompted us to examine whether β-TRCP interacts with VEGFR2 in vitro. Using coimmunoprecipitation, we found that both β-TRCP1 and β-TRCP2 interact with VEGFR2 (Fig. 1 D). The interaction between VEGFR2 and β-TRCP1 was abolished when the C-terminal WD40 repeat motif of β-TRCP1, which has been shown to mediate the interaction with most of its substrates (Wu et al., 2003), was mutated (Fig.1 D). Furthermore, we detected an interaction between endogenous VEGFR2 and endogenous β-TRCP1 in HMVECs (Fig. 1 E) and further demonstrated that phosphatase treatment abolished the interaction between VEGFR2 and β-TRCP1 (Fig. 1 F). In support of an important role for β-TRCP in regulating VEGFR2 abundance, depletion of either β-TRCP1 or β-TRCP2 led to up-regulation of VEGFR2 levels (Fig. 1 G). More importantly, depletion of β-TRCP did not affect significantly VEGFR2 mRNA levels (see Fig. 4 D), suggesting that the observed increase in VEGFR2 abundance was mainly through posttranscriptional mechanisms. In support of this notion, using cycloheximide, we demonstrated that the half-life of VEGFR2 was markedly extended after depletion of β-TRCP1 (Fig. 1, H and I).

Bottom Line: Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC.We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells.These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology and Angiogenesis, Department of Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
The incidence of human papillary thyroid cancer (PTC) is increasing and an aggressive subtype of this disease is resistant to treatment with vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. VEGFR2 promotes angiogenesis by triggering endothelial cell proliferation and migration. However, the molecular mechanisms governing VEGFR2 stability in vivo remain unknown. Additionally, whether VEGFR2 influences PTC cell migration is not clear. We show that the ubiquitin E3 ligase SCF(β-TRCP) promotes ubiquitination and destruction of VEGFR2 in a casein kinase I (CKI)-dependent manner. β-TRCP knockdown or CKI inhibition causes accumulation of VEGFR2, resulting in increased activity of signaling pathways downstream of VEGFR2. β-TRCP-depleted endothelial cells exhibit enhanced migration and angiogenesis in vitro. Furthermore, β-TRCP knockdown increased angiogenesis and vessel branching in zebrafish. Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC. We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells. These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

Show MeSH
Related in: MedlinePlus