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. 2014 Feb 14;289(7):4009-17.
doi: 10.1074/jbc.M113.520106. Epub 2013 Dec 20.

The CUL7/F-box and WD Repeat Domain Containing 8 (CUL7/Fbxw8) Ubiquitin Ligase Promotes Degradation of Hematopoietic Progenitor Kinase 1

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Free PMC article

The CUL7/F-box and WD Repeat Domain Containing 8 (CUL7/Fbxw8) Ubiquitin Ligase Promotes Degradation of Hematopoietic Progenitor Kinase 1

Hua Wang et al. J Biol Chem. .
Free PMC article

Abstract

HPK1, a member of mammalian Ste20-like serine/threonine kinases, is lost in >95% pancreatic cancer through proteasome-mediated degradation. However, the mechanism of HPK1 loss has not been defined. The aims of this study are to identify the ubiquitin ligase and to examine the mechanisms that targets HPK1 degradation. We found that the CUL7/Fbxw8 ubiquitin ligase targeted HPK1 for degradation via the 26 S proteasome. The ubiquitination of HPK1 required its kinase activity and autophosphorylation. Wild-type protein phosphatase 4 (PP4), but not the phosphatase-dead PP4 mutant, PP4-RL, inhibits the interaction of Fbxw8 with HPK1 and Fbxw8-mediated ubiquitination of HPK1. In addition, we showed that Thr-355 of HPK1 is a key PP4 dephosphorylation site, through which CUL7/Fbxw8 ubiquitin ligase and PP4 regulates HPK1 stability. Knockdown of Fbxw8 restores endogenous HPK1 protein expression and inhibits cell proliferation of pancreatic cancer cells. Our study demonstrated that targeted degradation of HPK1 by the CUL7/Fbxw8 ubiquitin ligase constitutes a negative-feedback loop to restrain the activity of HPK1 and that CUL7/Fbxw8 ubiquitin ligase promotes pancreatic cancer cell proliferation. CUL7/Fbxw8 ubiquitin ligase-mediated HPK1 degradation revealed a direct link and novel role of CUL7/Fbxw8 ubiquitin ligase in the MAPK pathway, which plays a critical role in cell proliferation and differentiation.

Keywords: CUL7 Ubiquitin Ligase; E3 Ubiquitin Ligase; Fbxw8; HPK1; MAP Kinases (MAPKs); PP4; Pancreatic Cancer; Proteasome; Protein Phosphatase; Serine Threonine Protein Kinase.

Figures

FIGURE 1.
FIGURE 1.
CUL7/Fbxw8 ubiquitin ligase mediates HPK1 degradation in pancreatic cancer. A and B, analysis of HPK1 protein interactions in Panc-1 cells by antibody array or immunoprecipitation. Panc-1/Flag-HPK1 stable cells (C1) were treated with 2 μm MG132 for 16 h, and then harvested. Cell extracts were used for antibody array analysis (A) or immunoprecipitation with M2 antibody followed by Western blotting using antibodies against HPK1, Skp1, CUL7, CUL1, or Fbxw8 (B). Panc-1 parental cells, which had no detectable HPK1 protein, were used as controls. C, 293T cells were transfected with plasmids expressing Myc-Fbxw8 and Flag-HPK1; 36 h after transfection, the cells were harvested for immunoprecipitation with anti-Myc or anti-Flag antibodies followed by Western blotting as indicated. D, endogenous HPK1 interacts with Fbxw8 in Jurkat T cells. HPK1 from Jurkat T cells was immunoprecipitated with an anti-HPK1 antibody. The immunoprecipitate was then immunoblotted with an anti-Fbxw8 antibody. E, G, and H, overexpression of Fbxw8 results in a dose-dependent decrease of wild-type HPK1 but not the kinase-dead HPK1-M46 and HGK in 293T cells. F, knockdown of Fbxw8 in 293T cells stabilizes HPK1 protein. I, Fbxw8 overexpression decreases the half-life of HPK1 protein in 293T cells.
FIGURE 2.
FIGURE 2.
Cotransfection of Fbxw8 results in the polyubiquitination of HPK1 protein mainly through K48 of ubiquitin. A, wild-type HPK1 and HA-Ub were cotransfected with either Myc-Fbxw8 or Flag-Fbxw7. Thirty-six hours after transfection, cells were harvested for immunoprecipitation with anti-HPK1 antibody followed by Western blotting as indicated. The inputs for the immunoprecipitation are shown at the bottom. B, Flag-HPK1 and Myc-Fbxw8 were cotransfected with wild-type His.Myc-Ub, His.Myc-Ub K48R, or His.Myc-Ub K63R into 293T cells. Nickel-nitrilotriacetic acid agarose beads were used to pull down the ubiquitinated HPK1 protein.
FIGURE 3.
FIGURE 3.
CUL7/Fbxw8 ubiquitin ligase-mediated degradation and ubiquitination of HPK1 is dependent on HPK1 kinase activity and the autophosphorylation of HPK1. A, kinase activities and autophosphorylation of wild-type HPK1 and HPK1 mutant proteins were measured by immunocomplex kinase assays with the myelin basic protein (MBP) used as a substrate. B–E, overexpression of Fbxw8 results in a dose-dependent decrease in HPK1-S159A and HPK1-T165A protein expression but has no effect on the stability of HPK1 S171A or HPK1 T175A. F, loss of HPK1 kinase activity and autophosphorylation in HPK1-M46 and HPK1-T171A results in a marked decrease in Fbxw8-mediated HPK1 ubiquitination compared with wild-type HPK1. Wild-type or mutant HPK1 and HA-Ub were cotransfected with Myc-Fbxw8. The cells were treated with 2.0 μm MG132 for 6 h before harvesting. Immunoprecipitation with anti-HPK1 antibody followed by Western blot as indicated.
FIGURE 4.
FIGURE 4.
PP4, but not the phosphatase-dead mutant PP4-RL, inhibits Fbxw8-mediated HPK1 ubiquitination and the interaction between Fbxw8 and HPK1. HEK293T cells were transfected with Flag-HPK1, HA-ubiquitin, Myc-Fbxw8, Flag-PP4, or HA-PP4-RL as shown, and cell lysates were prepared 42 h after transfection. HPK1 was immunoprecipitated with an anti-HPK1 antibody. The immunoprecipitates were then immunoblotted with an anti-HA antibody. The immunoprecipitated HPK1, Fbxw8, PP4, and PP4-RL were monitored by immunoblotting using an anti-HPK1, anti-Myc, anti-Flag, or anti-HA antibody, respectively. The inputs for the immunoprecipitation were shown at the bottom.
FIGURE 5.
FIGURE 5.
Threonine 355 of HPK1 is a key residue for PP4 dephosphorylation/stabilization of HPK1 and for CUL7/Fbxw8 ubiquitin ligase-mediated HPK1 degradation. A, schematic drawing showing the possible serine or threonine phosphorylation sites in HPK1 proline-rich region (amino acids 288–482) using PhosphoSitePlus program. B, kinase activities and autophosphorylation of wild-type HPK1, HPK1-T349A and HPK1-T355A were measured by immunocomplex kinase assays with the myelin basic protein (MBP) used as a substrate. C–F, proteasome inhibitor MG132 stabilizes wild-type HPK1, HPK1-S159A, and HPK1-T349A in a dose-dependent manner, but not HPK1-T355A. G, overexpression of Fbxw8 has little effect on the stability of HPK1-T355A. H and I, overexpression of PP4 results in a dose-dependent increase in wild-type HPK1 protein expression, but has little effect on the stability of HPK1-T355A.
FIGURE 6.
FIGURE 6.
PP4 dephosphorylation site is important for CUL7/Fbxw8 ubiquitin ligase-mediated HPK1 ubiquitination. A, okadaic acid (OA) increases threonine phosphorylation of HPK1-PR.HEK293T cells were transfected with wildtype HPK1 and HA-HPK1-PR. Forty-two hours after transfection, the cells were either treated with 50 nm OA or untreated as a control for 4 h before harvest. HA-HPK1-PR were immunoprecipitated with an anti-HA antibody and then immunoblotted with anti-phosphothreonine or anti-HA antibody (top). The input of HPK1 and HA-HPK1-PR were monitored by immunoblotting (bottom). B, Fbxw8 does not increase ubiquitination of HPK1-T355A. HEK293T cells were transfected with HA-ubiquitin and either Flag-HPK1-T355 alone or with Myc-Fbxw8. Forty-two hours after transfection, cell lysates were prepared. HPK1-T355 was immunoprecipitated with an anti-M2 antibody and then immunoblotted with an anti-HA antibody as indicated.
FIGURE 7.
FIGURE 7.
Knockdown of Fbxw8 restores endogenous HPK1 protein expression and inhibits cell proliferation in pancreatic cancer cells. A and B, Fbxw8 knockdown stable cell lines were generated from Panc-1 or Panc-28 cells and screened by QRT-PCR. C and D, knockdown of Fbxw8 restores endogenous HPK1 protein expression in Panc-1 and Panc-28 cells. E and F, Panc-1 or Panc-28 parental, shRNA control, and Fbxw8 knockdown stable cells were plated at an equal density and cultured under normal conditions for the indicated time periods. Cell numbers were counted and plotted. The growth curves have been repeated three times.

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