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Phosphorylation of RasGRP1 by Shc3 prevents RasGRP1 degradation and contributes to Ras/c-Jun activation in hepatocellular carcinoma

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Abstract

Ras guanine nucleotide-releasing protein 1 (RasGRP1), a Ras activator, is upregulated in hepatocellular carcinoma (HCC) and other kinds of cancer and is associated with the poor prognosis of patients. However, little is known about the underlying regulatory mechanisms of RasGRP1 in the context of cancer. Here, we report that RasGRP1 physically interacted with the adaptor protein Src homolog and collagen homolog 3 (Shc3). Moreover, RasGRP1 C-terminus domain (aa 607–797) bound to the central collagen-homology 1 (CH1) domain of Shc3. Subsequently, Shc3 enhanced the RasGRP1 tyrosine phosphorylation rate and stability by inhibiting its ubiquitination. Notably, the phosphorylation-mimicking mutants of RasGRP1, RasGRP1 Y704A, and Y748A, rescued the phosphorylation and ubiquitination levels of RasGRP1 in HCC cells. Further investigation showed that the RasGRP1 and Shc3 interaction induced activation of Ras and c-Jun, resulting in cell proliferation in vitro. Moreover, the regulation of Shc3/RasGRP1/Ras/c-Jun signal transduction was confirmed in vivo using the subcutaneous xenograft mouse model. Thus, we propose that continuous Shc3 overexpression may be a possible mechanism for maintaining RasGRP1 stability and that persistent activation of Ras/c-Jun signaling through the interaction of RasGRP1 and Shc3 is a key event increasing cell proliferation. Our findings suggest that the interaction of RasGRP1 and Shc3 plays an important role in HCC tumorigenesis and suggests the potential clinical usage of novel biomarkers and therapeutic targets in HCC.

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Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant No. NSFC81800165), and Tianjin First Central Hospital Science and Technology Fund (Grant No. 2019CM02).

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Authors and Affiliations

  1. Department of Pharmacy, Tianjin First Central Hospital, School of Medicine, Nankai University, Tianjin, 300192, China

    Xinran Zhang, Yuanyuan Guo, Meiling Yan, Ying Xiao, Yunzhuo Dong, Ruixia Zhang, Yinpeng Qin, Yishan Bu, Yi Zhang & Huier Gao

  2. Key Laboratory of Cancer Prevention and Therapy, Department of Pediatric Oncology, National Clinical Research Center for Cancer, Tianjin’s Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin Medical University, Tianjin, 300060, China

    Yun Liu

  3. Department of Critical Care Medicine, Tianjin First Central Hospital, Tianjin Institute of Emergency Medicine, Tianjin, 300192, China

    Rui Yang

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Contributions

XZ, YL, and HG: conceived and designed the project. XZ and YL: carried out the molecular and cellular biology experiment with assistance from RY, YG, MY, YX, YD, RZ, and YQ. YB and YZ: statistical analysis and artworks were performed with assistance from XZ. XZ and HG: the manuscript was written.

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11010_2023_4839_MOESM1_ESM.tif

Supplementary file1 A Western blot of RasGRP1 in Shc3-knockdown HCCLM3 and HepG2 cells. B The relative mRNA expression levels of RasGRP1 in Shc3-knockdown HCCLM3 and HepG2 cells. Data were normalized to scramble. Data are from three independent experiments. N.S., not significant, error bars denote mean±SD (TIF 2608 KB)

11010_2023_4839_MOESM2_ESM.tif

Supplementary file2 A The relative mRNA expression levels of Shc3 in Shc3-overexpressing Huh7 and HepG2 cells. B The relative mRNA expression levels of Shc3 in Shc3-knockdwon HCCLM3 and HepG2 cells. C The relative mRNA expression levels of RasGRP1 and Shc3 in Shc3-overexpressing Huh7 cells transfected with RasGRP1 siRNAs. D Western blot of Shc3 in Shc3-knockdown HepG2 cells. Data are from three independent experiment (TIF 7463 KB)

11010_2023_4839_MOESM3_ESM.tif

Supplementary file3 A Western blots of PCNA and β-actin (upper), and the relative intensity of PCNA (bottom) in Shc3-overexpressing Huh7 and HepG2 cells. B Western blots of PCNA and β-actin (upper), and the relative intensity of PCNA (bottom) in Shc3-knockdown HCCLM3 and HepG2 cells. Data are from three independent experiments. *P < 0.05, **P < 0.01, error bars denote mean±SD (TIF 9505 KB)

11010_2023_4839_MOESM4_ESM.tif

Supplementary file4 Tumor volumes of xenograft models in Shc3-overexpression group and pCDH group (n = 5). *P < 0.05, error bars denote mean±SD (TIF 1228 KB)

Supplementary file5 (DOC 50 KB)

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Zhang, X., Liu, Y., Yang, R. et al. Phosphorylation of RasGRP1 by Shc3 prevents RasGRP1 degradation and contributes to Ras/c-Jun activation in hepatocellular carcinoma. Mol Cell Biochem (2023). https://doi.org/10.1007/s11010-023-04839-4

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