4 results on '"Hu, Jiancheng"'
Search Results
2. The AMPK inhibitor overcomes the paradoxical effect of RAF inhibitors through blocking phospho-Ser-621 in the C terminus of CRAF.
- Author
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Yuan J, Ng WH, Yap J, Chia B, Huang X, Wang M, and Hu J
- Subjects
- 14-3-3 Proteins metabolism, Cell Line, Tumor, Dimerization, HEK293 Cells, Humans, Phosphorylation, Protein Binding, Proto-Oncogene Mas, Proto-Oncogene Proteins c-raf chemistry, Proto-Oncogene Proteins c-raf metabolism, Serine chemistry, Signal Transduction, Adenylate Kinase antagonists & inhibitors, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins c-raf antagonists & inhibitors, Serine metabolism
- Abstract
The dimerization-driven paradoxical activation of RAF proto-oncogene Ser/Thr kinase (RAF) is the predominant cause of drug resistance and toxicity in cancer therapies with RAF inhibitors. The scaffold protein 14-3-3, which binds to the RAF C terminus, is essential for RAF activation under physiological conditions, but the molecular basis is unclear. Here we investigated whether and how 14-3-3 regulates the dimerization-driven transactivation of the RAF isoform CRAF by RAF inhibitors and affects drug resistance and toxicity by virtue of the dominant role of CRAF in these processes. We demonstrated that 14-3-3 enhances the dimerization-driven transactivation of CRAF by stabilizing CRAF dimers. Further, we identified AMP-activated protein kinase (AMPK) and CRAF itself as two putative kinases that redundantly phosphorylate CRAF's C terminus and thereby control its association with 14-3-3. Next, we determined whether the combinatory inhibition of AMPK and CRAF could overcome the paradoxical effect of RAF inhibitors. We found that the AMPK inhibitor (AMPKi) not only blocked the RAF inhibitor-driven paradoxical activation of ERK signaling and cellular overgrowth in Ras-mutated cancer cells by blocking phosphorylation of Ser-621 in CRAF but also reduced the formation of drug-resistant clones of BRAF
V600E -mutated cancer cells. Last, we investigated whether 14-3-3 binding to the C terminus of CRAF is required for CRAF catalytic activity and observed that it was dispensable in vivo Altogether, our study unravels the molecular mechanism by which 14-3-3 regulates dimerization-driven RAF activation and identified AMPKi as a potential agent to counteract drug resistance and adverse effects of RAF inhibitors in cancer therapies., (© 2018 Yuan et al.)- Published
- 2018
- Full Text
- View/download PDF
3. Mutation that blocks ATP binding creates a pseudokinase stabilizing the scaffolding function of kinase suppressor of Ras, CRAF and BRAF.
- Author
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Hu J, Yu H, Kornev AP, Zhao J, Filbert EL, Taylor SS, and Shaw AS
- Subjects
- Enzyme Stability, Humans, Indenes pharmacology, Indoles pharmacology, Mutation, Protein Conformation drug effects, Protein Kinase Inhibitors pharmacology, Protein Kinases chemistry, Protein Kinases genetics, Protein Multimerization drug effects, Proto-Oncogene Proteins B-raf chemistry, Proto-Oncogene Proteins B-raf genetics, Proto-Oncogene Proteins c-raf chemistry, Proto-Oncogene Proteins c-raf genetics, Pyrazoles pharmacology, Sulfonamides pharmacology, ras Proteins genetics, Adenosine Triphosphate metabolism, Protein Kinases metabolism, Proto-Oncogene Proteins B-raf metabolism, Proto-Oncogene Proteins c-raf metabolism, ras Proteins metabolism
- Abstract
Because mutations in RAS and BRAF represent the most common mutations found in human tumors, identification of inhibitors has been a major goal. Surprisingly, new oncogenic BRAF specific inhibitors inhibit cells transformed with mutated BRAF but paradoxically stimulate the growth of cells transformed with RAS. Here, we show that the mechanism for activation is via drug-induced dimer formation between CRAF and kinase suppressor of Ras (KSR)1. To understand the function of KSR1, we generated a KSR1 mutant that cannot bind ATP but stabilizes the closed, active conformation of KSR1. Molecular modeling suggested that the mutant stabilizes the two hydrophobic spines critical for the closed active conformation. We, therefore, could use the mutant to discriminate between the scaffold versus kinase functions of KSR1. The KSR1 mutant bound constitutively to RAF and mitogen-activated protein kinase kinase (MEK) but could not reconstitute activity suggesting that the catalytic activity of KSR1 is required for its function. Analogous mutations in BRAF and CRAF allowed us to test the generality of the model. The mutation induced changes consistent with the active, closed conformation of both kinases and confirmed that BRAF functions distinctly from CRAF in the MAP kinase pathway. Not only does this work suggest that KSR1 may function as a kinase, we anticipate that the mutation that we generated may be broadly applicable to stabilize the closed conformation of other kinases many of which may also form dimers.
- Published
- 2011
- Full Text
- View/download PDF
4. Allosteric Activation of Functionally Asymmetric RAF Kinase Dimers
- Author
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Hu, Jiancheng, Stites, Edward C, Yu, Haiyang, Germino, Elizabeth A, Meharena, Hiruy S, Stork, Philip JS, Kornev, Alexandr P, Taylor, Susan S, and Shaw, Andrey S
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Cancer ,1.1 Normal biological development and functioning ,Underpinning research ,Allosteric Regulation ,Amino Acid Motifs ,Amino Acid Sequence ,Animals ,Cell Line ,Dimerization ,Enzyme Activation ,Humans ,Mice ,Models ,Molecular ,Molecular Sequence Data ,Mutation ,Phosphorylation ,Protein Conformation ,Protein Kinases ,Proto-Oncogene Proteins B-raf ,Proto-Oncogene Proteins c-raf ,Sequence Alignment ,Tryptophan ,raf Kinases ,Medical and Health Sciences ,Developmental Biology ,Biological sciences ,Biomedical and clinical sciences - Abstract
Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.
- Published
- 2013
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