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Kinase-independent role for CRAF-driving tumour radioresistance via CHK2.

Advani SJ, Camargo MF, Seguin L, Mielgo A, Anand S, Hicks AM, Aguilera J, Franovic A, Weis SM, Cheresh DA - Nat Commun (2015)

Bottom Line: Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance.CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response.Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Medicine and Applied Sciences at the UC San Diego Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

No MeSH data available.


Related in: MedlinePlus

CRAF pS338 regulates the formation of a complex between CRAF and the DNA repair kinase CHK2.(a) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy and immunoblotted with indicated antibodies. Data are representative of three independent experiments. (b) HCT-116 cells were exposed to 6 Gy. Lysates were immunoprecipitated for CHK2 pT68 and blotted for CRAF. For reciprocal pull down, lysates were immunoprecipitated and CRAF blotted for CHK2. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (c) HCT-116 cells were treated with KG5 and lysates were immunoprecipitated for CRAF or CHK2 and immunoblotted with indicated antibodies. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (d) Expression of CHK2 was silenced using siRNA in HCT-116 cells expressing wild-type (WT) CRAF or the phospho-mimetic CRAF S338D mutant. Cells were stained for γH2AX, and the number of γH2AX foci per cell was counted as a measure of DNA double strand breaks. Mean±s.e.m, *P=0.02 from two-sided t-test comparing S338D+si-CTRL versus S338D+si-CHK2 (n=3 fields each group). Data shown are representative of two different siRNAs for CHK2, for two independent experiments. (e) Schematic represents PAK1-mediated activation of CRAF pS338 in response to ionizing radiation and its recruitment of CHK2 leading to CHK2 activation that supports DNA repair and radioresistance.
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f4: CRAF pS338 regulates the formation of a complex between CRAF and the DNA repair kinase CHK2.(a) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy and immunoblotted with indicated antibodies. Data are representative of three independent experiments. (b) HCT-116 cells were exposed to 6 Gy. Lysates were immunoprecipitated for CHK2 pT68 and blotted for CRAF. For reciprocal pull down, lysates were immunoprecipitated and CRAF blotted for CHK2. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (c) HCT-116 cells were treated with KG5 and lysates were immunoprecipitated for CRAF or CHK2 and immunoblotted with indicated antibodies. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (d) Expression of CHK2 was silenced using siRNA in HCT-116 cells expressing wild-type (WT) CRAF or the phospho-mimetic CRAF S338D mutant. Cells were stained for γH2AX, and the number of γH2AX foci per cell was counted as a measure of DNA double strand breaks. Mean±s.e.m, *P=0.02 from two-sided t-test comparing S338D+si-CTRL versus S338D+si-CHK2 (n=3 fields each group). Data shown are representative of two different siRNAs for CHK2, for two independent experiments. (e) Schematic represents PAK1-mediated activation of CRAF pS338 in response to ionizing radiation and its recruitment of CHK2 leading to CHK2 activation that supports DNA repair and radioresistance.

Mentions: An orchestrated series of genes regulate the response to the DNA-damaging effects of radiation2627. To consider how CRAF pS338 might mediate radioprotection, RNA was extracted from U87 glioblastoma cells expressing WT CRAF or the K375M/S338D double mutant and analysed using a reverse transcription–PCR (RT–PCR) array involving 92 genes with known roles in response to DNA damage. Of the 92 genes examined, 6 showed a >2-fold increase in cells expressing the CRAF double mutant relative to WT. These include ATM, ATR, BRCA1, BRCA2, RAD17 and POLK, all of which were validated by RT–PCR (Supplementary Fig. 8). Since the checkpoint kinases CHK1/CHK2 are activated by ATR/ATM and their downstream activity is modulated by BRCA1/BRCA2 (refs 28, 29), we examined the effect of the K375M/S338D mutation on CHK1/CHK2 activity. While the radiation-induced increase in active CHK1 (pS345) was similar for cells expressing WT CRAF or the CRAF K375M/S338D double mutant, active CHK2 (pT68) was upregulated twofold in cells expressing the CRAF double mutant (Fig. 4a). These findings suggest that activation of CHK2, a well-known contributor to radioresistance30313233, may partly account for the radio-protective effect of CRAF S338 phosphorylation.


Kinase-independent role for CRAF-driving tumour radioresistance via CHK2.

Advani SJ, Camargo MF, Seguin L, Mielgo A, Anand S, Hicks AM, Aguilera J, Franovic A, Weis SM, Cheresh DA - Nat Commun (2015)

CRAF pS338 regulates the formation of a complex between CRAF and the DNA repair kinase CHK2.(a) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy and immunoblotted with indicated antibodies. Data are representative of three independent experiments. (b) HCT-116 cells were exposed to 6 Gy. Lysates were immunoprecipitated for CHK2 pT68 and blotted for CRAF. For reciprocal pull down, lysates were immunoprecipitated and CRAF blotted for CHK2. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (c) HCT-116 cells were treated with KG5 and lysates were immunoprecipitated for CRAF or CHK2 and immunoblotted with indicated antibodies. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (d) Expression of CHK2 was silenced using siRNA in HCT-116 cells expressing wild-type (WT) CRAF or the phospho-mimetic CRAF S338D mutant. Cells were stained for γH2AX, and the number of γH2AX foci per cell was counted as a measure of DNA double strand breaks. Mean±s.e.m, *P=0.02 from two-sided t-test comparing S338D+si-CTRL versus S338D+si-CHK2 (n=3 fields each group). Data shown are representative of two different siRNAs for CHK2, for two independent experiments. (e) Schematic represents PAK1-mediated activation of CRAF pS338 in response to ionizing radiation and its recruitment of CHK2 leading to CHK2 activation that supports DNA repair and radioresistance.
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Related In: Results  -  Collection

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f4: CRAF pS338 regulates the formation of a complex between CRAF and the DNA repair kinase CHK2.(a) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy and immunoblotted with indicated antibodies. Data are representative of three independent experiments. (b) HCT-116 cells were exposed to 6 Gy. Lysates were immunoprecipitated for CHK2 pT68 and blotted for CRAF. For reciprocal pull down, lysates were immunoprecipitated and CRAF blotted for CHK2. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (c) HCT-116 cells were treated with KG5 and lysates were immunoprecipitated for CRAF or CHK2 and immunoblotted with indicated antibodies. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (d) Expression of CHK2 was silenced using siRNA in HCT-116 cells expressing wild-type (WT) CRAF or the phospho-mimetic CRAF S338D mutant. Cells were stained for γH2AX, and the number of γH2AX foci per cell was counted as a measure of DNA double strand breaks. Mean±s.e.m, *P=0.02 from two-sided t-test comparing S338D+si-CTRL versus S338D+si-CHK2 (n=3 fields each group). Data shown are representative of two different siRNAs for CHK2, for two independent experiments. (e) Schematic represents PAK1-mediated activation of CRAF pS338 in response to ionizing radiation and its recruitment of CHK2 leading to CHK2 activation that supports DNA repair and radioresistance.
Mentions: An orchestrated series of genes regulate the response to the DNA-damaging effects of radiation2627. To consider how CRAF pS338 might mediate radioprotection, RNA was extracted from U87 glioblastoma cells expressing WT CRAF or the K375M/S338D double mutant and analysed using a reverse transcription–PCR (RT–PCR) array involving 92 genes with known roles in response to DNA damage. Of the 92 genes examined, 6 showed a >2-fold increase in cells expressing the CRAF double mutant relative to WT. These include ATM, ATR, BRCA1, BRCA2, RAD17 and POLK, all of which were validated by RT–PCR (Supplementary Fig. 8). Since the checkpoint kinases CHK1/CHK2 are activated by ATR/ATM and their downstream activity is modulated by BRCA1/BRCA2 (refs 28, 29), we examined the effect of the K375M/S338D mutation on CHK1/CHK2 activity. While the radiation-induced increase in active CHK1 (pS345) was similar for cells expressing WT CRAF or the CRAF K375M/S338D double mutant, active CHK2 (pT68) was upregulated twofold in cells expressing the CRAF double mutant (Fig. 4a). These findings suggest that activation of CHK2, a well-known contributor to radioresistance30313233, may partly account for the radio-protective effect of CRAF S338 phosphorylation.

Bottom Line: Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance.CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response.Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Medicine and Applied Sciences at the UC San Diego Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

No MeSH data available.


Related in: MedlinePlus