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Activation of RAF1 (c-RAF) by the Marine Alkaloid Lasonolide A Induces Rapid Premature Chromosome Condensation.

Jossé R, Zhang YW, Giroux V, Ghosh AK, Luo J, Pommier Y - Mar Drugs (2015)

Bottom Line: We found that LSA induced RAF1 phosphorylation on Serine 338 within minutes in human colorectal carcinoma HCT-116, ovarian carcinoma OVCAR-8, and Burkitt's lymphoma CA46 cell lines.RAF1 depletion by siRNAs attenuated LSA-induced PCC in HCT-116 and OVCAR-8 cells.Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC.

View Article: PubMed Central - PubMed

Affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute (CCR-NCI), NIH, Bethesda, MD 20892-9760, USA. rozenn.josse@gmail.com.

ABSTRACT
Lasonolide A (LSA), a potent antitumor polyketide from the marine sponge, Forcepia sp., induces rapid and reversible protein hyperphosphorylation and premature chromosome condensation (PCC) at nanomolar concentrations independent of cyclin-dependent kinases. To identify cellular targets of LSA, we screened 2951 shRNAs targeting a pool of human kinases and phosphatases (1140 RefSeqs) to identify genes that modulate PCC in response to LSA. This led to the identification of RAF1 (C-RAF) as a mediator of LSA-induced PCC, as shRNAs against RAF1 conferred resistance to LSA. We found that LSA induced RAF1 phosphorylation on Serine 338 within minutes in human colorectal carcinoma HCT-116, ovarian carcinoma OVCAR-8, and Burkitt's lymphoma CA46 cell lines. RAF1 depletion by siRNAs attenuated LSA-induced PCC in HCT-116 and OVCAR-8 cells. Furthermore, mouse embryonic fibroblasts (MEF) with homozygous deletion in Raf1, but not deletion in the related kinase Braf, were resistant to LSA-induced PCC. Complementation of Raf1-/- MEFs with wild-type human RAF1, but not with kinase-dead RAF1 mutant, restored LSA-induced PCC. Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC. Our findings implicate a previously unknown, MAPK-independent role of RAF1 in chromatin condensation and potent activation of this pathway by LSA.

No MeSH data available.


Related in: MedlinePlus

RAF1 and MYPT1, two candidates identified in the shRNA screen, are rapidly phosphorylated in response to LSA. (A–C) Time course induction of RAF1 and MYPT1 phosphorylation by LSA in HCT-116 colorectal cancer cells (A), OVCAR-8 ovarian cancer cells (B) and CA46 Burkitt’s lymphoma cells (C). Cells were exposed to 100 nM LSA for the indicated times. RAF1 activation was evaluated by its phosphorylation on S338. MYPT1 inhibition was evaluated by its inhibitory phosphorylation on T696 and by MLC2 phosphorylation on S19 due to its MYPT1-mediated dephosphorylation on Ser19.
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marinedrugs-13-03625-f002: RAF1 and MYPT1, two candidates identified in the shRNA screen, are rapidly phosphorylated in response to LSA. (A–C) Time course induction of RAF1 and MYPT1 phosphorylation by LSA in HCT-116 colorectal cancer cells (A), OVCAR-8 ovarian cancer cells (B) and CA46 Burkitt’s lymphoma cells (C). Cells were exposed to 100 nM LSA for the indicated times. RAF1 activation was evaluated by its phosphorylation on S338. MYPT1 inhibition was evaluated by its inhibitory phosphorylation on T696 and by MLC2 phosphorylation on S19 due to its MYPT1-mediated dephosphorylation on Ser19.

Mentions: Phosphorylation of S338 plays a pivotal role for RAF1 activation [16,26]. To determine the role of RAF1 in the cellular effects of LSA, we measured the kinetics of RAF1 phosphorylation on S338 in three cell lines (Figure 2). These include the HCT-116 cell line we used for the shRNA screen, the ovarian cancer cell line OVCAR-8, which is wild-type for the RAS pathway [27], and the Burkitt’s lymphoma cell line CA46, which we used in our initial study of LSA-induced PCC [13]. In all three-cell lines, LSA induced a marked phosphorylation of RAF1 within 15 min (Figure 2).


Activation of RAF1 (c-RAF) by the Marine Alkaloid Lasonolide A Induces Rapid Premature Chromosome Condensation.

Jossé R, Zhang YW, Giroux V, Ghosh AK, Luo J, Pommier Y - Mar Drugs (2015)

RAF1 and MYPT1, two candidates identified in the shRNA screen, are rapidly phosphorylated in response to LSA. (A–C) Time course induction of RAF1 and MYPT1 phosphorylation by LSA in HCT-116 colorectal cancer cells (A), OVCAR-8 ovarian cancer cells (B) and CA46 Burkitt’s lymphoma cells (C). Cells were exposed to 100 nM LSA for the indicated times. RAF1 activation was evaluated by its phosphorylation on S338. MYPT1 inhibition was evaluated by its inhibitory phosphorylation on T696 and by MLC2 phosphorylation on S19 due to its MYPT1-mediated dephosphorylation on Ser19.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4483648&req=5

marinedrugs-13-03625-f002: RAF1 and MYPT1, two candidates identified in the shRNA screen, are rapidly phosphorylated in response to LSA. (A–C) Time course induction of RAF1 and MYPT1 phosphorylation by LSA in HCT-116 colorectal cancer cells (A), OVCAR-8 ovarian cancer cells (B) and CA46 Burkitt’s lymphoma cells (C). Cells were exposed to 100 nM LSA for the indicated times. RAF1 activation was evaluated by its phosphorylation on S338. MYPT1 inhibition was evaluated by its inhibitory phosphorylation on T696 and by MLC2 phosphorylation on S19 due to its MYPT1-mediated dephosphorylation on Ser19.
Mentions: Phosphorylation of S338 plays a pivotal role for RAF1 activation [16,26]. To determine the role of RAF1 in the cellular effects of LSA, we measured the kinetics of RAF1 phosphorylation on S338 in three cell lines (Figure 2). These include the HCT-116 cell line we used for the shRNA screen, the ovarian cancer cell line OVCAR-8, which is wild-type for the RAS pathway [27], and the Burkitt’s lymphoma cell line CA46, which we used in our initial study of LSA-induced PCC [13]. In all three-cell lines, LSA induced a marked phosphorylation of RAF1 within 15 min (Figure 2).

Bottom Line: We found that LSA induced RAF1 phosphorylation on Serine 338 within minutes in human colorectal carcinoma HCT-116, ovarian carcinoma OVCAR-8, and Burkitt's lymphoma CA46 cell lines.RAF1 depletion by siRNAs attenuated LSA-induced PCC in HCT-116 and OVCAR-8 cells.Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC.

View Article: PubMed Central - PubMed

Affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute (CCR-NCI), NIH, Bethesda, MD 20892-9760, USA. rozenn.josse@gmail.com.

ABSTRACT
Lasonolide A (LSA), a potent antitumor polyketide from the marine sponge, Forcepia sp., induces rapid and reversible protein hyperphosphorylation and premature chromosome condensation (PCC) at nanomolar concentrations independent of cyclin-dependent kinases. To identify cellular targets of LSA, we screened 2951 shRNAs targeting a pool of human kinases and phosphatases (1140 RefSeqs) to identify genes that modulate PCC in response to LSA. This led to the identification of RAF1 (C-RAF) as a mediator of LSA-induced PCC, as shRNAs against RAF1 conferred resistance to LSA. We found that LSA induced RAF1 phosphorylation on Serine 338 within minutes in human colorectal carcinoma HCT-116, ovarian carcinoma OVCAR-8, and Burkitt's lymphoma CA46 cell lines. RAF1 depletion by siRNAs attenuated LSA-induced PCC in HCT-116 and OVCAR-8 cells. Furthermore, mouse embryonic fibroblasts (MEF) with homozygous deletion in Raf1, but not deletion in the related kinase Braf, were resistant to LSA-induced PCC. Complementation of Raf1-/- MEFs with wild-type human RAF1, but not with kinase-dead RAF1 mutant, restored LSA-induced PCC. Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC. Our findings implicate a previously unknown, MAPK-independent role of RAF1 in chromatin condensation and potent activation of this pathway by LSA.

No MeSH data available.


Related in: MedlinePlus