A Brichkina1, NTM Nguyen1, R Baskar1, S Wee1, J Gunaratne1, RC Robinson1,2 and DV Bulavin*,3
1 Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore 138673, Singapore;
2 Department of Biochemistry,
Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore
3 Institute for Research on Cancer and Aging of Nice (IRCAN),
INSERM, U1081-UMR CNRS 7284, University of Nice – Sophia Antipolis, Centre Antoine Lacassagne, Nice, France
*Correspondence should be addressed to Dmitry.Bulavin@unice.fr
Published in Cell Death and Differentiation on 27 May 2016.
The stress-induced p38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in multiple physiological processes, including cancer. In turn, p38MAPK phosphorylation at Thr180 and Tyr182 is a key regulatory mechanism for its activation and functions. Here we show that this mechanism is actively regulated through isomerisation of Pro224. Different cyclophilins can isomerise this proline residue and modulate the ability of upstream kinases to phosphorylate Thr180 and Tyr182.
In vivo mutation of Pro224 to Ile in endogenous p38MAPK significantly reduced its phosphorylation and activity. This resulted in attenuation of p38MAPK signalling, which in turn caused an enhanced apoptosis and sensitivity to a DNA-damaging drug, cisplatin. We further found a reduction in size and number of lesions in homozygous mice carrying the p38MAPK P224I substitution in a K-ras model of lung tumorigenesis. We propose that cyclophilin-dependent isomerisation of p38MAPK is an important novel mechanism in regulating p38MAPK phosphorylation and functions. Thus, inhibition of this process, including with drugs that are in clinical trials, may improve the efficacy of current anti-cancer therapeutic regimes.
Figure 7: Inactivation of p38MAPK due to lack of Pro224 isomerisation leads to a higher sensitivity of tumour cells to cisplatin treatment. (a) Wild-type MEFs or P224I MEFs were transformed with E1A and RasV12 oncogenes, and established stable cell lines were xenografted into nude mice subcutaneously (1 × 105 cells per flank, left side for wildtype cells, right side for knock-in cells). Once the tumours reach 1 cm in diameter (10–14 days), one group of mice was left untreated (at least three mice per group), and the
second group of mice was treated with cisplatin at a dose of 7 mg/kg, two times with a 4-days interval. Mice were killed once the tumour size of control mice reached about 2 cm in diameter. Solid tumours were excised, weighted and photographed. The graph represents an average weight of all tumours per genotype/treatment. (b) Histological sections of subcutaneously xenografted cells collected after cisplatin treatment as in Figure 7a. Paraffin sections were stained with anti-cleaved caspase-3 antibodies (CC3), the number of cells per field (×20 objective) was counted and presented as a graph. One representative image of CC3 staining is presented in Supplementary Figure S6b. (c) Wild type and homozygous P224I knock-in mice were crossed on a K-ras background to develop lung tumours and killed at an age of 10 weeks. Histological sections were stained with phospho-specific antibodies against p38 and Hsp27, and representative images are shown. Staining intensity of images taken at × 60 objective (n=10) was quantified using ImageJ program and presented in the graph. A representative image is shown in Supplementary Figure S6c. (d) Visible lung tumour lesions were micro-dissected from three pairs of wild type and P224I mice, and processed for mRNA expression analysis of p38 targets by qRT-PCR. (e) Wild type and P224I knock-in mice were crossed on a K-ras background to develop lung tumours and killed at the age of 10 weeks The numbers of lesions were counted and presented. (f) The relative tumour area was calculated after hematoxylin/eosin staining and the percentage relative to total lung area is presented. A representative image of the lung section is shown; note the dark hematoxylin/eosinpositive nodules (indicated with arrows) on the surface of the lungs representing tumour lesions. (g) Wild type and homozygous P224I knock-in mice were crossed on a K-ras background to develop lung tumours. At the age of 10 weeks, mice were injected with cisplatin. Apoptosis was analysed with an anti-cleaved caspase-3 antibody; the number of positive cells was counted and presented as a ratio per scored tumour area. One representative image is shown in Supplementary Figure S6d. NS stands for nonsignificant, *Po0.05, **Po0.01, ***Po0.001