Increase in proapoptotic activity of inhibitory PAS domain protein via phosphorylation by MK2
Shuya Kasai1,2, Mary J E Richardson1, Satoru Torii1,3
1Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
Abstract:
Inhibitory PAS domain protein (IPAS) is a bifunctional protein that downregulates hypoxic gene expression and exerts proapoptotic activity by preventing prosurvival activity of Bcl-xL and its related factors. Proapoptotic activity of IPAS is attenuated by the activation of the PINK1-Parkin pathway, and involved in neuronal degeneration in an experimental mouse model of Parkinson's disease. The current study shows that phosphorylation of IPAS at Ser184 by MAPK-activated protein kinase 2 (MK2 or MAPKAPK2) enhances the proapoptotic function of IPAS. Perinuclear clustering of mitochondria and activation of caspase-3 caused by the transient expression of EGFP-IPAS were increased by UVB irradiation. The C-terminal region of IPAS mediated the UVB susceptibility of IPAS. Increase in IPAS-induced mitochondrial clustering by UVB was completly inhibited by the p38 MAPK inhibitor SB203580. Mass spectrometry analysis of UVB-activated IPAS identified several phosphorylation sites in the C-terminal region containing p38 MAPK consensus phosphorylation sites at Ser219 and Ser223, and an MK2 consensus site at Ser184. Although mutations of Ser219 and Ser223 to Ala did not suppress the UVB-induced mitochondrial clustering, replacement of Ser184 with Ala blocked it. A phosphomimetic substitution at Ser184 enhanced mitochondrial clustering and activation of caspase-3 without UVB exposure. Furthermore, binding affinity to Bcl-xL was increased by the mutation. Treatment of PC12 cells with CoCl2 caused activation of MK2 and mitochondrial clustering. IPAS-dependent cell death induced by CoCl2 in PC12 cells was decreased by the treatment with the MK2 inhibitor MK2 inhibitor III and by siRNA-directed silencing of MK2.
Insights
Phosphorylation of Inhibitory PAS domain protein (IPAS) at Ser184 by MAPK-activated protein kinase 2 (MK2) enhances its proapoptotic function. This phosphorylation increases mitochondrial clustering and cell death, particularly under UVB exposure or CoCl2 treatment.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Inhibitory PAS domain protein (IPAS) is a bifunctional protein involved in regulating hypoxic gene expression and promoting apoptosis.
- IPAS's proapoptotic activity is linked to preventing prosurvival factors like Bcl-xL and is implicated in Parkinson's disease models.
- The PINK1-Parkin pathway can attenuate IPAS's proapoptotic function.
Purpose of the Study:
- To investigate the role of IPAS phosphorylation by MK2 in regulating its proapoptotic activity.
- To determine the impact of UVB irradiation and other stressors on IPAS function and localization.
- To elucidate the specific phosphorylation sites on IPAS crucial for its response to cellular stress.
Main Methods:
- Transient expression of EGFP-IPAS and its mutants in cells.
- UVB irradiation and CoCl2 treatment to induce cellular stress.
- Mitochondrial clustering assessment, caspase-3 activation assays, and mass spectrometry for phosphosite identification.
- Inhibition studies using SB203580 (p38 MAPK inhibitor) and MK2 inhibitor III, along with MK2 siRNA silencing.
Main Results:
- Phosphorylation of IPAS at Ser184 by MK2 significantly enhances its proapoptotic function.
- UVB irradiation increases IPAS-induced mitochondrial perinuclear clustering and caspase-3 activation, mediated by the C-terminal region.
- Mutation of Ser184 to Alanine blocks UVB-induced mitochondrial clustering, while a phosphomimetic substitution at Ser184 enhances clustering and Bcl-xL binding independently of UVB.
- CoCl2 treatment activates MK2, leading to mitochondrial clustering and IPAS-dependent cell death, which is reduced by MK2 inhibition or silencing.
Conclusions:
- MK2-mediated phosphorylation of IPAS at Ser184 is a key mechanism enhancing its proapoptotic activity.
- This phosphorylation event plays a critical role in cellular responses to stress, such as UVB irradiation and CoCl2 exposure.
- Targeting the MK2-IPAS interaction could offer therapeutic strategies for conditions involving aberrant apoptosis and neuronal degeneration.
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