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ABSTRACT
Accumulating evidence suggests that mitogen-activated protein kinases (MAPKs) regulate macroautophagy/autophagy. However, the involvement of dual-specificity protein phosphatases (DUSPs), endogenous inhibitors for MAPKs, in autophagy remains to be determined. Here we report that DUSP1/MKP-1, the founding member of the DUSP family, plays a critical role in regulating autophagy. Specifically, we demonstrate that DUSP1 knockdown by shRNA in human ovarian cancer CAOV3 cells and knockout in murine embryonic fibroblasts, increases both basal and rapamycin-increased autophagic flux. Overexpression of DUSP1 had the opposite effect. Importantly, knockout of Dusp1 promoted phosphorylation of ULK1 at Ser555, and BECN1/Beclin 1 at Ser15, and the association of PIK3C3/VPS34, ATG14, BECN1 and MAPK, leading to the activation of the autophagosome-initiating class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Furthermore, knockdown and pharmacological inhibitor studies indicated that DUSP1-mediated suppression of autophagy reflected inactivation of the MAPK1-MAPK3 members of the MAPK family. Knockdown of DUSP1 sensitized CAOV3 cells to rapamycin-induced antigrowth activity. Moreover, CAOV3-CR cells, a line that had acquired cisplatin resistance, exhibited an elevated DUSP1 level and were refractory to rapamycin-induced autophagy and cytostatic effects. Knockdown of DUSP1 in CAOV3-CR cells restored sensitivity to rapamycin. Collectively, this work identifies a previously unrecognized role for DUSP1 in regulating autophagy and suggests that suppression of DUSP1 may enhance the therapeutic activity of rapamycin.
Abbreviations
| BafA1 | = | bafilomycin A1 |
| BECN1 | = | Beclin 1, autophagy related |
| CREB | = | cAMP response element binding protein |
| DUSP1/MKP-1 | = | dual-specificity protein phosphatase 1 |
| MAPK | = | mitogen-activated protein kinase |
| MAP1LC3/LC3 | = | microtubule-associated protein 1 light chain 3 |
| MAP2K/MKK/MEK | = | mitogen-activated protein kinase kinase |
| MEFs | = | mouse embryonic fibroblasts |
| MTOR | = | mechanistic target of rapamycin (serine/threonine kinase) |
| MTT | = | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
| PIK3C3 | = | phosphoinositide-3-kinase, class 3 |
| PtdIns3K | = | class III phosphatidylinositol 3-kinase |
| SQSTM1/p62 | = | sequestosome 1 |
| shRNA | = | short hairpin RNA |
| siRNA | = | small interfering RNA |
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. James Hatfield for assistance with the electron microscopy and Dr. Yusen Liu for helpful discussion.
Funding
This work was supported, in part, by National Institutes of Health Grant 1R21CA178111 through the NCI.