Abstract
Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.
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Acknowledgements
We thank Drs Natlie G Ahn (University of Colorado), Piero Crespo (Consejo Superior de Investigaciones Cientı´ficas), Xosé R Bustelo (University of Salamanca), Robert J Kay (University of British Columbia) and Juan S Bonifacino (National Institute of Child Health and Human Development) for generously providing the plasmids. This work was supported by research grants from Ministry of Science and Technology of China (2007CB947100 to YC and 2010CB529506 to YP and ZW), National Natural Science Foundation of China (30830037 and 81021002 to YC and 30971660 to YP), Chinese Academy of Sciences (KSCX2-EW-R-08 to YC), and Shanghai Institutes for Biological Sciences (2009KIP207 to YP).
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(Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Hydrophobicity analysis of PAQR10 and PAQR11. (PDF 175 kb)
Supplementary information, Figure S2
Characterization of PAQR10/11 antibodies. (PDF 474 kb)
Supplementary information, Figure S3
PAQR10 but not AdipoR2 can activate ERK signaling. (PDF 90 kb)
Supplementary information, Figure S4
A Golgi-specific protein TGN38 does not affect ERK signaling. (PDF 120 kb)
Supplementary information, Figure S5
PAQR10-mediated stimulation of Egr-1 gene expression is inhibited by GFP-RBD and MEK inhibitor PD98059. (PDF 93 kb)
Supplementary information, Figure S6
Control experiments for confocal studies. (PDF 100 kb)
Supplementary information, Figure S7
PAQR10 increases Golgi localization of HRas, NRas, KRas4A but no KRas4B in the Golgi apparatus in MDCK cells. (PDF 136 kb)
Supplementary information, Figure S8
The distribution of HRas on the plasma membrane and Golgi apparatus is affected by PAQR10/PAQR11 overexpression. (PDF 65 kb)
Supplementary information, Figure S9
Interaction of PAQR10/PAQR11 with HRas, NRas and KRas4A. (PDF 395 kb)
Supplementary information, Figure S10
Analysis of PAQR10/PAQR11 siRNA efficiency. (PDF 236 kb)
Supplementary information, Figure S11
Analysis of RasGRP1 siRNA efficiency. (PDF 62 kb)
Supplementary information, Figure S12
Silencing of RasGRP1 affects activation of Ras in the Golgi apparatus by PAQR10/PAQR11 overexpression. (PDF 347 kb)
Supplementary information, Figure S13
Knockdown of RasGRP1 does not affect PAQR10/PAQR11-induced Golgi localization of HRas and the interaction between HRas and PAQR10/PAQR11. (PDF 171 kb)
Supplementary information, Figure S14
EGF treatment has no effect on the expression of PAQR10 and PAQR11. (PDF 284 kb)
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Jin, T., Ding, Q., Huang, H. et al. PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus. Cell Res 22, 661–676 (2012). https://doi.org/10.1038/cr.2011.161
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DOI: https://doi.org/10.1038/cr.2011.161
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