Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling.
Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays.
1] Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistokatu 6, FI-20520 Turku, Finland [2] Department of Pathology, University of Turku, FI-20520 Turku, Finland [3] Turku Doctoral Program of Biomedical Sciences (TuBS), Turku, Finland.
Otto
O
Kauko
1] Department of Mathematics and Statistics, University of Turku, FI-20014 Turku, Finland [2] Drug Research Doctoral Programme (DRDP), Turku, Finland.
Teemu Daniel
TD
Laajala
Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistokatu 6, FI-20520 Turku, Finland.
Mikael
M
Jumppanen
Institute for Molecular Medicine Finland, Tukholmankatu 8, FI-00290 Helsinki, Finland.
Petteri
P
Hintsanen
1] Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistokatu 6, FI-20520 Turku, Finland [2] Turku Centre for Computer Science, FI-20520 Turku, Finland.
Veronika
V
Suni
Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistokatu 6, FI-20520 Turku, Finland.
Pekka
P
Haapaniemi
1] Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistokatu 6, FI-20520 Turku, Finland [2] Van 't Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Garry
G
Corthals
Institute for Molecular Medicine Finland, Tukholmankatu 8, FI-00290 Helsinki, Finland.
Tero
T
Aittokallio
1] Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistokatu 6, FI-20520 Turku, Finland [2] Department of Pathology, University of Turku, FI-20520 Turku, Finland.
Jukka
J
Westermarck
1] Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistokatu 6, FI-20520 Turku, Finland [2] Faculty of Pharmacy, Meijo University, Yagotoyama 150, Tempaku, Nagoya 468-8503, Japan.
Susumu Y
SY
Imanishi
D019540:Area Under Curve; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D002851:Chromatography, High Pressure Liquid; D016000:Cluster Analysis; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D053208:Kaplan-Meier Estimate; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009369:Neoplasms / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012372:ROC Curve; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D001324:Autoantigens; C500266:CIP2A protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010748:Phosphopeptides; D034741:RNA, Small Interfering; C009495:titanium dioxide; D014025:Titanium; D054648:Protein Phosphatase 2; D018631:ras Proteins