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Kinome siRNA-phosphoproteomic screen identifies networks regulating AKT signaling

Abstract

To identify regulators of intracellular signaling, we targeted 541 kinases and kinase-related molecules with small interfering RNAs (siRNAs), and determined their effects on signaling with a functional proteomics reverse-phase protein array (RPPA) platform assessing 42 phospho and total proteins. The kinome-wide screen demonstrated a strong inverse correlation between phosphorylation of AKT and mitogen-activated protein kinase (MAPK) with 115 genes that, when targeted by siRNAs, demonstrated opposite effects on MAPK and AKT phosphorylation. Network-based analysis identified the MAPK subnetwork of genes along with p70S6K and FRAP1 as the most prominent targets that increased phosphorylation of AKT, a key regulator of cell survival. The regulatory loops induced by the MAPK pathway are dependent on tuberous sclerosis complex 2 but demonstrate a lesser dependence on p70S6K than the previously identified FRAP1 feedback loop. The siRNA screen also revealed novel bi-directionality in the AKT and GSK3 (Glycogen synthase kinase 3) interaction, whereby genetic ablation of GSK3 significantly blocks AKT phosphorylation, an unexpected observation as GSK3 has only been predicted to be downstream of AKT. This method uncovered novel modulators of AKT phosphorylation and facilitated the mapping of regulatory loops.

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Acknowledgements

We thank Drs Kwiatkowski and J Woodgett for the knock out TSC2 and GSK3 cells, respectively. This study was funded in part by the Kleberg Center for Molecular Markers, the Komen Foundation, Stand Up to Cancer/American Association for Cancer Research Dream Team Translational Cancer Research Grant, Grant No. SU2C-AACR- DT0209, NIH CCSG P30CA16672, NIH Foundation DPA86424-444938 to BD and GBM, NIH CCTS support to DS, NIH T90DK070109 fellowship to J-TT and SI, Komen fellowship KG101547 to KK and PTR, DOD BC044268 and NIH R01CA125109 to PTR, NIH P01CA099031 and P50CA083639 to GBM, and U54 CA112970 to PTR and GBM. LN was supported by the Seed Funding Program Collaborative Advances in Biomedical Computing (CAMC), funded by the John and Ann Doerr Fund for Computational Biomedicine.

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Correspondence to P T Ram.

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Lu, Y., Muller, M., Smith, D. et al. Kinome siRNA-phosphoproteomic screen identifies networks regulating AKT signaling. Oncogene 30, 4567–4577 (2011). https://doi.org/10.1038/onc.2011.164

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