title: 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.
authors.affiliation: Department of Systems Biology, UT MD Anderson Cancer Center, Houston, TX 77054, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Lu
authors.forename: M
authors.initials: M
authors.lastname: Muller
authors.forename: D
authors.initials: D
authors.lastname: Smith
authors.forename: B
authors.initials: B
authors.lastname: Dutta
authors.forename: K
authors.initials: K
authors.lastname: Komurov
authors.forename: S
authors.initials: S
authors.lastname: Iadevaia
authors.forename: D
authors.initials: D
authors.lastname: Ruths
authors.forename: J T
authors.initials: JT
authors.lastname: Tseng
authors.forename: S
authors.initials: S
authors.lastname: Yu
authors.forename: Q
authors.initials: Q
authors.lastname: Yu
authors.forename: L
authors.initials: L
authors.lastname: Nakhleh
authors.forename: G
authors.initials: G
authors.lastname: Balazsi
authors.forename: J
authors.initials: J
authors.lastname: Donnelly
authors.forename: M
authors.initials: M
authors.lastname: Schurdak
authors.forename: S
authors.initials: S
authors.lastname: Morgan-Lappe
authors.forename: S
authors.initials: S
authors.lastname: Fesik
authors.forename: P T
authors.initials: PT
authors.lastname: Ram
authors.forename: G B
authors.initials: GB
authors.lastname: Mills
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D006801:Humans; D053858:Metabolic Networks and Pathways / Q000235:genetics / Q000502:physiology*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D034741:RNA, Small Interfering; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D051057:Proto-Oncogene Proteins c-akt