One of the unique characteristics of cellular signaling pathways is that a common signaling pathway can selectively regulate multiple cellular functions of a hormone; however, this selective downstream control through a common signaling pathway is poorly understood. Here we show that the insulin-dependent AKT pathway uses temporal patterns multiplexing for selective regulation of downstream molecules. Pulse and sustained insulin stimulations were simultaneously encoded into transient and sustained AKT phosphorylation, respectively. The downstream molecules, including ribosomal protein S6 kinase (S6K), glucose-6-phosphatase (G6Pase), and glycogen synthase kinase-3β (GSK3β) selectively decoded transient, sustained, and both transient and sustained AKT phosphorylation, respectively. Selective downstream decoding is mediated by the molecules' network structures and kinetics. Our results demonstrate that the AKT pathway can multiplex distinct patterns of blood insulin, such as pulse-like additional and sustained-like basal secretions, and the downstream molecules selectively decode secretion patterns of insulin.

Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flows through a network, through signaling pathways that involve 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network reveals that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We thus provide a versatile method of reconstruction of signal flow through the network using phosphoproteome and metabolome data.

The regulation of glucose metabolism by pulse stimulations of insulin is compared with the effect of ramp stimulations. Specific network motifs mediate the differential response to these temporal patterns of stimulations that mimic in vivo patterns of insulin secretion.

auto regresslve with exogenous input (ARX) model of the MAPKs IEG path way which is generally used to anatyze frequeney dependent relauonships betwecn mputs (upstrearns) and outpuLs (downstreams) We measured dee phosphorylation level of ERK, p38 JNK, CREB and ex pTession level of the EGRI c Fos upon NGF IIACAP and EGF stimuh fOr 3 hours by 3mmutes mtervals usmg Quanttatsve lmagc Cytometry whtch allows us to obtain high pTecitsion time semes daLa Based on these data we Lonstruct ARX models which were abteto reproduce the measured data We analyLed a frequency respo]tse function based on the model and found that the on]y ERK aLtivity was important tor EGRI expTession whereas ERK and CERB aLtiv ity were important for c Fos exp]ession Furthermore wc found that EGRI exprestsion