The mitogen-activated protein (MAP) kinase pathway is comprised of a three-tiered kinase cascade. The distributive kinetic mechanism of two-site MAP kinase phosphorylation inherently generates a nonlinear switch-like response. However, a linear graded response of MAP kinase has also been observed in mammalian cells, and its molecular mechanism remains unclear. To dissect these input-output behaviors, we quantitatively measured the kinetic parameters involved in the MEK (MAPK/ERK kinase)-ERK MAP kinase signaling module in HeLa cells. Using a numerical analysis based on experimentally determined parameters, we predicted in silico and validated in vivo that ERK is processively phosphorylated in HeLa cells. Finally, we identified molecular crowding as a critical factor that converts distributive phosphorylation into processive phosphorylation. We proposed the term quasi-processive phosphorylation to describe this mode of ERK phosphorylation that is operated under the physiological condition of molecular crowding. The generality of this phenomenon may provide a new paradigm for a diverse set of biochemical reactions including multiple posttranslational modifications. M itogen-activated protein (MAP) kinase cascades are evolutionarily conserved signaling pathways that are involved in the control of physiological and pathological cellular processes including cell proliferation, survival, differentiation, apoptosis, and tumorigenesis (1-4). Each MAP kinase pathway contains a three-tiered kinase cascade consisting of a MAP kinase kinase kinase, a MAP kinase kinase, and the MAP kinase, which are sequentially activated in this order. MAP kinases, which are activated by dual phosphorylation of conserved threonine and tyrosine residues within the activation loop, phosphorylate their targets on serine or threonine residues (1, 3, 5). Five distinct groups of MAP kinases have been characterized in mammals. Among the five groups, the most studied is the Raf/MEK/ERK MAP kinase cascade (hereafter called the ERK MAP kinase cascade), which is activated by mitogenic ligands such as growth factors, cytokines, and phorbol esters.Ferrell and coworkers found that in Xenopus oocytes the ERK MAP kinase pathway responds to increasing levels of progesterone in an all-or-none or "switch-like" manner, in which individual cells in the population exhibit either "on" or "off" status (6, 7). This property of ERK MAP kinase system befits to determine allor-none irreversible responses including cell-cycle progression, neuronal differentiation, and T cell selection (6-10). The switchlike responses can arise from both positive feedback via protein synthesis and dual phosphorylation steps of the MAP kinase, which is called the "distributive phosphorylation model" (11-15) (Fig. 1A). The distributive model of ERK phosphorylation results in an increase in the cooperativity of this system, and consequently contributes to a switch-like input-output response.In different cellular contexts, however, the ERK MAP kinase cascade exhibits "graded" response, in wh...

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.

Summary The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex, and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss-of-function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 down-regulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.

SummaryCellular signaling processes can exhibit pronounced cell-to-cell variability in genetically identical cells. This affects how individual cells respond differentially to the same environmental stimulus. However, the origins of cell-to-cell variability in cellular signaling systems remain poorly understood. Here, we measure the dynamics of phosphorylated MEK and ERK across cell populations and quantify the levels of population heterogeneity over time using high-throughput image cytometry. We use a statistical modeling framework to show that extrinsic noise, particularly that from upstream MEK, is the dominant factor causing cell-to-cell variability in ERK phosphorylation, rather than stochasticity in the phosphorylation/dephosphorylation of ERK. We furthermore show that without extrinsic noise in the core module, variable (including noisy) signals would be faithfully reproduced downstream, but the within-module extrinsic variability distorts these signals and leads to a drastic reduction in the mutual information between incoming signal and ERK activity.