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...

EGF-induced activation of ERK has been extensively studied by both experimental and theoretical approaches. Here, we used a simulation model based mostly on experimentally determined parameters to study the ERK-mediated negative feedback regulation of the Ras guanine nucleotide exchange factor, son of sevenless (SOS). Because SOS1 is phosphorylated at multiple serine residues upon stimulation, we evaluated the role of the multiplicity by building two simulation models, which we termed the decisive and cooperative phosphorylation models. The two models were constrained by the duration of Ras activation and basal phosphorylation level of SOS1. Possible solutions were found only in the decisive model wherein at least three, and probably more than four, phosphorylation sites decisively suppress the SOS activity. Thus, the combination of experimental approaches and the model analysis has suggested an unexpected role of multiple phosphorylations of SOS1 in the negative regulation.

The high-affinity receptor for IgE (Fc epsilon RI) has a tetrameric structure structure composed of one alpha, one beta, and two disulfide-linked gamma subunits, of which the alpha subunit binds IgE with high affinity. A recombinant soluble form of the ectodomain of the human Fc epsilon RI alpha subunit (rsFc epsilon RI alpha) was recently generated by gene engineering and was verified to bind IgE with an affinity as high as that of native Fc epsilon RI on the cell surface. rsFc epsilon RI alpha was prepared on a large scale in order to analyze its biological function. rsFc epsilon RI alpha completely inhibited IgE binding to the cell surface, resulting in abrogation of the chemical mediator release from RBL-2H3 cells. Furthermore it completely abolished the passive cutaneous anaphylaxis (PCA) response by trapping IgE specifically when it was administered into rats prior to IgE sensitization. Even after IgE sensitization, treatment of rsFc epsilon RI alpha substantially reduced the PCA response. It was finally shown that rsFc epsilon RI alpha inhibited IgE binding to human peripheral blood basophils and the histamine release from them. In this paper we address the ability of rsFc epsilon RI alpha to specifically prevent the IgE-mediated allergic reaction.

Interleukin-11 (IL-11) is a pleiotropic cytokine that supports various types of hematopoietic cell growth and is involved in bone resorption. We report here the involvement of recombinant human IL-11 (rHuIL-11) in osteoblast differentiation in mouse mesenchymal progenitor cells, C3H10T1/2. rHuIL-11 alone increased alkaline phosphatase (ALP) activity and upregulated expression levels of osteocalcin (OC), bone sialo protein (BSP), and parathyroid hormone receptor (PTHR) mRNA. rHuIL-11 had no effect on expression of type II collagen, peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2), adipocyte fatty acid-binding protein P2 (aP2), and myogenic MyoD protein (MyoD). Recombinant human bone morphogenetic protein (rHuBMP)-2 increased ALP activity and mRNA expression of these genes except for MyoD. The expression patterns of ALP activity and osteoblast-specific or chondrocyte-specific genes suggest that rHuIL-11 may be involved in early differentiation of osteoblasts at a step earlier than that which is affected by rHuBMP-2. In support of this hypothesis, combined treatment with rHuIL-11 and rHuBMP-2 synergistically increased ALP activity and mRNA expression of OC and type II collagen, rHuIL-11 also abrogated the increased levels of PPAR-gamma2, aP2 mRNA caused by rHuBMP-2. Our results suggest that rHuIL-11 alone and in combination with rHuBMP-2 can induce osteoblastic differentiation of progenitor cells and plays an important role in osteogenesis.

X-ray spectromicroscopy with a full-field imaging technique is a powerful method for chemical analysis of heterogeneous complex materials with a nano-scale spatial resolution. For imaging optics, an X-ray reflective optical system has excellent capabilities with highly efficient, achromatic, and long-working-distance properties. An advanced Kirkpatrick–Baez geometry that combines four independent mirrors with elliptic and hyperbolic shapes in both horizontal and vertical directions was developed for this purpose, although the complexity of the system has a limited applicable range. Here, we present an optical system consisting of two monolithic imaging mirrors. Elliptic and hyperbolic shapes were formed on a single substrate to achieve both high resolution and sufficient stability. The mirrors were finished with a ~1-nm shape accuracy using elastic emission machining. The performance was tested at SPring-8 with a photon energy of approximately 10 keV. We could clearly resolve 50-nm features in a Siemens star without chromatic aberration and with high stability over 20 h. We applied this system to X-ray absorption fine structure spectromicroscopy and identified elements and chemical states in specimens of zinc and tungsten micron-size particles.