Processive phosphorylation of ERK MAP kinase in mammalian cells

Abstract

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...

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