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2007 Scientific Report

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VARI |

VARI | 2007 Identification of phosphatases that regulate chemoresistance Our research has shown that a large percentage of phosphatases and their regulatory subunits contribute to cell survival. This is a previously unrecognized general role for phosphatases as negative regulators of apoptosis, and it is important because phosphatases may no longer be simply viewed as enzymes that oppose the action of kinases. This research also identified a number of phosphatases whose loss of function results in chemoresistance, implicating these proteins as potential tumor suppressors. In our RNAi study, 5% of all phosphatases were shown to act in this way; an example is MK-STYX. Down-regulation of MK-STYX resulted in dramatic cellular resistance to cisplatin-, Taxol- or etoposide-induced cell death, which is consistent with up-regulated survival signals in these cells (Fig. 1). Also, MK-STYX is located at 7q11.23, a chromosome region mutated in colon cancer. MK-STYX is similar to MKP-1, which inactivates MAPKs; MK-STYX, however, is predicted to be a catalytically inactive phosphatase. Our observations suggest that MK-STYX acts against cell survival by sequestering pro-survival signaling components in a way analogous to the “substrate-trapping” effects of catalytically inactive phosphatases. Figure 1A. Figure 1B. 37 Figure 1. Identification of MK-STYX as a potential tumor suppressor phosphatase. Cells were transfected with control siRNA or MK-STYX siRNA for 48 h and then were treated for an additional 24 h with solvent control (–) or 50 μM cisplatin (+). Cell viability was visualized by A) crystal violet stain and B) cleavage of full-length PARP measured by western blot analysis.

Van Andel Research Institute | Scientific Report Graded MAPK signaling and switch-like c-Fos induction We also take a systems biology approach to understanding two key molecular pathways, Ras/MAPK and PI3K/mTOR. In the Ras/MAPK pathway, growth factors activate the small G protein Ras, which recruits Raf to the plasma membrane where it is activated and phosphorylates MEK1/2, which in turn phosphorylates ERK1/2-MAPKs. Activated ERK1/2 phosphorylates additional kinases (such as RSK) and specific transcription factors (such as c-Fos and Elk-1) that are important in cellular proliferation, differentiation, and survival. One project in the lab involved the question of whether the evolutionarily conserved MAPK pathway exhibits a switch-like or a graded response in mammalian cells. Ultrasensitive switch-like responses control cell-fate decisions in many biological settings, and the regulation of kinase activity is one way in which such behavior can be initiated. Signaling molecules switch between two discontinuous, stable states with no intermediate; this is referred to as a bistable response (Fig. 2, top panel). Given the irreversible, all-or-none nature of many cell behaviors, including cell cycle control and apoptosis, significant effort has been focused on identifying the cellular mechanisms underlying bistability. Our research and that of others has provided solid evidence for graded MAPK signaling in mammalian cells (Fig. 2, lower panel); that is, as agonist concentration increases, single-cell kinase activity increases proportionally. Yet we have also found that the proliferative response to growth factor stimulation is switch-like, demonstrating that the ultrasensitive step in the MAPK pathway occurs at the level of MAPK nuclear concentration and switch-like c-Fos induction. Although c-Fos induction and cell cycle entry in mammalian cells is switch-like, graded MAPK activation could have an important role in cell survival, since many MAPK targets regulating cell survival are in the cytoplasm. 38 Figure 2. Figure 2. Total cell population MAPK measurements. Single cells exhibiting a bistable (all-or-none) response or graded response (linear). From left: Wolters, Ludema, Kraker, Looyenga, MacKeigan, Nelson, Sian

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