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  • Rat pheochromocytoma PC cells have been widely


    Rat pheochromocytoma PC12 cells have been widely used as a cell system to study intracellular signaling leading to neurite outgrowth. The advantages of using PC12 cells in the CHK studies are the fact that they naturally express the rat form of CHK and the observation that down-regulation of CHK function was previously shown to impair neurite outgrowth [15] and MAPK [16] activation following NGF treatment in these cells. In this study, we confirmed these findings using RNAi technology (Fig. 2C). The importance of MAPK stimulation in PC12 cells is currently well established. Although Erk1/2 is rapidly activated in PC12 cells in response to many different factors (EGF, cAMP, phorbol esters, etc.), this activation is transient; in contrast, compounds able to induce neuritogenesis (like NGF) cause sustained MAPK activation [40]. Thus, most studies distinguish between transient activation of the MAPK pathway, elicited by mitogenic signals, and prolonged activation, leading to Fosinopril sodium arrest and subsequent differentiation [27]. In this study, we aimed to reveal the mechanism by which CHK is capable of inducing sustained activation of Erk1/2. To study CHK-elicited signaling, we needed to specifically activate CHK. In one of our previous reports, we described an increase in CHK activity elicited by its aggregation with a tyrosine kinase receptor, namely ErbB2 [12]. However, the receptors known to harbor CHK induce multiple and redundant signaling pathways. Thus, we decided to use instead exogenous expression of CHK as a specific stimulus to increase CHK activity in the cells studied. Following that, we used Erk1/2 as a point to begin our studies and we moved toward the molecules upstream of Erk1/2 to identify the “point-of-entry” of CHK into the MAPK pathway. As a result of our work, we identified the Ras-mediated pathway as the one transducing the signal to MAPK from activated CHK. Importantly, in our studies we show for the first time dissociation between the activation of the MAPK pathway by CHK and the inhibition of Src-family kinases by this enzyme, which highlights a substantial functional difference between CHK and Csk. Based on our results of CHK-elicited effects on the MAPK signal transduction pathway, we propose that activated CHK, aside from phosphorylating Src-family kinases, induces a complex formation between Grb2 and SHP-2, which in turn positively affects signaling through the Ras/Raf/MEK1/2/Erk1/2 pathway (see Fig. 7 for a schematic representation of CHK-mediated signaling towards Erk1/2). The biological importance of the signaling pathway(s) elicited by CHK is not fully comprehended. Ras, identified here as a signal transducer acting downstream to CHK (Fig. 4C and D), is a potent oncogene and mutated forms are found in approximately 30% of all human cancers [41]. In contrast to its growth promoting effects in cultured murine fibroblasts, Ras can induce growth arrest in human fibroblasts and the differentiation of cells of neuronal origin. For example, microinjection of active Ras in PC12 cells causes neurite outgrowth [42]. Also, NGF- and bFGF-induced neurite formation in PC12 cells are blocked by microinjection of anti-Ras antibodies [43] or by expression of a dominant-negative Ras mutant (S17NRas) [44]. Sustained Ras activation occurs when PC12 cells are treated with NGF or bFGF, both of which induce neurite outgrowth, but not in the cells treated with EGF which acts as a mitogen for these cells [45]. Based on these facts, the increased activity of Ras following CHK expression suggests the involvement of CHK in neurite outgrowth and neuronal differentiation. The emerging question is what pathway uses CHK to activate Ras? Our studies indicate participation of the SHP-2 phosphatase in this signaling. SHP-2 is a ubiquitous cytosolic phosphatase with a high level of expression in neural tissue [46]. In a recent study, the molecular mechanism of SHP-2-mediated effects on the MAPK kinase pathway upon stimulation with EGFR was shown to involve an increased half-life of activated Ras (Ras:GTP) in the cell. SHP-2 was reported to mediate this increase by interfering with the process of Ras inactivation (conversion to the GDP-bound form) catalyzed by GTPase-activating protein (RasGAP) via inhibition of the tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane [39]. SHP-2 was also shown to interact with autophosphorylated RTKs (e.g. TrkA) [47], which in turn induce the phosphorylation and activation of SHP-2. Furthermore, in a recent study, it has been shown that SHP-2 regulates Src-family kinase activity by negatively controlling Csk function, which results in Ras/Erk activation [48]. However, many details of the molecular mechanisms of SHP-2-mediated signaling remain an enigma. Here, we show that exogenous expression of CHK induced an aggregation of Grb2 and SHP-2 (Fig. 5A and C), which together form a complex known to positively regulate Ras activity [49]. Of note, SHP-2 activity is required for the appropriate transduction of signals originating from a number of cell surface receptors [50], including EGFR [39] and NGFR [51], [52]. Interference with SHP-2 activity, in vitro, inhibits NGF-induced neurite outgrowth in PC12 cells [51], results similar to our Fosinopril sodium reports with CHK [15], [16]. This suggests that, aside from inhibition of Src, the function of CHK in neural cells is to cooperate with SHP-2 in the regulation of cellular differentiation and neurite outgrowth.