MEK1 and MEK2 regulate distinct functions by sorting ERK2 to different intracellular compartments.

In this study, we provide novel insight into the mechanism of how ERK2 can be sorted to different intracellular compartments and thereby mediate different responses. MEK1-activated ERK2 accumulated in the nucleus and induced proliferation. Conversely, MEK2-activated ERK2 was retained in the cytoplasm and allowed survival. Localization was a determinant for ERK2 functions since MEK1 switched from providing proliferation to be a mediator of survival when ERK2 was routed to the cytoplasm by the attachment of a nuclear export site. MEK1-mediated ERK2 nuclear translocation and proliferation were shown to depend on phosphorylation of S298 and T292 sites in the MEK1 proline-rich domain. These sites are phosphorylated on cellular adhesion in MEK1 but not MEK2. Whereas p21-activated kinase phosphorylates S298 and thus enhances the MEK1-ERK2 association, ERK2 phosphorylates T292, leading to release of active ERK2 from MEK1. On the basis of these results, we propose that the requirement of adhesion for cells to proliferate in response to growth factors, in part, may be explained by the MEK1 S298/T292 control of ERK2 nuclear translocation. In addition, we suggest that ERK2 intracellular localization determines whether growth factors mediate proliferation or survival and that the sorting occurs in an adhesion-dependent manner.

High molecular weight DNA fragments are processed by caspase sensitive or caspase independent pathways in cultures of cerebellar granule neurons.

Many recent reports on internucleosomal DNA fragments have appeared, however, little is known about the mechanisms of the generation of their upstream high molecular weight (HMW) fragments. Caspases are a family of proteases with important functions in the execution of apoptotic cell death. The caspase-sensitivity of the formation of HMW fragments was therefore investigated using a specific caspase-3 inhibitor (Ac-DEVD-cmk) and a general caspase inhibitor (boc-D-fmk). Apoptosis inducing factor (AIF) can translocate to the nucleus and generate HMW fragments independently of caspase. Cultures of cerebellar granule neurons (CGNs) were therefore exposed to glutamate (100 micro M) or deprived of potassium and serum to induce apoptosis, or treated with a high concentration of calcium ionophore A23187 (1 micro M) to induce necrosis. Fragmentation of DNA into two classes of HMW fragments (>680 and 50-300 kbp) was observed after treatment with glutamate or A23187. Traces of approximately 50-kbp fragments were detectable after the K(+)/serum-deprivation. The amount of >680-kbp HMW fragments increased (i.e. their further degradation was inhibited) and cell death was reduced in the presence of Ac-DEVD-cmk or boc-D-fmk following glutamate treatment. Only boc-D-fmk treatment resulted in a similar accumulation of >680-kbp HMW fragments and reduced cell death after K(+)/serum-deprivation. No such changes were observed with caspase inhibitors after A23187 treatment. AIF redistribution was observed following glutamate treatment and K(+)/serum-deprivation. Thus, even in a simple cell culture of CGNs, HMW fragments are formed by diverse mechanisms: the degradation of DNA may be sensitive to different caspases or be caspase and AIF independent.