Phosphorylation of PP2Ac by PKC is a key regulatory step in the PP2A-switch-dependent AKT dephosphorylation that leads to apoptosis.

BACKGROUND: Although GqPCR activation often leads to cell survival by activating the PI3K/AKT pathway, it was previously shown that in several cell types AKT activity is reduced and leads to JNK activation and apoptosis. The mechanism of AKT inactivation in these cells involves an IGBP1-coupled PP2Ac switch that induces the dephosphorylation and inactivation of both PI3K and AKT. However, the machinery involved in the initiation of PP2A switch is not known. METHODS: We used phospho-mass spectrometry to identify the phosphorylation site of PP2Ac, and raised specific antibodies to follow the regulation of this phosphorylation. Other phosphorylations were monitored by commercial antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by a TUNEL assay as well as PARP1 cleavage using SDS-PAGE and Western blotting. RESULTS: We identified Ser24 as a phosphorylation site in PP2Ac. The phosphorylation is mediated mainly by classical PKCs (PKCα and PKCß) but not by novel PKCs (PKCδ and PKCε). By replacing the phosphorylated residue with either unphosphorylatable or phosphomimetic residues (S24A and S24E), we found that this phosphorylation event is necessary and sufficient to mediate the PP2A switch, which ultimately induces AKT inactivation, and a robust JNK-dependent apoptosis. CONCLUSION: Our results show that the PP2A switch is induced by PKC-mediated phosphorylation of Ser24-PP2Ac and that this phosphorylation leads to apoptosis upon GqPCR induction of various cells. We propose that this mechanism may provide an unexpected way to treat some cancer types or problems in the endocrine machinery.

Retention of ERK in the cytoplasm mediates the pluripotency of embryonic stem cells.

The dynamic subcellular localization of ERK1/2 plays an important role in regulating cell fate. Differentiation of mouse embryonic stem cells (mESCs) involves inductive stimulation of ERK1/2, and therefore, inhibitors of the ERK cascade are used to maintain pluripotency. Interestingly, we found that in pluripotent mESCs, ERK1/2 do not translocate to the nucleus either before or after stimulation. This inhibition of nuclear translocation may be dependent on a lack of stimulated ERK1/2 interaction with importin7 rather than a lack of ERK1/2 phosphorylation activating translocation. At late stages of naive-to-primed transition, the action of the translocating machinery is restored, leading to elevation in ERK1/2-importin7 interaction and their nuclear translocation. Importantly, forcing ERK2 into the naive cells' nuclei accelerates their early differentiation, while prevention of the translocation restores stem cells' pluripotency. These results indicate that prevention of nuclear ERK1/2 translocation serves as a safety mechanism for keeping pluripotency of mESCs.

Nuclear ERK: Mechanism of Translocation, Substrates, and Role in Cancer.

The extracellular signal-regulated kinases 1/2 (ERK) are central signaling components that regulate stimulated cellular processes such as proliferation and differentiation. When dysregulated, these kinases participate in the induction and maintenance of various pathologies, primarily cancer. While ERK is localized in the cytoplasm of resting cells, many of its substrates are nuclear, and indeed, extracellular stimulation induces a rapid and robust nuclear translocation of ERK. Similarly to other signaling components that shuttle to the nucleus upon stimulation, ERK does not use the canonical importinmechanism of nuclear translocation. Rather, it has its own unique nuclear translocation signal (NTS) that interacts with importin7 to allow stimulated shuttling via the nuclear pores. Prevention of the nuclear translocation inhibits proliferation of B-Raf- and N/K-Ras-transformed cancers. This effect is distinct from the one achieved by catalytic Raf and MEK inhibitors used clinically, as cells treated with the translocation inhibitors develop resistance much more slowly. In this review, we describe the mechanism of ERK translocation, present all its nuclear substrates, discuss its role in cancer and compare its translocation to the translocation of other signaling components. We also present proof of principle data for the use of nuclear ERK translocation as an anti-cancer target. It is likely that the prevention of nuclear ERK translocation will eventually serve as a way to combat Ras and Raf transformed cancers with less side-effects than the currently used drugs.