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Abstract 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 PKCβ) 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.

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 PKCβ) 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. [ABSTRACT FROM AUTHOR]

The response of cells to extracellular signals is mediated by a variety of intracellular signaling pathways that determine stimulus-dependent cell fates. One such pathway is the cJun-N-terminal Kinase (JNK) cascade, which is mainly involved in stress-related processes. The cascade transmits its signals via a sequential activation of protein kinases, organized into three to five tiers. Proper regulation is essential for securing a proper cell fate after stimulation, and the mechanisms that regulate this cascade may involve the following: (1) Activatory or inhibitory phosphorylations, which induce or abolish signal transmission. (2) Regulatory dephosphorylation by various phosphatases. (3) Scaffold proteins that bring distinct components of the cascade in close proximity to each other. (4) Dynamic change of subcellular localization of the cascade's components. (5) Degradation of some of the components. In this review, we cover these regulatory mechanisms and emphasize the mechanism by which the JNK cascade transmits apoptotic signals. We also describe the newly discovered PP2A switch, which is an important mechanism for JNK activation that induces apoptosis downstream of the Gq protein coupled receptors. Since the JNK cascade is involved in many cellular processes that determine cell fate, addressing its regulatory mechanisms might reveal new ways to treat JNK-dependent pathologies. [ABSTRACT FROM AUTHOR]

The response of cells to extracellular signals is mediated by a variety of intracellular signaling pathways that determine stimulus-dependent cell fates. One such pathway is the cJun-N-terminal Kinase (JNK) cascade, which is mainly involved in stress-related processes. The cascade transmits its signals via a sequential activation of protein kinases, organized into three to five tiers. Proper regulation is essential for securing a proper cell fate after stimulation, and the mechanisms that regulate this cascade may involve the following: (1) Activatory or inhibitory phosphorylations, which induce or abolish signal transmission. (2) Regulatory dephosphorylation by various phosphatases. (3) Scaffold proteins that bring distinct components of the cascade in close proximity to each other. (4) Dynamic change of subcellular localization of the cascade’s components. (5) Degradation of some of the components. In this review, we cover these regulatory mechanisms and emphasize the mechanism by which the JNK cascade transmits apoptotic signals. We also describe the newly discovered PP2A switch, which is an important mechanism for JNK activation that induces apoptosis downstream of the Gq protein coupled receptors. Since the JNK cascade is involved in many cellular processes that determine cell fate, addressing its regulatory mechanisms might reveal new ways to treat JNK-dependent pathologies.