The B55α subunit of PP2A drives a p53-dependent metabolic adaptation to glutamine deprivation.

Glutamine is an essential nutrient for cancer cell survival and proliferation, yet the signaling pathways that sense glutamine levels remain uncharacterized. Here, we report that the protein phosphatase 2A (PP2A)-associated protein, α4, plays a conserved role in glutamine sensing. α4 promotes assembly of an adaptive PP2A complex containing the B55α regulatory subunit via providing the catalytic subunit upon glutamine deprivation. Moreover, B55α is specifically induced upon glutamine deprivation in a ROS-dependent manner to activate p53 and promote cell survival. B55α activates p53 through direct interaction and dephosphorylation of EDD, a negative regulator of p53. Importantly, the B55α-EDD-p53 pathway is essential for cancer cell survival and tumor growth under low glutamine conditions in vitro and in vivo. This study delineates a previously unidentified signaling pathway that senses glutamine levels as well as provides important evidence that protein phosphatase complexes are actively involved in signal transduction.

Ceramide starves cells to death by downregulating nutrient transporter proteins.

Ceramide induces cell death in response to many stimuli. Its mechanism of action, however, is not completely understood. Ceramide induces autophagy in mammalian cells maintained in rich media and nutrient permease downregulation in yeast. These observations suggested to us that ceramide might kill mammalian cells by limiting cellular access to extracellular nutrients. Consistent with this proposal, physiologically relevant concentrations of ceramide produced a profound and specific downregulation of nutrient transporter proteins in mammalian cells. Blocking ceramide-induced nutrient transporter loss or supplementation with the cell-permeable nutrient, methyl pyruvate, reversed ceramide-dependent toxicity. Conversely, cells became more sensitive to ceramide when nutrient stress was increased by acutely limiting extracellular nutrients, inhibiting autophagy, or deleting AMP-activated protein kinase (AMPK). Observations that ceramide can trigger either apoptosis or caspase-independent cell death may be explained by this model. We found that methyl pyruvate (MP) also protected cells from ceramide-induced, nonapoptotic death consistent with the idea that severe bioenergetic stress was responsible. Taken together, these studies suggest that the cellular metabolic state is an important arbiter of the cellular response to ceramide. In fact, increasing nutrient demand by incubating cells in high levels of growth factor sensitized cells to ceramide. On the other hand, gradually adapting cells to tolerate low levels of extracellular nutrients completely blocked ceramide-induced death. In sum, these results support a model where ceramide kills cells by inducing intracellular nutrient limitation subsequent to nutrient transporter downregulation.

TIPRL Inhibits Protein Phosphatase 4 Activity and Promotes H2AX Phosphorylation in the DNA Damage Response.

Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response.