Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1a and 1b), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1a or -1b increased the synthesis and secretion of [ 3 H]glycerol-labeled lipids under either basal-or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL 1 (S f . 100) and VLDL 2 (S f 20-100). Expression of lipin-1a or -1b increased secretion efficiency and decreased intracellular degradation of [35 S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [ 3 H]glycerolipids and [ 35 S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1a not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1a mutant failed to promote

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival.

Cyclin D1 plays a critical role in controlling the G 1 /S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2␣ (eIF2␣) kinases. We examined the effect of activation of the eIF2␣ kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2␣ phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3␤ and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2␣ phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2␣ phosphorylation during short, but not prolonged, periods of stress.

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