Albert Gubern

Cell Cycle Control and HIV-1 Susceptibility Are Linked by CDK6-Dependent CDK2 Phosphorylation of SAMHD1 in Myeloid and Lymphoid Cells

Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain–containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67+) primary CD4+ T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability.

Group IVA Phospholipase A2 Is Necessary for the Biogenesis of Lipid Droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A2 inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A2 (cPLA2α). Knocking down cPLA2α expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA2α fusion protein (EGFP-cPLA2) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA2α at Ser505. Transfection of a S505A mutant cPLA2α showed that phosphorylation at Ser505 is key for enzyme activity and LD formation. cPLA2α contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA2α inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA2α in the formation of nascent LD from the endoplasmic reticulum.

Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group via phospholipase A2

This work investigates the metabolic origin of triacylglycerol (TAG) formed during lipid droplet (LD) biogenesis induced by stress. Cytotoxic inhibitors of fatty acid synthase induced TAG synthesis and LD biogenesis in CHO-K1 cells, in the absence of external sources of fatty acids. TAG synthesis was required for LD biogenesis and was sensitive to inhibition and down-regulation of the expression of group VIA phospholipase A2 (iPLA2-VIA). Induction of stress with acidic pH, C2-ceramide, tunicamycin, or deprivation of glucose also stimulated TAG synthesis and LD formation in a manner dependent on iPLA2-VIA. Overexpression of the enzyme enhanced TAG synthesis from endogenous fatty acids and LD occurrence. During stress, LD biogenesis but not TAG synthesis required phosphorylation and activation of group IVA PLA2 (cPLA2α). The results demonstrate that iPLA2-VIA provides fatty acids for TAG synthesis while cPLA2α allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates.

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A2 (cPLA2α). This work dissects the pathway leading to cPLA2α activation and LD biogenesis. Both processes were Ca2+-independent, as they took place after pharmacological blockade of Ca2+ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA2α, which abrogates its Ca2+ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca2+ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA2α at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA2α at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA2α and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.

Cell Survival during Complete Nutrient Deprivation Depends on Lipid Droplet-fueled β-Oxidation of Fatty Acids

Background: Cellular stress leading to cell death induces the formation of lipid droplets. Results: Nutrient deprivation induces LD biogenesis and mobilization, fueling fatty acid oxidation to sustain cell viability. Conclusion: β-Oxidation requires biogenesis and mobilization of LD. Significance: The role of LD in cell survival and β-oxidation might provide new potential targets for antitumor therapy. Cells exposed to stress of different origins synthesize triacylglycerols and generate lipid droplets (LD), but the physiological relevance of this response is uncertain. Using complete nutrient deprivation of cells in culture as a simple model of stress, we have addressed whether LD biogenesis has a protective role in cells committed to die. Complete nutrient deprivation induced the biogenesis of LD in human LN18 glioblastoma and HeLa cells and also in CHO and rat primary astrocytes. In all cell types, death was associated with LD depletion and was accelerated by blocking LD biogenesis after pharmacological inhibition of Group IVA phospholipase A2 (cPLA2α) or down-regulation of ceramide kinase. Nutrient deprivation also induced β-oxidation of fatty acids that was sensitive to cPLA2α inhibition, and cell survival in these conditions became strictly dependent on fatty acid catabolism. These results show that, during nutrient deprivation, cell viability is sustained by β-oxidation of fatty acids that requires biogenesis and mobilization of LD.

The p57 CDKi integrates stress signals into cell‐cycle progression to promote cell survival upon stress

The p57Kip2 cyclin‐dependent kinase inhibitor (CDKi) has been implicated in embryogenesis, stem‐cell senescence and pathologies, but little is known of its role in cell cycle control. Here, we show that p57Kip2 is targeted by the p38 stress‐activated protein kinase (SAPK). Phosphorylation of p57Kip2 at T143 by p38 enhances its association with and inhibition of Cdk2, which results in cell‐cycle delay upon stress. Genetic inactivation of the SAPK or the CDKi abolishes cell‐cycle delay upon osmostress and results in decreased cell viability. Oxidative stress and ionomycin also induce p38‐mediated phosphorylation of p57 and cells lacking p38 or p57 display reduced viability to these stresses. Therefore, cell survival to various stresses depends on p57 phosphorylation by p38 that inhibits CDK activity. Together, these findings provide a novel molecular mechanism by which cells can delay cell cycle progression to maximize cell survival upon stress.

The p38 SAPK is recruited to chromatin via its interaction with transcription factors.

In mammals, the stress-activated protein kinase (SAPK) p38 coordinates a rapid and complex transcriptional program to adapt to sudden changes in the extracellular environment. Although a number of genes have been reported to be under the control of p38, the basic mechanisms of transcriptional regulation by this SAPK remain uncharacterized. Here we show that in response to osmotic shock, anisomycin- or TNFα-activated p38 SAPK is recruited to stress-induced genes. The MAPKK MKK6 is also found at stress-responsive promoters. The recruitment of RNA polymerase II complex to the target promoters requires p38 activity. Moreover, when tethered to DNA as a LexA fusion protein, p38 activates transcription in a stress-regulated manner. Thus, p38 activity allows for recruitment of RNA polymerase and transcription initiation. p38 directly phosphorylates and interacts with the transcription factor Elk1. p38 activity is necessary for the recruitment of Elk1 to the c-Fos promoter, and knocking down Elk1 by siRNAs compromises both p38 recruitment to the c-Fos promoter and c-Fos transcriptional up-regulation upon osmostress. In addition, p38 recruitment to the osmoinducible gene Cox2 and the TNFα target gene IL8 is mediated by the transcription factors AP1 and NFκB, respectively. Therefore, anchoring of active SAPK to target genes is mediated by transcription factors. The presence of active p38 at open reading frames also suggests the involvement of the SAPK in elongation. Taken together, SAPK recruitment to target genes appears to be a broad mechanism to regulate transcription that has been preserved from yeast to mammals.

NAADP mediates ATP-induced Ca2+ signals in astrocytes.

Intracellular Ca2+ signals provide astrocytes with a specific form of excitability that enables them to regulate synaptic transmission. In this study, we demonstrate that NAADP‐AM, a membrane‐permeant analogue of the new second messenger nicotinic acid‐adenine dinucleotide phosphate (NAADP), mobilizes Ca2+ in astrocytes and that the response is blocked by Ned‐19, an antagonist of NAADP signalling. We also show that NAADP receptors are expressed in lysosome‐related acidic vesicles. Pharmacological disruption of either NAADP or lysosomal signalling reduced Ca2+ responses induced by ATP and endothelin‐1, but not by bradykinin. Furthermore, ATP increased endogenous NAADP levels. Overall, our data provide evidence for NAADP being an intracellular messenger for agonist‐mediated calcium signalling in astrocytes.

A novel G1 checkpoint mediated by the p57 CDK inhibitor and p38 SAPK promotes cell survival upon stress

Comment on: Joaquin M, et al. EMBO J 2012; 31:2952-64.