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Macrophage autophagy is a highly anti-atherogenic process that promotes the catabolism of cytosolic lipid droplets (LDs) to maintain cellular lipid homeostasis. Selective autophagy relies on tags such as ubiquitin and a set of selectivity factors including selective autophagy receptors (SARs) to label specific cargo for degradation. Originally described in yeast cells, “lipophagy” refers to the degradation of LDs by autophagy. Yet, how LDs are targeted for autophagy is poorly defined. Here, we employed mass spectrometry to identify lipophagy factors within the macrophage foam cell LD proteome. In addition to structural proteins (e.g., PLIN2), metabolic enzymes (e.g., ACSL) and neutral lipases (e.g., PNPLA2), we found the association of proteins related to the ubiquitination machinery (e.g., AUP1) and autophagy (e.g., HMGB, YWHA/14-3-3 proteins). The functional role of candidate lipophagy factors (a total of 91) was tested using a custom siRNA array combined with high-content cholesterol efflux assays. We observed that knocking down several of these genes, including Hmgb1, Hmgb2, Hspa5, and Scarb2, significantly reduced cholesterol efflux, and SARs SQSTM1/p62, NBR1 and OPTN localized to LDs, suggesting a role for these in lipophagy. Using yeast lipophagy assays, we established a genetic requirement for several candidate lipophagy factors in lipophagy, including HSPA5, UBE2G2 and AUP1. Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Targeting these to selectively enhance lipophagy to promote cholesterol efflux in foam cells may represent a novel strategy to treat atherosclerosis. Abbreviations: ADGRL3: adhesion G protein-coupled receptor L3; agLDL: aggregated low density lipoprotein; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BMDM: bone-marrow derived macrophages; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; BSA: bovine serum albumin; CALCOCO2: calcium binding and coiled-coil domain 2; CIRBP: cold inducible RNA binding protein; COLGALT1: collagen beta(1-O)galactosyltransferase 1; CORO1A: coronin 1A; DMA: deletion mutant array; Faa4: long chain fatty acyl-CoA synthetase; FBS: fetal bovine serum; FUS: fused in sarcoma; HMGB1: high mobility group box 1; HMGB2: high mobility group box 2: HSP90AA1: heat shock protein 90: alpha (cytosolic): class A member 1; HSPA5: heat shock protein family A (Hsp70) member 5; HSPA8: heat shock protein 8; HSPB1: heat shock protein 1; HSPH1: heat shock 105kDa/110kDa protein 1; LDAH: lipid droplet associated hydrolase; LIPA: lysosomal acid lipase A; LIR: LC3-interacting region; MACROH2A1: macroH2A.1 histone; MAP1LC3: microtubule-associated protein 1 light chain 3; MCOLN1: mucolipin 1; NBR1: NBR1, autophagy cargo receptor; NPC2: NPC intracellular cholesterol transporter 2; OPTN: optineurin; P/S: penicillin-streptomycin; PLIN2: perilipin 2; PLIN3: perilipin 3; PNPLA2: patatin like phospholipase domain containing 2; RAB: RAB, member RAS oncogene family; RBBP7, retinoblastoma binding protein 7, chromatin remodeling factor; SAR: selective autophagy receptor; SCARB2: scavenger receptor class B, member 2; SGA: synthetic genetic array; SQSTM1: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; TFEB: transcription factor EB; TOLLIP: toll interacting protein; UBE2G2: ubiquitin conjugating enzyme E2 G2; UVRAG: UV radiation resistance associated gene; VDAC2: voltage dependent anion channel 2; VIM: vimentin

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.

Under environmental stress, such as glucose deprivation, cells form stress granules - the accumulation of cytoplasmic aggregates of repressed translational initiation complexes, proteins, and stalled mRNAs. Recent research implicates stress granules in various diseases, such as neurodegenerative diseases, but the exact regulators responsible for the assembly and disassembly of stress granules are unknown. An important aspect of stress granule formation is the presence of post-translational modifications on core proteins. One of those modifications is lysine acetylation, which is regulated by either a lysine acetyltransferase (KAT) or a lysine deacetylase (KDAC) enzyme. This work deciphers the impact of lysine acetylation on an essential protein found in Saccharomyces cerevisiae stress granules, poly(A) binding protein (Pab1). We demonstrated that an acetylation mimic of the lysine residue in the position 131 reduces stress granule formation upon glucose deprivation and other stressors such as ethanol, raffinose, and vanillin. We present genetic evidence that the enzyme Rpd3 is the primary candidate for deacetylation of Pab1-K131. Further, our electromobility shift assay studies suggest that the acetylation of Pab1-K131 negatively impacts poly(A) RNA binding. Due to the conserved nature of stress granules, therapeutics targeting the activity of KATs and KDAC enzymes may be a promising route to modulate stress granule dynamics in the disease state.

Cellular metabolism is tightly regulated by many signaling pathways and processes, including lysine acetylation of proteins. While lysine acetylation of metabolic enzymes can directly influence enzyme activity, there is growing evidence that lysine acetylation can also impact protein localization. As the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 has been implicated in a variety of metabolic processes, we have explored whether NuA4 controls the localization and/or protein levels of metabolic proteins. We performed a high-throughput microscopy screen of over 360 GFP-tagged metabolic proteins and identified 23 proteins whose localization and/or abundance changed upon deletion of the NuA4 scaffolding subunit, EAF1. Within this, three proteins were required for glycogen synthesis and 14 proteins were associated with the mitochondria. We determined that in eaf1Δ cells the transcription of glycogen biosynthesis genes is upregulated resulting in increased proteins and glycogen production. Further, in the absence of EAF1, mitochondria are highly fused, increasing in volume approximately 3-fold, and are chaotically distributed but remain functional. Both the increased glycogen synthesis and mitochondrial elongation in eaf1Δ cells are dependent on Bcy1, the yeast regulatory subunit of PKA. Surprisingly, in the absence of EAF1, Bcy1 localization changes from being nuclear to cytoplasmic and PKA activity is altered. We found that NuA4-dependent localization of Bcy1 is dependent on a lysine residue at position 313 of Bcy1. However, the glycogen accumulation and mitochondrial elongation phenotypes of eaf1Δ, while dependent on Bcy1, were not fully dependent on Bcy1-K313 acetylation state and subcellular localization of Bcy1. As NuA4 is highly conserved with the human Tip60 complex, our work may inform human disease biology, revealing new avenues to investigate the role of Tip60 in metabolic diseases., Author summary Metabolism, how cells process nutrients and energy substrates, is dramatically altered in many diseases. For example, during tumorigenesis, changes in cellular metabolism allow for the rapid cellular growth and division. One way to control metabolism is by moving the proteins involved in metabolism to different parts of the cell. Similar to an assembly line, when all the “parts” (metabolic proteins) are brought together, efficiency can be increased. In contrast, if one or more parts are shipped to the wrong place (mislocalized), production is decreased, and an alternative production process may even be necessary to meet demands. Here we ask whether Tip60, a protein complex implicated in many diseases, affects the location of metabolic proteins in the cell. We use budding yeast as a model system to assess the effect of disrupting yeast Tip60 on the location of over 360 metabolic proteins. We found that Tip60 controls the location of many metabolic proteins and in the absence of Tip60 there is an increase in the mitochondria and in cellular carbohydrate storage (the metabolic ‘hubs’ of the cell). This work suggests that yeast Tip60 functions as a “brake” on cellular metabolism and provides novel potential roles of Tip60 in metabolic diseases.

Eukaryotic cells form stress granules under a variety of stresses, however the signaling pathways regulating their formation remain largely unknown. We have determined that the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 is required for stress granule formation upon glucose deprivation but not heat stress. Further, the Tip60 complex, the human homolog of the NuA4 complex, is required for stress granule formation in cancer cell lines. Surprisingly, the impact of NuA4 on glucose-deprived stress granule formation is partially mediated through regulation of acetyl-CoA levels, which are elevated in NuA4 mutants. While elevated acetyl-CoA levels suppress the formation of glucose-deprived stress granules, decreased acetyl-CoA levels enhance stress granule formation upon glucose deprivation. Further our work suggests that NuA4 regulates acetyl-CoA levels through the Acetyl-CoA carboxylase Acc1. Altogether this work establishes both NuA4 and the metabolite acetyl-CoA as critical signaling pathways regulating the formation of glucose-deprived stress granules., Author summary In response to environmental stress, such as nutrient limitations or toxic chemicals, cells must quickly counteract these threats in order to survive. One way cells fight environmental challenges is through the formation of stress granules, which are aggregates of proteins and mRNA within the cytoplasm. Though their formation is essential for survival under multiple conditions, when stress granules are inappropriately formed they become causative for diseases such as amyotrophic lateral sclerosis and fragile X syndrome. Further stress granules contribute to chemotherapy resistance of cancer cells by promoting survival. Therefore it is critical to understand how stress granules are formed and disassembled. Here using the budding yeast Saccharomyces cerevisiae we determine that an enzyme called NuA4 is contributing to stress granule formation upon glucose deprivation. Why is this important? We determine that Tip60, the equivalent of NuA4 in mammalian cells, is also regulating stress granule formation in cancer cells. As there is a growing number of drugs that target this class of enzyme, there is the possibility that these drugs may reduce stress granule formation offering a novel therapeutic approach to treat numerous diseases.

c-Myc transcriptional activity in cells is dampened by the Mad family of transcriptional repressors. The expression of one member, hMad4, is increased in growth-arrested states such as quiescence or replicative senescence; hMad4 mRNA levels in replicatively senescent fibroblasts are about twice those seen in their young contact-inhibited quiescent counterparts. Moreover, the repression of hMad4 transcription following serum stimulation observed in quiescent young fibroblasts is lost in senescent cells. This loss results in persistent expression of hMad4, which leads to an inability to switch from an hMad4/Max complex to a c-Myc/Max complex on selected c-Myc target genes following serum stimulation. We have located an initiator element (Inr), a candidate for Miz-1 binding, in the hMad4 promoter. In reporter assays, Miz-1 enhances reporter GFP expression; this enhancement is inhibited by co-expressing c-Myc. Thus hMad4, as does its murine counterpart, contains the Inr element through which Miz-1 activates its expression; but this action is inhibited in the presence of c-Myc. This inhibition may explain the down-regulation of hMad4, corresponding to the up-regulation of c-Myc, in young serum-starved quiescent fibroblasts upon serum stimulation. However, this reciprocal change does not occur in replicatively senescent fibroblasts upon serum stimulation; instead, hMad4 persists in the presence of high levels of c-Myc activation. Our results suggest that: (1) replicative senescence-specific factors may block c-Myc inhibition of Miz-1 activation of hMad4 expression; and (2) the continual presence of hMad4 protein may transcriptionally repress selected c-Myc target genes, whose functions are key to the signaling pathways leading to apoptosis inhibition and permanent exit of cell cycle traverse in normal human fibroblasts. J. Cell. Biochem. © 2005 Wiley-Liss, Inc.

Telomerase is a reverse transcriptase that uses an integral RNA molecule to add de novo G-rich repeats onto telomeric DNA, or onto nontelomeric DNA generated during chromosome fragmentation and breakage events. A telomerase-mediated DNA substrate cleavage activity has been reported in ciliates and yeasts. Nucleolytic cleavage may serve a proofreading function, enhance processivity or ensure that nontemplate telomerase RNA sequences are not copied into DNA. We identified and characterized a human telomerase-mediated nucleolytic cleavage activity using enzyme reconstituted in a rabbit reticulocyte lysate in vitro transcription/translation system and native enzyme extracted from cells. We found that telomerase catalyzed the removal of nucleotides from DNA substrates including those that can form a mismatch with the RNA template or that contain nontelomeric sequences located 3' to a telomeric sequence. Unlike Tetrahymena telomerase, human telomerase catalyzed the removal of more than one nucleotide (up to 13) from telomeric primers. DNA substrates predicted to align at the 3'-end of the RNA template were not cleaved, consistent with cleavage being dictated by the template 5'-end. We also found some differences in the nuclease activity between RRL-reconstituted human telomerase and native enzyme.

The catalytic subunit of telomerase (TERT) contains conserved reverse transcriptase-like motifs but N- and C-terminal regions unique to telomerases. Despite weak sequence conservation, the C terminus of TERTs from various organisms has been implicated in telomerase-specific functions, including telomerase activity, functional multimerization with other TERT molecules, enzyme processivity and telomere length maintenance. We studied hTERT proteins containing small C-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase activity, hTERT multimerization and processivity. Using sequence alignment of five vertebrate TERTs and Arabidopsis thaliana TERT, we identified blocks of highly conserved amino acids that were required for human telomerase activity and functional hTERT complementation. We adapted the non-PCR-based telomerase elongation assay to characterize telomerase expressed and reconstituted in the in vitro transcription/translation rabbit reticulocyte lysate system. Using this assay, we found that the hTERT C terminus, like the C terminus of Saccharomyces cerevisiae TERT, contributes to successive nucleotide addition within a single 6-base telomeric repeat (type I processivity). Certain mutations in the hTERT C terminus also reduced the repetitive addition of multiple telomeric repeats (type II processivity). Our results suggest a functionally conserved role for the TERT C terminus in telomerase enzyme processivity.

Telomerase is a specialized RNA template-containing reverse transcriptase that mediates telomere repeat synthesis at chromosome ends. The maintenance of telomere length and integrity is essential for cell survival. Telomerase is active in most immortal and tumor cells, whereas the majority of normal human cells demonstrate no detectable activity and undergo telomere shortening. The identification of a possible role for telomerase in cellular aging and cancer has led to numerous studies designed to characterize this ribonucleoprotein enzyme. Inhibiting telomerase activity in immortal human cells reduces cellular proliferative capacity and can lead to cell death. Identifying mechanisms to specifically inhibit telomerase activity in malignant cells could thus be of great therapeutic value in the treatment of cancer. In this review, we summarize the current understanding of the mechanism of action of human telomerase. The biochemical characterization of telomerase is necessary for the design and evaluation of antitelomerase therapies. Different strategies are currently under investigation to design inhibitors that target the reverse transcriptase and RNA components of the telomerase complex. Recent advances in the design of these inhibitors and their properties are discussed.

Functional human telomerase complexes are minimally composed of the human telomerase RNA (hTR) and a catalytic subunit (human telomerase reverse transcriptase [hTERT]) containing reverse transcriptase (RT)-like motifs. The N terminus of TERT proteins is unique to the telomerase family and has been implicated in catalysis, telomerase RNA binding, and telomerase multimerization, and conserved motifs have been identified by alignment of TERT sequences from multiple organisms. We studied hTERT proteins containing N-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase catalytic activity, hTR binding, and hTERT multimerization. Using multiple sequence alignment, we identified two vertebrate-conserved TERT N-terminal regions containing vertebrate-specific residues that were required for human telomerase activity. We identified two RNA interaction domains, RID1 and RID2, the latter containing a vertebrate-specific RNA binding motif. Mutations in RID2 reduced the association of hTR with hTERT by 50 to 70%. Inactive mutants defective in RID2-mediated hTR binding failed to complement an inactive hTERT mutant containing an RT motif substitution to reconstitute activity. Our results suggest that functional hTERT complementation requires intact RID2 and RT domains on the same hTERT molecule and is dependent on hTR and the N terminus.

Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets.

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell death in mammalian and fission yeast cells, suggesting that Vpr may affect a conserved cellular process. It is unclear, however, whether Vpr-induced yeast cell death mimics Vpr-mediated apoptosis in mammalian cells. We have recently identified a number of Vpr suppressors that not only suppress Vpr-induced cell death in fission yeast, but also block Vpr-induced apoptosis in mammalian cells. These findings suggest that Vpr-induced cell death in yeast may resemble some of the apoptotic processes of mammalian cells. The goal of this study was to develop and validate a fission yeast model system for future studies of apoptosis. Similar to Vpr-induced apoptosis in mammalian cells, we show here that Vpr in fission yeast promotes phosphatidylserine externalization and induces hyperpolarization of mitochondria, leading to changes of mitochondrial membrane potential. Moreover, Vpr triggers production of reactive oxygen species (ROS), indicating that the apoptotic-like cell death might be mediated by ROS. Interestingly, Vpr induces unique morphologic changes in mitochondria that may provide a simple marker for measuring the apoptotic-like process in fission yeast. To verify this possibility, we tested two Vpr suppressors (EF2 and Hsp16) that suppress Vpr-induced apoptosis in mammalian cells in addition to a newly identified Vpr suppressor (Skp1). All three proteins abolished cell death mediated by Vpr and restored normal mitochondrial morphology in the yeast cells. In conclusion, Vpr-induced cell death in fission yeast resembles the mammalian apoptotic process. Fission yeast may thus potentially be used as a simple model organism for the future study of the apoptotic-like process induced by Vpr and other proapoptotic agents.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a virion-associated accessory protein with an average length of 96 amino acids and a calculated molecular mass of 12.7 kDa (14). Increasing evidence suggests that Vpr plays an important role in the viral life cycle and pathogenesis of HIV-1. For example, Vpr is required both in vitro and in vivo for efficient viral infection of nondividing cells such as monocytes and macrophages (21-23). Extracellular addition of Vpr to latently infected T lymphocytes markedly increased HIV-1 replication (32). Rhesus monkeys, chimpanzees, and human subjects infected with Vpr-defective viruses demonstrate slow disease progression, often accompanied by reversion of the mutated vpr genes back to the wild-type phenotype (17, 19, 30, 56, 65). Vpr displays several distinct activities in host cells. One of these activities is to lock host cells into the G2 phase of the cell cycle, known as the G2 arrest (22, 24, 51, 52). The cell cycle G2 arrest induced by Vpr is thought to suppress human immune functions by preventing T-cell clonal expansion (50) and to provide an optimized cellular environment for maximal levels of viral replication (19). Therefore, further understanding of Vpr-induced cell cycle G2 arrest could provide additional insights into molecular actions of Vpr in augmenting viral replication and modulation of host immune responses. Vpr induces cell cycle G2 arrest in eukaryotic cells ranged from fission yeast (Schizosaccharomyces pombe) to mammalian cells, suggesting the cellular pathway(s) targeted by Vpr must be highly conserved. Indeed, previous studies have demonstrated that Vpr induces G2 arrest by promoting hyperphosphorylation of fission yeast Cdc2 or human Cdk1, a kinase that determines onset of mitosis in all eukaryotes (22, 51, 64). Vpr exerts its inhibitory effect through T14 and Y15 of Cdk1 and Y15 of Cdc2, since the expression of nonphosphorylated mutants, T14A Y15F of Cdk1 or Y15F of Cdc2, prevents Vpr-induced G2 arrest (12, 22). Furthermore, Vpr inhibits the Cdc25 phosphatase (3, 13) and activates Wee1 kinase (13, 58) to promote phosphorylation of Cdc2/Cdk1 during induction of G2 arrest. Cdc25 normally dephosphorylates Cdc2/Cdk1 to promote mitosis, whereas Wee1 kinase phosphorylates Cdc2/Cdk1 that prevents entry of mitosis. Consistent with the roles of Wee1 and Cdc25 in Vpr-induced G2 arrest, proteins that are involved in the regulation of Cdc25 or Wee1 have been identified to either enhance or inhibit Vpr-induced G2 arrest. Fission yeast Wos2, which is a human p23 homologue and a Wee1 inhibitor (45), has been shown to be a multicopy Vpr suppressor (13). A Cdc25 inhibitor rad24 (36), which is the human 14-3-3 homologue, enhances Vpr-induced G2 arrest when overproduced in fission yeast (13). It is thus clear that a regulated balance between the Wee1 kinase and the Cdc25 phosphatase is critical to determine the Cdc2 activity that in turn regulates the G2/M transition (13, 18, 41). Upon DNA damage or inhibition of DNA replication, cellular DNA checkpoint responds to these cellular crises by inducing cell cycle G2 arrest. The G2 arrest is achieved by shuffling Cdc25 to the cytoplasm, where Cdc25 is no longer able to access Cdc2 and is further degraded by proteolysis (2, 49, 54). In fission yeast, as well as in human cells, the nuclear exclusion of Cdc25 is dependent on Rad24/14-3-3 proteins and Cdc25 phosphorylation, which is mediated through the ATR/ATM-activated Chk1/Chk2 kinases (27, 36, 49, 54, 61). Typically, 14-3-3 binds to phosphorylated Cdc25 that propels it from the nucleus to the cytoplasm, where it is undergoes proteasome-mediated degradation (2, 49, 54). Consistent with this model, a mutant allele of cdc25 that cannot bind to 14-3-3 proteins or a mutant Cdc25 that cannot be phosphorylated at its nine serine/threonine sites remains nuclear, thus allowing cells to enter mitosis in spite of treatment with agents that activate the mitotic DNA checkpoints (61). Similar to the regulation of Cdc25 in cellular DNA checkpoint responses, Vpr appears to prevent Cdc25C from entering the nucleus (25). Furthermore, Vpr binds to Cdc25C and 14-3-3 in human cells (18, 26), providing a possible mechanistic basis for the effect of Vpr on Cdc25 and the cell cycle G2/M regulation. However, whether the upstream kinases such as Chk1 or Cds1/Chk2, which normally phosphorylate Cdc25 during the DNA checkpoints, are specifically responsible for the phosphorylation of Cdc25 and subsequent nuclear exclusion and protein degradation is at present unknown. Whether Chk1 or Cds1/Chk2 is responsible for the G2 arrest induced by Vpr is controversial. In fission yeast, mutations in both chk1 and cds1, which are thought to be part of the checkpoints (6, 16, 60), do not block Vpr-induced G2 arrest (13, 41). However, reports from mammalian studies showed that Vpr activates Chk1 for the G2 induction (67, 68). Considering that mitotic DNA checkpoints are highly conserved between mammalian and fission yeast cells, it is unclear at the moment why, given that activation of human Chk1 by Vpr is at least partially required for G2 arrest, deletion of chk1, cds1, or chk1/cds1 (homologues of Chk1/Chk2) does not block Vpr-induced G2 arrest in fission yeast (12, 13). One possibility is that other kinase(s), i.e., other than Chk1 or Cds1/Chk2, are involved in the phosphorylation of Cdc25 during the G2 induction by Vpr. The fission yeast Srk1 kinase (for Sty1-regulated kinase 1) and its mammalian counterpart, MAPKAP kinase-2 (MK2), have recently been implicated as a third possible kinase, in addition to Chk1 and Chk2, to regulate Cdc25 (35, 38). The Srk1 phosphorylates Cdc25 by direct interaction at the same phosphorylation site as Chk1 and Cds1, and that overexpression of srk1 causes cell cycle arrest in a cell lacking both Cds1 and Chk1 (35). Importantly, Srk1 does not regulate Cdc25 in response to the DNA damage or replication checkpoints but only under the normal growth conditions or in response to nongenotoxic environmental stress (35). Similarly, Ser216 of Cdc25C has also been shown to be the optimal phosphorylation site by MK2 and depletion of MK2 by small interfering RNA (siRNA) abolished UV-induced Cdc25C phosphorylation (38). Because of the controversy regarding the involvement of Chk1/Chk2 and the other regulators of Cdc25, it is possible that Vpr may inhibit Cdc25 by phosphorylation through a different kinase such as Srk1/MK2. Thus, a more detailed knowledge of the mechanisms involving Cdc25 should provide new insight about the cellular response to vpr gene expression during the induction of cell cycle G2 arrest. The goal of the present study was to use the fission yeast model to further delineate the molecular regulation of Cdc25 in response to vpr gene expression. We hypothesized that Vpr induces cell cycle G2 arrest at least in part through an Srk1/MK2-mediated regulatory pathway. The results described here confirm this hypothesis. Our data in fission yeast have further shown that, as in mammalian cells and the DNA checkpoints, Vpr also promote nuclear exclusion of Cdc25 through a Rad24/14-3-3-dependent mechanism. However, Chk1/Cds1, the fission yeast homologues of human Chk1 and Chk2, are not involved in the Cdc25 nuclear exclusion. Instead, Srk1/MK2 is one of the kinases modulated by Vpr to inhibit Cdc25 for the induction of cell cycle G2 arrest.