Involvement of dihydroceramide desaturase in cell cycle progression in human neuroblastoma cells

The role of dihydroceramide desaturase as a key enzyme in the de novo pathway of ceramide generation was investigated in human neuroblastoma cells (SMS-KCNR). A novel assay using water-soluble analogs of dihydroceramide, dihydroceramidoids (D-erythro-dhCCPS analogs), was used to measure desaturase activity in situ. Conversion of D-erythro-2-N-[12'-(1'-pyridinium)-dodecanoyl]-4,5-dihydrosphingosine bromide (C(12)-dhCCPS) to its 4,5-desaturated counterpart, D-erythro-2-N-[12'-(1'-pyridinium)dodecanoyl]sphingosine bromide (C(12)-CCPS), was determined by liquid chromatography/mass spectrometry analysis. The validity of the assay was confirmed using C(8)-cyclopropenylceramide, a competitive inhibitor of dihydroceramide desaturase. A human homolog (DEGS-1) of the Drosophila melanogaster des-1 gene was recently identified and reported to have desaturase activity. Transfection of SMS-KCNR cells with small interfering RNA to DEGS-1 significantly blocked the conversion of C(12)-dhCCPS to C(12)-CCPS. The associated accumulation of endogenous dihydroceramides confirmed DEGS-1 as the main active dihydroceramide desaturase in these cells. The partial loss of DEGS-1 inhibited cell growth, with cell cycle arrest at G(0)/G(1). This was accompanied by a significant decrease in the amount of phosphorylated retinoblastoma protein. This hypophosphorylation was inhibited by tautomycin and not by okadaic acid, suggesting the involvement of protein phosphatase 1. Additionally, we found that treatment of SMS-KCNR cells with fenretinide inhibited desaturase activity in a dose-dependent manner. An increase in dihydroceramides (but not ceramides) paralleled this process as measured by liquid chromatography/mass spectrometry. There were no effects on the mRNA or protein levels of DEGS-1, suggesting that fenretinide acts at the post-translational level as an inhibitor of this enzyme. Tautomycin was also able to block the hypophosphorylation of the retinoblastoma protein observed upon fenretinide treatment. These findings suggest a novel biological function for dihydroceramides.     

Ablation of Dihydroceramide Desaturase Confers Resistance to Etoposide-Induced Apoptosis In Vitro

Sphingolipid biosynthesis is potently upregulated by factors associated with cellular stress, including numerous chemotherapeutics, inflammatory cytokines, and glucocorticoids. Dihydroceramide desaturase 1 (Des1), the third enzyme in the highly conserved pathway driving sphingolipid biosynthesis, introduces the 4,5-trans-double bond that typifies most higher-order sphingolipids. Surprisingly, recent studies have shown that certain chemotherapeutics and other drugs inhibit Des1, giving rise to a number of sphingolipids that lack the characteristic double bond. In order to assess the effect of an altered sphingolipid profile (via Des1 inhibition) on cell function, we generated isogenic mouse embryonic fibroblasts lacking both Des1 alleles. Lipidomic profiling revealed that these cells contained higher levels of dihydroceramide than wild-type fibroblasts and that complex sphingolipids were comprised predominantly of the saturated backbone (e.g. sphinganine vs. sphingosine, dihydrosphingomyelin vs. sphingomyelin, etc.). Des1 ablation activated pro-survival and anabolic signaling intermediates (e.g. Akt/PKB, mTOR, MAPK, etc.) and provided protection from apoptosis caused by etoposide, a chemotherapeutic that induces sphingolipid synthesis by upregulating several sphingolipid biosynthesizing enzymes. These data reveal that the double bond present in most sphingolipids has a profound impact on cell survival pathways, and that the manipulation of Des1 could have important effects on apoptosis.     

Dihydroceramide desaturase and dihydrosphingolipids: debutant players in the sphingolipid arena

Sphingolipids are a wide family of lipids that share common sphingoid backbones, including (2S,3R)-2-amino-4-octadecane-1,3-diol (dihydrosphingosine) and (2S,3R,4E)-2-amino-4-octadecene-1,3-diol (sphingosine). The metabolism and biological functions of sphingolipids derived from sphingosine have been the subject of many reviews. In contrast, dihydrosphingolipids have received poor attention, mainly due to their supposed lack of biological activity. However, the reported biological effects of active site directed dihydroceramide desaturase inhibitors and the involvement of dihydrosphingolipids in the response of cells to known therapeutic agents support that dihydrosphingolipids are not inert but are in fact biologically active and underscore the importance of elucidating further the metabolic pathways and cell signaling networks involved in the biological activities of dihydrosphingolipids. Dihydroceramide desaturase is the enzyme involved in the conversion of dihydroceramide into ceramide and it is crucial in the regulation of the balance between sphingolipids and dihydrosphingolipids. Furthermore, given the enzyme requirement for O? and the NAD(P)H cofactor, the cellular redox balance and dihydroceramide desaturase activity may reciprocally influence each other. In this review both dihydroceramide desaturase and the biological functions of dihydrosphingolipids are addressed and perspectives on this field are discussed.     

Metabolism of the unnatural anticancer lipid safingol,L-threo-dihydrosphingosine,in cultured cells

We studied the metabolism of radioactively labeled safingol (l-threo-dihydrosphingosine) in primary cultured neurons, B104 neuroblastoma cells, and Swiss 3T3 fibroblasts, and compared it to that of its natural stereoisomer d-erythro-dihydrosphingosine. Both sphingoid bases are used as biosynthetic precursors for complex sphingolipids, albeit to different rates. Whereas a considerable amount of the natural sphingoid base is also directed to the catabolic pathway (20-66%, cell type dependent), only a minor amount of the nonnatural safingol is subjected to catabolic cleavage, most of it being N-acylated to the respective stereochemical variant of dihydroceramide. Interestingly, N-acylation of safingol to l-threo-dihydroceramide is less sensitive to fumonisin B1 than the formation of the natural d-erythro-dihydroceramide. In addition, safingol-derived l-threo-dihydroceramide, unlike its physiologic counterpart, is not desaturated. Most of it either accumulates in the cells (up to 50%) or is used as a biosynthetic precursor of the respective dihydrosphingomyelin (up to 45%). About 5% is, however, glucosylated and channeled into the glycosphingolipid biosynthetic pathway. Our results demonstrate that, despite its nonnatural stereochemistry, safingol is recognized and metabolized preferentially by enzymes of the sphingolipid biosynthetic pathway. Furthermore, our data suggest that the cytotoxic potential of safingol is reduced rather than enhanced via its metabolic conversion.     

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C(16:0)-, C(24:0)-, C(24:1)-dihydroceramide (dhCer) and led to a depletion of C(24:0)-, C(24:1)-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using (13)C,(15)N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC(50) of 78.9 +/- 1.5 muM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib.     

Role for de novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae

The recent findings of sphingolipids as potential mediators of yeast heat stress responses led us to investigate their possible role in the heat-induced cell cycle arrest and subsequent recovery. The sphingolipid-deficient yeast strain 7R4 was found to lack the cell cycle arrest seen in the isogenic wild type. Furthermore, strain lcb1-100, which harbors a temperature-sensitive serine palmitoyltransferase, lacked increased de novo generated sphingoid bases upon heat stress. Importantly, this strain was found to lack the transient heat-induced G0/G1 arrest. These results indicate a role for sphingolipids and specifically those generated in the de novo pathway in the cell cycle arrest response to heat. To determine the bioactive sphingolipid regulating this response, an analysis of key mutants in the sphingolipid biosynthetic and degradation pathways was performed. Strains deleted in sphingoid base kinases, sphingoid phosphate phosphatase, lyase, or dihydrosphingosine hydroxylase were found to display the cell cycle arrest. Also, the knockout of a fatty acyl elongation enzyme, which severely attenuates ceramide production, displayed the arrest. These experiments suggested that the active species for cell cycle arrest were the sphingoid bases. In further support of these findings, exogenous phytosphingosine (10 microM) was found to induce transient arrest. Stearylamine did not induce an arrest, demonstrating chemical specificity, and L-erythro- was not as potent as D-erythro-dihydrosphingosine showing stereospecificity. To investigate a possible arrest mechanism, we studied the hyperstable Cln3 (Cln3-1) strain LDW6A that has been previously shown to be resistant to heat stress-induced cell cycle arrest. The strain containing Cln3-1 was found to be resistant to cell cycle arrest induced by exogenous phytosphingosine, indicating that Cln3 acts downstream of the sphingoid bases in this response. Interestingly, cell cycle recovery from the transient arrest was found to be dependent upon the sphingoid base kinases (LCB4, LCB5). Overall, this combination of genetic and pharmacologic results demonstrates a role for de novo sphingoid base biosynthesis by serine palmitoyltransferase in the transient G0/G1 arrest mediated through Cln3 via a novel mechanism.     

3-Deoxy-3,4-dehydro analogs of XM462. Preparation and activity on sphingolipid metabolism and cell fate

Three analogs of the dihydroceramide desaturase inhibitor XM462 are reported. The compounds inhibit both dihydroceramide desaturase and acid ceramidase, but with different potencies depending on the N-acyl moiety. Other enzymes of sphingolipid metabolism, such as neutral ceramidase, acid sphingomyelinase, acid glucosylceramide hydrolase, sphingomyelin synthase and glucosylceramide synthase, are not affected. The effect on the sphingolipidome of the two best inhibitors, namely (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)octanamide (RBM2-1B) and (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)pivalamide (RBM2-1D), is in accordance with the results obtained in the enzyme assays. These two compounds reduce cell viability in A549 and HCT116 cell lines with similar potencies and both induced apoptotic cell death to similar levels than C8-Cer in HCT116 cells. The possible therapeutic implications of the activities of these compounds are discussed.     

Multiple sphingolipid abnormalities following cerebral microendothelial hypoxia

Hypoxia has been previously shown to inhibit the dihydroceramide (DHC) desaturase, leading to the accumulation of DHC. In this study, we used metabolic labeling with [3H]‐palmitate, HPLC/MS/MS analysis, and specific inhibitors to show numerous sphingolipid changes after oxygen deprivation in cerebral microendothelial cells. The increased DHC, particularly long‐chain forms, was observed in both whole cells and detergent‐resistant membranes. This was reversed by reoxygenation and blocked by the de novo sphingolipid synthesis inhibitor myriocin, but not by the neutral sphingomyelinase inhibitor GW‐4869. Furthermore, oxygen deprivation of microendothelial cells increased levels of dihydro‐sphingosine (DH‐Sph), DH‐sphingosine1‐phosphate (DH‐S1P), DH‐sphingomyelin (DH‐SM), DH‐glucosylceramide (DH‐GlcCer), and S1P levels. In vitro assays revealed no changes in the activity of sphingomyelinases or sphingomyelin synthase, but resulted in reduced S1P lyase activity and 40% increase in glucosylceramide synthase (GCS) activity, which was reversed by reoxygenation. Inhibition of the de novo sphingolipid pathway (myriocin) or GCS (EtPoD4) induced endothelial barrier dysfunction and increased caspase 3‐mediated cell death in response to hypoxia. Our findings suggest that hypoxia induces synthesis of S1P and multiple dihydro‐sphingolipids, including DHC, DH‐SM, DH‐GlcCer, DH‐Sph and DH‐S1P, which may be involved in ameliorating the effects of stroke .

     

Dihydroceramide accumulation and reactive oxygen species are distinct and nonessential events in 4-HPR-mediated leukemia cell death

4-(Hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid with a strong apoptotic effect towards different cancer cell lines in vitro, and it is currently tested in clinical trials. Increases of reactive oxygen species (ROS) and modulation of endogenous sphingolipid levels are well-described events observed upon 4-HPR treatment, but there is still a lack of understanding of their relationship and their contribution to cell death. LC-MS analysis of sphingolipids revealed that in human leukemia CCRF-CEM and Jurkat cells, 4-HPR induced dihydroceramide but not ceramide accumulation even at sublethal concentrations. Myriocin prevented the 4-HPR-induced dihydroceramide accumulation, but it did not prevent the loss of viability and increase of intracellular ROS production. On the other hand, ascorbic acid, Trolox, and vitamin E reversed 4-HPR effects on cell death but not dihydroceramide accumulation. NDGA, described as a lipoxygenase inhibitor, exerted a significantly higher antioxidant activity than vitamin E and abrogated 4-HPR-mediated ROS. It did not however rescue cellular viability. Taken together, this study demonstrates that early changes observed upon 4-HPR treatment, i.e., sphingolipid modulation and ROS production, are mechanistically independent events. Furthermore, the results indicate that 4-HPR-driven cell death may occur even in the absence of dihydroceramide or ROS accumulation. These observations should be taken into account for an improved design of drug combinations.     

Cell cycle progression and cell polarity require sphingolipid biosynthesis in Aspergillus nidulans

Sphingolipids are major components of the plasma membrane of eukaryotic cells and were once thought of merely as structural components of the membrane. We have investigated effects of inhibiting sphingolipid biosynthesis, both in germinating spores and growing hyphae of Aspergillus nidulans. In germinating spores, genetic or pharmacological inactivation of inositol phosphorylceramide (IPC) synthase arrests the cell cycle in G(1) and also prevents polarized growth during spore germination. However, inactivation of IPC synthase not only eliminates sphingolipid biosynthesis but also leads to a marked accumulation of ceramide, its upstream intermediate. We therefore inactivated serine palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway, to determine effects of inhibiting sphingolipid biosynthesis without an accumulation of ceramide. This inactivation also prevented polarized growth but did not affect nuclear division of germinating spores. To see if sphingolipid biosynthesis is required to maintain polarized growth, and not just to establish polarity, we inhibited sphingolipid biosynthesis in cells in which polarity was already established. This inhibition rapidly abolished normal cell polarity and promoted cell tip branching, which normally never occurs. Cell tip branching was closely associated with dramatic changes in the normally highly polarized actin cytoskeleton and found to be dependent on actin function. The results indicate that sphingolipids are essential for the establishment and maintenance of cell polarity via control of the actin cytoskeleton and that accumulation of ceramide is likely responsible for arresting the cell cycle in G(1).     

The dihydroceramide desaturase is not essential for cell viability in Schizosaccharomyces pombe

Recent studies have identified a new family of desaturase-like polypeptide sequences in many higher eukaryotes. Functional characterisation of one member of this family, from Schizosaccharomyces pombe, revealed the enzyme to be a sphingolipid desaturase. This S. pombe gene designated SDCB3b8.07c was identified as the dihydroceramide Delta(4)-desaturase, responsible for the synthesis of sphingosine. Homologous recombination was used to disrupt the endogenous S. pombe dihydroceramide Delta(4)-desaturase. Surprisingly, this had no effect on cell viability, indicating that sphingosine may not be crucial for normal S. pombe functions. This observation has implications for our understanding of the role of sphingosine and its phosphorylated metabolite sphingosine-1-phosphate in lower eukaryotes.     

Neutral sphingomyelinase-2 mediates growth arrest by retinoic acid through modulation of ribosomal S6 kinase

All-trans-retinoic acid (ATRA) induces growth arrest of many cell types. Previous studies have reported that ATRA can modulate cellular sphingolipids, but the role of sphingolipids in the ATRA response is not clear. Using MCF-7 cells as a model system, we show that ATRA stimulates an increase in ceramide levels followed by G(0)/G(1) growth arrest. Notably, induction of nSMase2 was the primary effect of ATRA on the sphingolipid network and was both time- and dose-dependent. Importantly, pretreatment with nSMase2 siRNA significantly inhibited ATRA effects on ceramide levels and growth arrest. In contrast, nSMase2 overexpression was sufficient to increase ceramide levels and induce G(0)/G(1) growth arrest of asynchronous MCF-7 cells. Surprisingly, neither ATRA stimulation nor nSMase2 overexpression had significant effects on classical cell cycle regulators such as p21/WAF1 or retinoblastoma. In contrast, ATRA suppressed phosphorylation of ribosomal S6 kinase (S6K) and its downstream targets S6 and eIF4B. Importantly, these effects were significantly inhibited by nSMase2 siRNA. Reciprocally, nSMase2 overexpression was sufficient to suppress S6K phosphorylation and signaling. Notably, neither ATRA effects nor nSMase2 effects on S6K phosphorylation required the ceramide-activated protein phosphatase PP2A, previously identified as important for S6K regulation. Finally, nSMase2 overexpression was sufficient to decrease translation as measured by methionine incorporation and analysis of polyribosome profiles. Taken together, these results implicate nSMase2 as a major component of ATRA-induced growth arrest of MCF-7 cells and identify S6K as a novel downstream target of nSMase2.     

Lipophagy prevents activity‐dependent neurodegeneration due to dihydroceramide accumulation in vivo

Dihydroceramide desaturases are evolutionarily conserved enzymes that convert dihydroceramide (dhCer) to ceramide (Cer). While elevated Cer levels cause neurodegenerative diseases, the neuronal activity of its direct precursor, dhCer, remains unclear. We show that knockout of the fly dhCer desaturase gene, infertile crescent (ifc), results in larval lethality with increased dhCer and decreased Cer levels. Light stimulation leads to ROS increase and apoptotic cell death in ifc‐KO photoreceptors, resulting in activity‐dependent neurodegeneration. Lipid‐containing Atg8/LC3‐positive puncta accumulate in ifc‐KO photoreceptors, suggesting lipophagy activation. Further enhancing lipophagy reduces lipid droplet accumulation and rescues ifc‐KO defects, indicating that lipophagy plays a protective role. Reducing dhCer synthesis prevents photoreceptor degeneration and rescues ifc‐KO lethality, while supplementing downstream sphingolipids does not. These results pinpoint that dhCer accumulation is responsible for ifc‐KO defects. Human dhCer desaturase rescues ifc‐KO larval lethality, and rapamycin reverses defects caused by dhCer accumulation in human neuroblastoma cells, suggesting evolutionarily conserved functions. This study demonstrates a novel requirement for dhCer desaturase in neuronal maintenance in vivo and shows that lipophagy activation prevents activity‐dependent degeneration caused by dhCer accumulation.     

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2^-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2^-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2^-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.     

Entamoeba invadens: Sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6 h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous d-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.     

Production of α-Galactosylceramide by a Prominent Member of the Human Gut Microbiota

While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer_Bf), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer_Bf has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.     

Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast

The PKC1-associated mitogen-activated protein (MAP) kinase pathway of Saccharomyces cerevisiae regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. Activation of PKC1 occurs via the GTPase RHO1 and the kinase pair PKH1 and PKH2. Here we report that YPK1 and YPK2, an essential pair of homologous kinases and proposed downstream effectors of PKH and sphingolipids, are also regulators of the PKC1-controlled MAP kinase cascade. ypk mutants display random distribution of the actin cytoskeleton and severely reduced activation of the MAP kinase MPK1. Upregulation of the RHO1 GTPase switch or the PKC1 effector MAP kinase pathway suppresses the growth and actin defects of ypk cells. ypk lethality is also suppressed by overexpression of an uncharacterized gene termed TUS1. TUS1 is a novel RHO1 exchange factor that contributes to cell wall integrity-mediated modulation of RHO1 activity. Thus, TUS1 and the YPKs add to the growing complexity of RHO1 and PKC1 regulation in the cell integrity signaling pathway. Furthermore, our findings suggest that the YPKs are a missing link between sphingolipid signaling and the cell integrity pathway.