The role and mechanism of diacylglycerol-protein kinase C1 signaling in melanogenesis by Cryptococcus neoformans.

The fungus Cryptococcus neoformans is an opportunistic human pathogen that causes a life-threatening meningoencephalitis by expression of virulence factors such as melanin, a black pigment produced by the cell wall-associated enzyme laccase. In previous studies (Heung, L. J., Luberto, C., Plowden, A., Hannun, Y. A., and Del Poeta, M. (2004) J. Biol. Chem. 279, 21144-21153) we proposed that the sphingolipid enzyme inositol-phosphoryl ceramide synthase 1 (Ipc1) regulates melanin production through the generation of diacylglycerol (DAG), which was found to activate in vitro protein kinase C1 (Pkc1). Here, we investigated the molecular mechanisms by which DAG regulates Pkc1 in vivo and the effect of this regulation on laccase activity and melanin synthesis. To this end we deleted the putative DAG binding C1 domain of C. neoformans Pkc1 and found that the C1 deletion abolished the activation of Pkc1 by DAG. Deletion of the C1 domain repressed laccase activity and, consequently, melanin production. Finally, we show that these biological effects observed in the C1 deletion mutant are mediated by alteration of cell wall integrity and displacement of laccase from the cell wall. These studies define novel molecular mechanisms addressing Pkc1-laccase regulation by the sphingolipid pathway of C. neoformans, with important implications for understanding and targeting the Ipc1-Pkc1-laccase cascade as a regulator of virulence of this important human pathogen.

Cryptococcus neoformans is a fungal pathogen that infects humans via the respiratory tract. Dissemination of the infection leads to development of a life-threatening meningoencephalitis, particularly in immunocompromised patients. An important factor that enables C. neoformans to cause disease is the pigment melanin, which is proposed to protect the fungus from the host immune response (1)(2)(3) and function as an immunomodulator (4). Laccase, a cell wall-associated enzyme encoded by the CNLAC1 gene (5,6), catalyzes the formation of melanin from exogenous diphenolic substrates (7). A recent study by Noverr et al. (8) has demonstrated that laccase is required for the dissemination of C. neoformans from the lung to the brain, thus suggesting a role for melanin not only in the establishment of infection but also in the progression of disease.
Despite the importance of laccase and melanin to the pathogenicity of C. neoformans, relatively little is known about the molecular mechanisms by which they are regulated. A few signaling molecules, including the mitogen-activated protein kinase signaling cascade component STE12 (9,10) and cyclophilin A (11), have proposed roles in the regulation of laccase. The G␣ protein-cAMP-protein kinase A pathway has also been shown to regulate melanin production by C. neoformans (12)(13)(14). More recently, we have identified a novel regulatory pathway initiated by the sphingolipid enzyme inositol-phosphoryl ceramide synthase (Ipc1), 1 which transfers an inositol-phosphoryl moiety from phosphatidylinositol to phytoceramide to produce inositol-phosphorylceramide (IPC) and diacylglycerol (DAG). We found that the effects of Ipc1 on melanin production are potentially propagated through the generation of DAG and the subsequent activation of protein kinase C 1 (Pkc1) (15).
The sphingolipid pathway is an important source of cell signaling molecules not only in mammalian cells but also in fungal cells. For example, the sphingolipid pathway has crucial roles in the heat stress response (16,17) and endocytosis (18,19) in the yeast Saccharomyces cerevisiae. The roles and mechanisms of sphingolipid-metabolizing enzymes in pathogenic fungi, however, have not been extensively explored. The putative Ipc1-DAG-Pkc1 pathway is the first evidence that the sphingolipid pathway is crucial to the regulation of fungal virulence. Additionally, the existence of this pathway establishes a key role for DAG generated from sphingolipid metabolism in eukaryotic signal transduction.
C. neoformans Pkc1 contains a putative DAG binding domain, or C1 domain, which is highly homologous to the C1 domain of DAG-dependent mammalian PKCs (15). The role of the C1 domain as a mediator of DAG-Pkc1 signaling in fungal organisms is not well defined. Whereas biochemical data suggests that C. neoformans Pkc1 is activated by DAG (15), fungal Pkc1 homologs from S. cerevisiae and Candida albicans are not regulated by DAG (20 -22). Mammalian PKC isoforms regulated by DAG have conserved residues in the C1 domain required for DAG activation (Pro 11 , Gly 23 , and Gln 27 ) as well as five hydrophobic residues surrounding the DAG-binding site (positions 8, 13, 20, 22, and 24) (23). The three DAG-binding site residues and four of the five hydrophobic residues are * This work was supported in part by the Burroughs Wellcome Fund, National Institutes of Health Grant AI56168 (to M. D. P.), and Centers of Biomedical Research Excellence Program of the National Center for Research Resources Grant RR17677 Project 2 (to M. D. P.) and Project 6 (to C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  conserved in the C1 domain of C. neoformans Pkc1 (15) but largely absent from the C1 domains of S. cerevisiae and C. albicans Pkc1 enzymes (24). Thus, C. neoformans Pkc1 presents a unique opportunity to study the role of the C1 domain as a site of DAG regulation in a fungal system.
In the present study we investigated the in vivo mechanism of DAG-Pkc1 signaling and the role of this regulation in laccase activity and melanin formation. We found that the C1 domain is required for the activation of Pkc1, and in vivo the C1 deletion decreases laccase activity and melanin production in C. neoformans. We further established that through the regulation of cell wall integrity, the Ipc1-DAG-Pkc1 pathway may modulate the proper localization of the laccase enzyme to the fungal cell wall.

MATERIALS AND METHODS
Strains, Growth Media, and Reagents-C. neoformans var. grubii serotype A strain H99 (wild type) and C. neoformans strain GAL7::IPC1 (25) were routinely grown in yeast extract/peptone/dextrose medium or Difco TM yeast nitrogen base (YNB) plus 2% glucose. Yeast extract/ peptone (YP) or YNB media supplemented with 2% galactose or 2% glucose was used for the up-and down-regulation of IPC1 expression in the GAL7::IPC1 strain. All chemical reagents were obtained from Sigma unless otherwise noted.
Lipid Extraction-Approximately 5 ϫ 10 9 cells were collected from overnight liquid cultures, and lipids were extracted by the method of Bligh and Dyer (26). The amount (nmol) of inorganic phosphate (P i ) in each sample was measured as previously described (27). Triton-X-100/ lipid mixed micelles were prepared (28) with lipids at 8 mol% (based on P i measurements) and used in the in vitro kinase assay with recombinant C. neoformans Pkc1 protein, which was expressed in S. cerevisiae and immunoprecipitated as described previously (15).
Tagging of PKC1 Gene-To facilitate the study of endogenously expressed Pkc1 in Cryptococcus, the PKC1 gene was tagged with the V5 epitope in both wild type and GAL7::IPC1 strains. To produce the PKC1::V5 construct, a fragment of the 3Ј-untranslated region of the PKC1 locus was amplified using the primers PKCTAG3 (5Ј-CAT GAT ATC CAC GAC GAA GAG CAT GAG CAT GGA C-3Ј) and PKCTAG4 (5Ј-CTA CTC GAG GAG TGT GTG TGT CAT GTC TAG AGG C-3Ј, which contains a XhoI site (boldface and underlined)). This 3Ј-untranslated region (UTR) fragment was subcloned into pCR®2.1-TOPO® vector (Invitrogen), digested with EcoRI, blunted, digested with XhoI, and ligated into pCR2.1-TOPO-NAT1 (15), which had been digested with EcoRV and XhoI, thereby yielding pNAT1-3Ј-UTR. This plasmid contains the nourseothricin acetyltransferase 1 gene (NAT1) that confers resistance to the antibiotic nourseothricin (Werner BioAgents, Jena-Cospeda, Germany). The 3Ј end of the PKC1 gene was amplified using the primers PKCTAG1 (5Ј-CAT GAG CTC CAA TCC GAG AAA CGT GTG TTT TTG-3Ј, which contains a SacI site (boldface and underlined)) and PKCTAG2 (5Ј-CTA GGA TCC CTA CGT AGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC GGC TTG TGC TGC AGC CCA AGG AGC-3Ј, which contains a BamHI site (boldface and underlined) and V5 epitope tag (italics)). This PKC1::V5 fragment was digested with SacI and BamHI and ligated into pNAT1-3Ј-UTR region digested with SacI and BamHI. The resulting pPKC1::V5 was transformed into C. neoformans strains H99 and GAL7::IPC1 by biolistics as described previously (29), and transformants were selected on yeast extract/peptone/dextrose plates containing 100 mg/liter nourseothricin. Genomic DNA was extracted from stable transformants, digested with EcoRV, and used for Southern blot analysis.
Deletion of the C1 Domain of Pkc1-A ⌬C1-PKC1 construct was produced by amplifying fragments of the PKC1 gene upstream and downstream of the C1 domain using the primers 5ЈC1-up (5Ј-CAT AGA GCT CCG ATA TGA CGC ACC ACT GAC C-3Ј) and 3ЈC1-up (5Ј-GCG CAA GCT TAC CAT TCA TTT CGT AGA CAT C-3Ј), which contain SacI and HindIII sites, respectively (boldface and underlined), and C1-down (5Ј-CGT CAA GCT TGG TAT GAC TAT GGA GAT GGC C-3Ј), which contains a HindIII site (boldface and underlined) and PKCTAG2 (see sequence above), which contains a BamHI site and V5 epitope tag. The two fragments were subcloned separately into pCR®2.1-TOPO® vector (Invitrogen). Next, pPKC1::V5 (see above) was digested with SacI and BamHI to excise the PKC1::V5 fragment, and the remaining vector was ligated with the C1-up and C1-down fragments that had been digested from pCR®2.1-TOPO® vector. The resulting p⌬C1-PKC1::V5 was transformed into C. neoformans H99 and GAL7::IPC1 strains by biolistics, and transformants were selected on yeast extract/peptone/ dextrose plates containing 100 mg/liter nourseothricin. Genomic DNA was extracted from stable transformants, digested with EcoRI, and used in Southern blot analysis.
To prepare antibody-bead complexes, 2 l of anti-V5 antibody (Invitrogen) were combined with 50 l of protein G-Sepharose 4 Fast Flow (50% slurry) (Amersham Biosciences) in 1-ml final volume of modified radioimmune precipitation assay buffer and incubated for 1 h at 4°C on a rocking platform. The antibody-bead complexes were washed twice with modified radioimmune precipitation assay buffer and collected by centrifugation at 10,000 ϫ g for 1 min at 4°C. To pre-clear the cell lysate, an aliquot containing 30 g of total protein was placed in 1-ml final volume of modified radioimmune precipitation assay buffer with 200 l of protein G-Sepharose (50% slurry), rocked at 4°C for 1 h, and then centrifuged at 10,000 ϫ g for 1 min at 4°C. The precleared supernatant was added to the antibody-bead complexes, and the immunoprecipitation was carried out for 16 h at 4°C on a rocking platform.
The activities of immunoprecipitated Pkc1 and ⌬C1-PKC1 enzymes were measured as previously described (15). Briefly, the enzymes conjugated to protein G-Sepharose 4 Fast Flow were washed twice with kinase buffer (50 mM Tris, pH 7.4, 10 mM sodium orthovanadate, 10 mM sodium fluoride, 5 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, and 0.01% each of leupeptin, aprotinin, and trypsin-chymotrypsin inhibitor). The reaction was started by the addition of the reaction mixture (20 mM Tris, pH 7.4, 10 mM MgCl 2 , 0.2 mg/ml histone type III-SS (Sigma H-4524), 1ϫ Triton-X-100/lipid mixed micelles, 50 M unlabeled ATP, 5 Ci/reaction of [␥-32 P]ATP). Triton-X-100/lipid mixed micelles were prepared as previously described (28) with 2.5 mol% dipalmitoylglycerol (Avanti Polar Lipids, Alabaster, AL). The reaction was carried out at 30°C for 10 min and terminated by the addition of 4ϫ Laemmli buffer. Samples were boiled for 5 min, loaded in a 12% SDS-polyacrylamide gel, and separated by SDS-PAGE. Gels were fixed in a 50% methanol, 10% glacial acetic acid solution, rehydrated, and dried on gel blot paper. Radioactively labeled phosphorylated histone was visualized by autoradiography and quantitated by the PhosphorImager system and Im-ageQuant analysis (Amersham Biosciences). Specific activity was defined as pmol of histone phosphorylated/min/mg of lysate, although it may vary according to the level of expression of the recombinant protein. The moles of phosphorylated histone were calculated using a standard curve of reaction mix containing both unlabeled ATP and [␥-32 P]ATP, which had been spotted onto gel blot paper and scanned by phosphorimaging concurrently with the dried gels. The amount of total lysate corresponding to the amount of recombinant immunoprecipitated Pkc1 used in the assay was determined by running an aliquot of the immunoprecipitate with a standard curve of total lysate containing the recombinant protein on Western.
Spheroplast Measurements-Spheroplasting of C. neoformans cells was performed as previously described (31). Briefly, cells were grown in YNB supplemented with 2% glucose or galactose for 16 h. The pellet was washed 3 times with 0.5 M NaCl, 50 mM EDTA, resuspended in a 5% ␤-mercaptoethanol solution in sterile water, and incubated at 37°C for 1 h. Cells were pelleted and resuspended in SCE solution (1 M sorbitol, 0.1 M sodium citrate, pH 5.8, 0.01 M EDTA) containing lysing enzymes from Trichoderma harzianum (Sigma) at 10 mg/ml and incubated at 37°C for 2 h with occasional mixing. Spheroplasts were counted using differential interference contrast on a Zeiss Axioskop microscope. At least 4 fields of ϳ50 cells each were counted.
Calcofluor Staining and Transmission Electron Microscopy-C. neoformans strains were grown in YNB supplemented with 2% galactose and stained with Calcofluor™ White M2R (Molecular Probes, Eugene, OR). Cells were observed by UV light microscopy.
For transmission electron microscopy cells were pelleted and fixed in 2% cacodylate glutaraldehyde for 1 h, rinsed overnight in cacodylate buffer, post-fixed in 2% aqueous osmium tetroxide for 1 h, and rinsed in distilled water. Samples were then dehydrated in ascending concentrations of ethanol: 50% EtOH for 5 min, 70% EtOH for 15 min, 95% EtOH for 15 min, and twice in 100% EtOH for 15 min each. Transition to infiltration was begun using the intermediate fluid, propylene oxide, for 2 changes of 15 min each. Samples were first infiltrated with a 1:1 solution of propylene oxide and EmBed 812 embedding resin for 1 h and continued with a 1:2 solution of propylene oxide and EmBed 812 overnight. Samples were embedded in pure EmBed 812 and cured in a 60°C oven for 48 h. Thin sections of 70 nm were cut using a Reichert ultramicrotome and collected on copper grids. The heavy metal salts of uranyl acetate and lead citrate were used for staining, and the sections were examined using a JEOL JEM1210 electron microscope. Supplies for electron microscopy were obtained from Electron Microscopy Sciences (Fort Washington, PA).
Cell Wall Extraction-Crude cell wall preparations were made similar to the method used by Zhu et al. (5) with minor modifications. Briefly, cells were collected and resuspended in lysis buffer (50 mM Tris-Cl, pH 7.5 and 1 mM phenylmethylsulfonyl fluoride). Acid-washed glass beads (Sigma) were added in a volume equal to 3 ⁄4 of the cell suspension, and the cells were homogenized in a Mini-Beadbeater (Bio-Spec Products, Bartlesville, OK) 3 times for 45 s at 4°C. Total cell lysate was aspirated from the beads and transferred to a new tube. Beads were washed once with lysis buffer, and the wash liquid was added to the tube containing the cell lysate. Microscopic observation confirmed complete lysis of cells. The lysate was then centrifuged at 16,000 ϫ g for 30 min at 4°C. The pellet was designated the cell wall fraction and separated from the supernatant. The cell wall fraction was resuspended in 10 mM sodium phosphate, pH 7.0, with 2% SDS and boiled for 10 min. After trichloroacetic acid precipitation of both cell wall and supernatant fractions, protein pellets were resuspended in Laemmli buffer by boiling for 10 min.
Capsule Induction-C. neoformans strains IPC1/PKC1::V5 and IPC1/⌬C1-PKC1::V5 were grown in YNB media supplemented with 2% glucose for 24 h in a 30°C shaker. Cells were pelleted, resuspended, and inoculated into induction media (3 g/liter BBL TM Sabouraud liquid broth modified antibiotic medium 13 (BD Biosciences), 50 mM MOPS, pH 7.3) at 2.5 ϫ 10 5 cells/ml. Cultures were grown for 40 h in a 30°C shaker, stained with india ink, and observed by light microscopy. To measure capsule size, pictures were taken of five different fields of each sample at 40ϫ magnification, and the areas of the capsules of 20 cells from each field (100 total cells) were measured using LabWorks software (UVP, Inc., Upland, CA).
Western Blot Analysis-Protein samples were loaded on SDS-polyacrylamide gels, separated by electrophoresis, and transferred onto Immun-Blot TM polyvinylidene difluoride membranes (Bio-Rad). Pkc1 and ⌬C1-PKC1 proteins tagged with the V5 epitope were detected using anti-V5 antibody at 1:2000 dilution in 5% milk, 1ϫ phosphate-buffered saline and 0.1% Tween 20. Laccase was detected using anti-laccase monoclonal antibody clone G3P4D3 (a gift from Dr. Peter Williamson, University of Illinois at Chicago College of Medicine) at 2.6 g/ml in 3% bovine serum albumin, 1ϫ phosphate-buffered saline and 0.1% Tween 20.
Statistical Analysis-Statistical analysis was performed using Student's t test.

Lipids Modulated by Ipc1
Regulate Activation of C. neoformans Pkc1-Because our previous studies indicated that Ipc1 modulates DAG and phytoceramide levels in C. neoformans cells and that Pkc1 activity is regulated by DAG and phytoceramide in vitro (15), we next investigated whether modulation of Ipc1 in C. neoformans cells regulates the activity of Pkc1. Therefore, Ipc1 expression was modulated in the GAL7::IPC1 strain by growing cells in the presence of glucose (repressing conditions) or galactose (inducing conditions). As a control the wild type strain H99 was grown under the same conditions. Next, total lipids were extracted from each of the cultures, and their effect on the activity of recombinant C. neoformans Pkc1 was measured by in vitro kinase assay (Fig. 1). When Pkc1 was treated with lipids from cells in which Ipc1 was down-regulated (GAL7::IPC1 grown on glucose), kinase activity decreased by 30.7% relative to treatment with lipids from the wild type cells grown in glucose (p Ͻ 0.05). Conversely, when Pkc1 was treated with lipids from cells in which Ipc1 was up-regulated (GAL7::IPC1 grown on galactose), kinase activity increased by 21.4% compared with treatment with lipids from wild type cells grown on galactose. No difference in Pkc1 activity was observed between treatment with lipids from the wild type cells grown in glucose or galactose. Therefore, the lipids that are modulated by Ipc1 in vivo regulate the activity of C. neoformans Pkc1.
Deletion of the C1 Domain Abolishes Activation of Pkc1 by DAG-Next, we wanted to determine whether the C1 domain plays a role in the activation of Pkc1 by DAG. The Pkc1 and ⌬C1-PKC1 proteins were immunoprecipitated, and their abilities to phosphorylate the substrate histone in the presence and absence of dipalmitoylglycerol (DPG) were measured (Fig. 3A).
In the presence of DPG, the activity of wild type Pkc1 significantly increased by more than 2-fold (p Ͻ 0.05), whereas there was no significant increase in activity of ⌬C1-PKC1 in the presence of DPG. The specific activities of the Pkc1 and ⌬C1-PKC1 proteins were similar in the absence of lipids at 14.2 Ϯ 4.1 and 17.1 Ϯ 3.7 pmol of histone phosphorylated/min/mg of lysate protein, respectively. Therefore, the C1 domain of Pkc1 mediates activation by DAG similar to mammalian PKC.
Deletion of the C1 Domain Significantly Represses Laccase Activity and Melanin Production in C. neoformans-Because the Ipc1-DAG-Pkc1 pathway regulates melanin production by C. neoformans (15) and the data above suggest that the C1 domain is required for the activation of Pkc1 by DAG, we investigated the effect of deletion of the C1 domain on melanin formation. First, the laccase activities of the parental strains H99 (WT) and GAL7::IPC1 and of the ⌬C1 mutant strains IPC1/⌬C1-PKC1 and GAL7::IPC1/⌬C1-PKC1 were measured (Fig. 4A). Laccase activity of IPC1/⌬C1-PKC1 was reduced by ϳ65% compared with the parental strain H99 (WT) (p Ͻ 0.03). Similarly, laccase activity of the GAL7::IPC1/⌬C1-PKC1 strain was lower by ϳ34% than the parental strain GAL7::IPC1 (p Ͻ 0.03). Under these inducing (galactose) conditions, the laccase activity of GAL7::IPC1 was higher than that of wild type H99 due to the up-regulation of Ipc1, as expected (p Ͻ 0.01).
To confirm this biochemical regulation at the biological level, melanin production was measured by growing cells on galactose plates containing L-3,4-dihydroxyphenylalanine, a substrate for the laccase enzyme. As illustrated in Fig. 4B, the strain IPC1/⌬C1-PKC1 produced less melanin pigment compared with the parental wild type strain H99 (WT), and GAL7::IPC1/⌬C1-PKC1 produced less melanin than parental strain GAL7::IPC1. Under these conditions, the GAL7::IPC1 strain, in which IPC1 is up-regulated, produced more melanin than the wild type, in which IPC1 is not modulated, as expected. The decreases in both laccase activity and melanin production were confirmed using a second independent  ⌬C1 Mutants Exhibit Defects in Cell Wall Integrity-Because laccase is a cell wall-associated enzyme (5) and in S. cerevisiae Pkc1-mediated pathways regulate cell wall integrity (32), we investigated whether the Ipc1-Pkc1 pathway in C. neoformans may regulate laccase activity by retaining the enzyme in the cell wall. To determine whether the Ipc1-Pkc1 pathway plays a role in cell wall integrity, the wild type, GAL7::IPC1, and their ⌬C1 mutant strains were tested for sensitivity to lysing enzymes, which digest components of the cell wall. After treat-ment with lysing enzymes, the number of spheroplasts, or cells lacking the cell wall, was counted. Down-regulation of Ipc1 in the GAL7::IPC1 strain increased the number of spheroplasts by 76.2% (p Ͻ 0.01), whereas up-regulation of Ipc1 caused a 62.8% decrease in the number of spheroplasts compared with the wild type strain (p Ͻ 0.01). There was no difference in the number of spheroplasts between the wild type strains grown in glucose versus galactose media (Fig. 5A). When the C1 domain was deleted from Pkc1 in the wild type strain, the number of spheroplasts increased by 64.9% (p Ͻ 0.01). When the C1 domain was deleted in the GAL7::IPC1 strain, the decrease in spheroplasts caused by the up-regulation of Ipc1 was abolished, and the number of spheroplasts returned to the wild type level (Fig. 5B) (p Ͻ 0.05). Therefore, modulation of Ipc1 regulates the susceptibility of C. neoformans cell wall to digestion, and deletion of the C1 domain increases this susceptibility.
To further test for cell wall defects, the ability of C. neoformans strains to grow in the presence of the detergent SDS was measured. Serial dilutions of the ⌬C1 mutants and their parental strains were spotted onto YP plates containing 0.05% SDS and 2% of either glucose or galactose (Fig. 6). The results show that up-regulation of Ipc1 in the GAL7::IPC1 strain increased growth in the presence of SDS compared with the wild type strain (compare lane 3 to lane 1 in galactose). Conversely, down-regulation of Ipc1 in the GAL7::IPC1 strain repressed growth compared with the wild type strain (compare lane 3 to lane 1 in galactose). In the wild type strain, cells exhibited reduced growth when the C1 domain is deleted from Pkc1 (compare lanes 2 and 1) in either glucose or galactose media. As expected, deletion of the C1 domain in the GAL7::IPC1 strain reduced growth under the condition of Ipc1 up-regulation (compare lane 4 to lane 3 in galactose). Interestingly, the C1 deletion slightly enhanced growth when Ipc1 is down-regulated (compare lanes 4 to 3 in glucose). On plates lacking SDS, all strains had similar growth in either glucose or galactose (data not shown).
⌬C1 Mutants Display Changes in Cell Wall and Capsule Morphology-To examine directly whether ⌬C1 mutants exhibit defects in the cell wall structure, C. neoformans wild type and IPC1/⌬C1-PKC1 cells were stained with calcofluor white, which detects chitin, a component of fungal cell walls (33), and cells were observed under UV light microscope (Fig. 7). By counting ϳ270 cells in at least four different fields, we found that 21.4 Ϯ 0.9% of budding cells of the wild type strain exhib-

DAG-Pkc1 Signaling in Fungal Melanogenesis
ited intense staining within the neck of the emerging bud. In contrast, only 15.9 Ϯ 0.67% of ⌬C1 budding cells showed a similar staining.
Because the capsule of C. neoformans is anchored to cell wall components (34), we investigated whether the ⌬C1 mutants exhibit any changes in capsule formation. Using india ink staining, we observed that overall capsule size was decreased by ϳ42% in the IPC1/⌬C1-PKC1 mutant strain compared with the parental IPC1/PKC1 strain (data not shown).
To further investigate whether structural alteration of the capsule was associated with the absence of the C1 domain, the wild type, GAL7::IPC1, IPC1/⌬C1-PKC1, and GAL7::IPC1/ ⌬C1-PKC1 strains were examined by transmission electron microscopy. As shown in Fig. 8, the ⌬C1 mutants exhibited reduced density and length of capsule microfibrils radiating from the cell wall compared with the parental strains. No significant differences in cell wall size were observed between the ⌬C1 mutants and their parental strains by transmission electron microscopy. All together, these results suggest that signal transduction through the C1 domain regulates capsule formation and may modulate components of the cell wall.
Deletion of the C1 Domain Disrupts Localization of Laccase to the Cell Wall-C. neoformans cells were lysed, and a crude cell wall fraction was separated from the remaining supernatant. The localization of laccase to each fraction was detected by Western blot using antibodies directed against the laccase enzyme (Fig. 9). In the wild type strain, the majority of laccase was associated with the cell wall. However, when the C1 domain was deleted, laccase was primarily localized in the supernatant. The total level of laccase protein did not change significantly between the wild type and IPC1/⌬C1-PKC1 strains. Samples were normalized to total cellular protein measured by Bio-Rad protein assay before fractionation. Equal loading of cellular proteins was confirmed by Ponceau staining of the immunoblot membrane (data not shown). DISCUSSION In the present study the role and mechanism of the C1 domain, a putative DAG binding domain of Pkc1, in mediating the regulation of melanin production of C. neoformans by the sphingolipid enzyme Ipc1 and its product DAG were investigated. The results indicate that deletion of the C1 domain disrupts the regulation of Pkc1 by Ipc1 and DAG and causes a significant decrease in laccase activity and, consequently, melanogenesis. Furthermore, we establish a role for the Ipc1-DAG-Pkc1 pathway in cell wall maintenance and provide evidence that signal transduction through this pathway is required for proper localization of laccase to the cell wall. All together, the data suggest that the C1 domain is the platform through which Ipc1, via DAG, regulates Pkc1 and controls melanin production in C. neoformans.
Previous data suggested that regulation of Pkc1 is under the control of Ipc1, since levels of DAG and phytoceramide are modulated by Ipc1 and since in vitro biochemical studies show that C. neoformans Pkc1 is activated by DAG (a product of Ipc1) and also inhibited by phytoceramide (a substrate for Ipc1) (15). The studies presented here confirm that signal transduction through an Ipc1-DAG-Pkc1 pathway does occur in vivo. When Ipc1 is down-and up-regulated in C. neoformans cells, the lipids modulated in vivo inhibit and activate Pkc1, respectively (Fig. 1). The inhibition of Pkc1 upon treatment with lipids extracted from cells in which Ipc1 was down-regulated (GAL7::IPC1 in glucose) is statistically significant, whereas the activation of Pkc1 upon treatment with lipids extracted from cells in which Ipc1 was up-regulated (GAL7::IPC1 in galactose) was not statistically significant although consistently higher than the control in three independent determinations. It must be considered that the assay utilized a total neutral lipids extract, and thus, the lipid molecules affecting Pkc1 activity (phytoceramide and DAG) constitute only a small proportion of the total lipids. Previously, we determined that down-regulation of Ipc1 decreases DAG and increases phytoceramide levels, synergistically leading to a significant inhibition of Pkc1. Interestingly, when Ipc1 is up-regulated, DAG levels are increased, but phytoceramide levels do not change from wild type levels (15). Therefore, under the conditions in which Ipc1 is up-regulated, the activation of Pkc1 is only due to the increase in DAG molecules, the effect of which may be diluted within the total lipid extract. Overall, the results from this experiment support the hypothesis that Ipc1 regulates Pkc1 via lipid metabolites in living cells.
The existence of in vivo DAG-Pkc1 signaling was further investigated by selective deletion of the C1 domain from the C. neoformans PKC1 locus to express endogenous Pkc1 protein that lacked only the residues comprising the C1 domain (Fig.  2). Deletion of the C1 domain abolished the activation of Pkc1 by DAG (Fig. 3) and resulted in significant decreases of laccase activity and production of melanin pigment (Fig. 4), indicating that transduction through the Ipc1-DAG-Pkc1 cascade regulates melanogenesis in vivo.
The mechanism by which the Ipc1-DAG-Pkc1 pathway regulates the laccase enzyme was then examined. Because laccase is a cell wall-associated enzyme (5) and Pkc1 regulates cell wall integrity in yeasts (32), we hypothesized that the Ipc1-DAG-Pkc1 pathway regulates laccase activity by maintaining localization of the enzyme to the cell wall.
We first investigated whether the Ipc1-DAG-Pkc1 pathway plays a role in the regulation of cell wall integrity in C. neoformans. We found that the sensitivity of C. neoformans to cell wall-digesting enzymes is modulated by Ipc1 (Fig. 5A), and importantly, deletion of the C1 domain increased this susceptibility to digestion (Fig. 5B). Another phenotype of cells with wall defects is decreased growth in the presence of SDS. We found that modulation of Ipc1 regulates growth in the presence of SDS, and deletion of the C1 domain increases the sensitivity to SDS (Fig. 6). Additionally, direct observation of cells stained for chitin in the cell wall revealed that deletion of the C1 domain causes a slight decrease in chitin deposition, particularly in the septum dividing the mother cell from the bud (Fig.  7). Although the ⌬C1 mutant strains did not exhibit any obvious defects in budding or division, the changes in septal chitin suggest that deletion of the C1 domain does cause some perturbation in the composition of the cell wall. Examination of cells by transmission electron microscopy did not reveal any significant changes in cell wall size. However, this observation again does not preclude any ultrastructural changes in the cell wall. Previously, it was observed in S. cerevisiae that downregulation of Ipc1 causes defects in chitin deposition (35) and that Pkc1 may regulate chitin synthase III (Chs3p) (36). Therefore, the Ipc1-DAG-Pkc1 pathway may control integrity of the cell wall through the synthesis of cell wall components without affecting its overall size. The mechanism by which laccase associates with components of the cell wall is not clear, although studies by Zhu et al. (5) indicate that laccase is covalently attached to the cell wall, potentially through a disulfide or thioester bond. Interestingly, a recent study by Reese and Doering (34) demonstrated that ␣-1,3-glucan, a component of C. neoformans cell walls, is required for anchoring of the capsule (34). We observed that upon induction of capsule formation, the ⌬C1 cells produce a smaller capsule than the wild type strain, as visualized by india ink staining (data not shown). Additional examination of cells using transmission electron microscopy verified that the ⌬C1 mutants have decreased length and density of capsule microfibrils compared with the parental strains (Fig. 8). In S. cerevisiae Pkc1 regulates synthesis of cell wall ␤-glucans (37) (␣glucans are not found in the S. cerevisiae cell wall (38)). Therefore, in C. neoformans Pkc1 may enable proper association of capsular material with the cell wall by regulating production of the cell wall components that anchor the capsule. As a parallel, the Ipc1-DAG-Pkc1 pathway may also regulate the synthesis of cell wall components that serve as an anchor for the laccase enzyme. Further investigation will be required to determine which components of the cell wall are regulated by the Ipc1-DAG-Pkc1 pathway in C. neoformans. However, we do find that disruption of the Ipc1-DAG-Pkc1 pathway prevents localization of laccase to the cell wall ( Fig. 9), suggesting that alterations of the cell wall by the Ipc1-DAG-Pkc1 pathway affect the proper anchoring of the laccase enzyme. Because melanin production diminishes when laccase is not associated with the cell wall, our study provides additional information on the biology of this enzyme, indicating that localization of laccase to the cell wall is required for its function and is regulated by the Ipc1-DAG-Pkc1 pathway.
The present work does not exclude other mechanisms by which the Ipc1-DAG-Pkc1 pathway may regulate laccase. Because Pkc1 is a kinase, there is the possibility that laccase is directly bound and phosphorylated by Pkc1. Previous characterization of the laccase enzyme did not reveal that it is a phosphorylated protein (6). Additionally, unpublished data from our laboratory suggest that laccase does not co-immunoprecipitate with Pkc1 and is not phosphorylated in vitro by Pkc1 (data not shown). The investigation into the molecular mechanism by which Pkc1 anchors laccase to the cell wall is the focus of our future studies.
Although the results indicate that the Ipc1-DAG-Pkc1 pathway plays a significant role in the regulation of the cell wall and, consequently, melanin production in C. neoformans, we made several observations that substantiate the existence of additional mechanisms which help modulate these biological effects. Most evident is that deletion of the C1 domain did not completely abolish melanin formation by fungal cells (Fig. 4), suggesting other pathways work in tandem with the Ipc1-DAG-Pkc1 pathway to regulate melanogenesis. Indeed, other studies have established a role for the G␣ protein-cAMP-protein kinase A pathway in melanin production by C. neoformans (12)(13)(14). Whether this pathway interacts with the Ipc1-DAG-Pkc1 pathway awaits further characterization. We also found that laccase activity of the GAL7::IPC1/⌬C1-PKC1 strain, although decreased compared with its parental strain, is still ϳ2-fold higher than that of the IPC1/⌬C1-PKC1, a trend that is reflected in the levels of melanin production (Fig. 4). Additionally, the susceptibility of the GAL7::IPC1/⌬C1-PKC1 strain to cell wall digestion is less than that of the IPC1/⌬C1-PKC1 strain (Fig. 5), and the GAL7::IPC1/⌬C1-PKC1 strain is less sensitive to SDS than the IPC1/⌬C1-PKC1 strain (see Fig. 6, lanes 2 and 4 in  galactose). The only difference between these two strains is that Ipc1 is up-regulated in the GAL7::IPC1/⌬C1-PKC1 strain, since the assays were performed under inducing (galactose) conditions. These data indicate that either the inhibition of Pkc1 by phytoceramide occurs partially through the C1 domain or that a second mechanism is mediating the effects of Ipc1 up-regulation on cell wall and melanogenesis in a C1 domain-independent manner. The first hypothesis is supported by previous studies which found that the inhibitory effect of phytoceramide on Pkc1 was partially diminished after deletion of the C1 domain (15). The second hypothesis is supported by studies in S. cerevisiae which show that genes, such as ECM18, ECM40, and FIG 2 involved in cell wall synthesis and organization, are dysregulated in the absence of sphingolipids (39). Seeing as Ipc1 catalyzes the formation of the complex sphingolipid IPC, it is intriguing to hypothesize that a difference in IPC levels contributes to the phenotypes observed in the two strains.
Other evidence supporting the inhibition of Pkc1 by phytoceramide through the C1 domain is provided by the interesting observation that under Ipc1-repressing (glucose) conditions, the GAL7::IPC1/⌬C1-PKC1 strain exhibited increased rather than decreased growth in the presence of SDS compared with the parental GAL7::IPC1 strain (see Fig. 6, rows 3 and 4 in glucose). This result is contrary to what might be expected if the regulation of Pkc1 by Ipc1 was only mediated by DAG through the C1 domain. In previous studies we found that when Ipc1 is down-regulated, both DAG and phytoceramide levels are modulated (DAG is decreased, and phytoceramide is increased) (15). Under these conditions one would predict that Pkc1 would be synergistically inhibited, since phytoceramide also has a negative effect on Pkc1 activity (15) through as yet  (42) and transfers an inositol-phosphoryl group from phosphatidylinositol (PI) to phytoceramide, thereby producing the complex sphingolipid IPC and DAG. DAG activates Pkc1 through the C1 domain (shown in green). Activation of Pkc1 maintains the structure of the cell wall, which enables proper association of laccase to the cell wall. In the cell wall laccase produces melanin pigment, which is deposited in granules in the cell wall adjacent to the plasma membrane (43). Maintenance of the cell wall by Pkc1 also allows proper formation of capsule microfibrils. Additional regulation of Pkc1 may occur through repression by phytoceramide (15). When the C1 domain is deleted, DAG is unable to activate Pkc1. As a result, the structure of the cell wall is altered, decreasing the localization of laccase to the cell wall and leading to a reduction in melanin production. Capsule microfibrils are also shorter and less concentrated in the ⌬C1-PKC1 mutant. unknown molecular mechanisms. However, if phytoceramide exerts any of its inhibitory effects through the C1 domain, deletion of the C1 domain would relieve this inhibition, which could explain why the SDS sensitivity of yeast cells in which Ipc1 is down-regulated would be greater when Pkc1 is fulllength versus ⌬C1-PKC1 (Fig. 6). Partial regulation of Pkc1 by phytoceramide through the C1 domain is supported by studies in mammalian cells which suggest that regulation of PKCs by ceramide may occur through the C1 (40) or the C2 domain (41). As mentioned above, our earlier in vitro data indicated that inhibition of recombinant C. neoformans Pkc1 by ceramides is slightly attenuated when the C1 domain is deleted (15). This previous result in combination with the data presented here would suggest that phytoceramide exerts some, although not all, of its inhibitory effects through the C1 domain in vivo and, therefore, is an additional mechanism by which Ipc1 can regulate the phenotypes attributed to its modulation. Clearly, the biochemical mechanism(s) by which phytoceramide regulates Pkc1 warrants further investigation.
In conclusion, this study demonstrates that the C1 domain of Pkc1 plays an important role in signal transduction initiated by the sphingolipid pathway. The results suggest that activation of Pkc1 by DAG through the C1 domain is a critical signaling event for the proper localization of laccase to the cell wall and that the perturbation of laccase-cell wall association results in alteration of melanin production (a model is presented in Fig. 10). Because melanin is an important virulence factor of C. neoformans, the results of this study have significant implications for understanding the mechanisms by which C. neoformans regulates its pathogenicity, a crucial step toward the development of new anti-fungal therapies.