Calcineurin-binding protein Cbp1 directs the specificity of calcineurin-dependent hyphal elongation during mating in Cryptococcus neoformans

Abstract

Mating and virulence of the human fungal pathogen Cryptococcus neoformans are controlled by calcineurin, a serine-threonine-specific calcium-activated phosphatase that is the target of the immunosuppressive drugs cyclosporine A and FK506. In previous studies, a calcineurin binding protein (Cbp1, Rcn1, Dscr1/Csp1-3/MCIP1-3) that is conserved from yeasts to humans has been identified, but whether this protein functions to regulate calcineurin activity or facilitate calcineurin function as a signaling effector has been unclear. Here we show that, like calcineurin, Cbp1 is required for mating in C. neoformans. By contrast, Cbp1 plays no role in promoting calcineurin-dependent growth at 37 degrees C and is not essential for haploid fruiting. Site-directed mutagenesis studies provide evidence that tandem phosphorylation and dephosphorylation of two serine residues in the conserved SP repeat motif are critical for Cbp1 function. Epistasis analysis supports models in which Cbp1 functions coordinately with calcineurin to direct hyphal elongation during mating. Taken together, these findings provide insights into the roles of Cbp1 as an accessory subunit or effector of calcineurin-specific signaling pathways, which may be features conserved among the calcipressins to govern calcineurin signaling in immune cells, cardiomyocytes, and neurons of multicellular eukaryotes.

FIG. 1.

Disruption of the CBP1 gene by homologous recombination. (A) The CBP1 gene was disrupted by the insertion of the 1.7-kb nourseothricin dominant drug resistance cassette into the coding region of CBP1, as described in Materials and Methods. (B) Disruptants were identified by PCR with primers flanking the cassette insertion site within the CBP1 coding region. A representative PCR result is shown for both wild-type (CBP1) and disrupted (cbp1) alleles. Sizes are indicated at left in kilobases.

FIG. 2.

Growth of cbp1 mutant is sensitive to exogenous calcium but not elevated temperature and regulation of CBP1 transcription is calcineurin-independent. (A). The isogenic wild-type CBP1 (JEC21) and the cbp1 mutant strain were serially diluted and spotted onto YPD medium for 72 h at 25°C or 37°C. (B) The isogenic wild-type CBP1 (JEC21) and cbp1 and cnb1 mutant strains were serially diluted and spotted onto YPD medium with 0, 100, or 200 mM CaCl₂ for 72 h at 25°C. (C and D) Northern blot analysis of total RNA isolated from isogenic wild-type (WT, strain JEC21), cnb1, and cbp1 mutant strains grown: (C) at 37°C in the presence or absence of 1 μg/ml FK506 for 4 h, or (D) at 25°C. Following hybridization with the CBP1 probe, the blots were reprobed with GPI10 (C) or CNB1 (D) for additional loading and hybridization controls. The 25 S rRNA stained with ethidium bromide serves as a loading control for D.

FIG. 3.

Cbp1 is necessary for mating. (A) Deletion of the CBP1 gene results in bilateral sterility. Isogenic a and α CBP1 wild-type (JEC20 and JEC21) and cbp1 strains were coincubated on V8 agar at 25°C for 7 days, in the presence or absence of 1 μg/ml FK506. (B) Isogenic a and α CBP1 wild-type and cbp1 mutant strains were grown in confrontation on filament agar for 72 h at 25°C to evaluate the role of Cbp1 in responses to pheromone. (C) The α CBP1 wild-type and cbp1 mutant strains were grown on filament agar for 72 h at 25°C to evaluate the role of Cbp1 in monokaryotic fruiting. For each coculture and confrontation, the colony edge was photographed at 100× magnification.

FIG. 4.

SP repeat of Cbp1 is required for hyphal elongation. (A) Alignment of the central peptide sequence motif containing the SP repeat for Cbp1 (C. neoformans), Rcn1 (S. cerevisiae), and DSCR1/MCIP1 (human). Amino acid positions for the conserved serines within the serine-proline repeat of C. neoformans Cbp1 are indicated. (B) Expression of Cbp1 complements to restore mating in a cbp1 mutant. Isogenic MATa and MATα cbp1 ura5 strains were transformed with constructs containing the CBP1 alleles in the pPM8 shuttle vector. Transformants were coincubated on nitrogen-limiting agar (V8) and photographed after incubation at 25°C for 7 days. (ΔSPR denotes the allele with deletion of the serine-proline repeat.) (C) Overexpression of Cbp1 fails to restore mating in a calcineurin mutant. Isogenic MATa and MATα wild-type (CNB1) and cnb1 ura5 strains were transformed with a construct containing the CBP1 allele in the pPM8 shuttle vector and cocultured on V8 medium for 7 days at 25°C. A wild-type cross (WT) is shown for comparison. Colony edges were photographed at 100× magnification.

FIG. 5.

Phosphorylation of S142, and tandem phosphorylation/dephosphorylation of S138, is necessary for Cbp1 function. Transformation with the S142E CBP1 allele, but not the S138E or S138E+S142E allele, restores mating in a bilateral cbp1 cross. Transformants were coincubated on mating medium (V8) and photographed at 100× magnification after incubation at 25°C for 7 days.

FIG. 6.

Influence of SP repeat phosphorylation on Cbp1 stability. (A) Phosphorylation of SP repeat regulates Cbp1 stability. Immunoblot analysis to detect FLAG-tagged Cbp1 fusions from total protein (10 μg) isolated from bilateral crosses of transformants containing CBP1::FLAG alleles cocultured on V8 medium for 48 h at 25°C. (B) Northern blot analysis of total RNA isolated from isogenic cbp1 ura5 transformants containing CBP1::FLAG alleles and grown to mid-log phase in selection medium at 25°C. Total RNA from wild-type and cnb1 ura5 strains were included as controls. Hybridization with the CBP1 probe as described for Fig. 2. The 17S rRNA stained with ethidium bromide serves as a loading control.

FIG. 7.

Influence of SP repeat phosphorylation on Cbp1-calcineurin interaction. (A) Phosphorylation status of SP repeat does not mediate the interaction of Cbp1 with calcineurin. Cotransformation of two-hybrid yeast reporter strain expressing calcineurin B (Y187) with plasmids expressing the Gal4 DNA-binding domain alone or fused to calcineurin A and the Gal4 activation domain alone or fused to Cbp1 (alleles listed on x-axis). Interaction values are shown in Miller units. (B) Mixed-allele mating assay. Isogenic MATα and MATa cbp1 ura5 strains were transformed with constructs containing the CBP1 alleles in the pPM8 shuttle vector. Transformants were coincubated in mixed-allele pairs on V8 agar and photographed after incubation at 25°C for 3 days. +, filamentation in mating assay; −, absence of filamentation.

FIG. 8.

Cbp1 is a positive regulator/effector of calcineurin. (A) Overexpression of activated calcineurin partially overcomes loss of Cbp1 function. Isogenic a and α cbp1 strains were transformed with plasmids expressing the CNA1ΔCam-AI calcineurin A constitutive truncation mutant or CBP1 and coincubated in bilateral crosses on V8 agar at 25°C for 7 days. For each coculture and confrontation, the colony edge was photographed at 100× magnification. (B) Overexpression of Cbp1 or Cbp1^S142E restores growth of calcium-sensitive cbp1 mutants. Transformants (cna1 or cbp1) harboring plasmids expressing Cbp1, Cbp1^S142E, or Cbp1^S142A, or constitutive calcineurin A, Cna1-ΔCam-AI, were inoculated at low density (OD₆₀₀ = 1) into YPD with or without CaCl₂ (100 or 200 mM) and incubated for 18 h at 25°C prior to culture density determination at OD₆₀₀, each in duplicate.

FIG. 9.

Model of calcineurin regulation by Cbp1 to promote mating-specific hyphal elongation. Calcineurin-associated Cbp1 is phosphorylated at serine 142 by mitogen-activated protein kinase, creating a priming site for phosphorylation at serine 138 by GSK-3. Similar to the model for protein phosphatase 1/I-2, GSK-3 associates with the calcineurin/Cbp1 complex and phosphorylates serine 138, leading to the activation of calcineurin function in mating-specific filamentation via substrate targeting of the complex. After phosphorylation of serine 138, GSK-3 loses binding affinity to the calcineurin/Cbp1 complex and dissociates. Calcineurin reverses phosphorylation at serine 138, leading to the return to the inactive state. The specific kinases involved in the phosphorylation of serines 138 and 142, and the putative subcellular targeting compartment and substrate(s) are not yet known. This model incorporates observations of Cbp1 and calcineurin function in C. neoformans and closely resembles the mode of regulation of the mammalian and yeast protein phosphatase 1/I-2(Glc8)/GSK-3 complexes, with significant similarity to the proposed model for regulation of calcineurin by Rcn1 in S. cerevisiae (15, 25, 39, 45).