Calcineurin is a highly conserved and ubiquitously expressed Ca2+- and calmodulin-dependent protein phosphatase. The in vivo role of calcineurin, however, is not fully understood. Here, we show that disruption of the calcineurin gene (ppb1(+)) in fission yeast results in a drastic chloride ion (Cl-)-sensitive growth defect and that a high copy number of a novel gene pmp1(+) suppresses this defect. pmp1(+) encodes a phosphatase, most closely related to mitogen-activated protein (MAP) kinase phosphatases of the CL100/MKP-1 family. Pmp1 and calcineurin share an essential function in Cl- homeostasis, cytokinesis and cell viability. Pmp1 phosphatase dephosphorylates Pmk1, the third MAP kinase in fission yeast, in vitro and in vivo, and is bound to Pmk1 in vivo, strongly suggesting that Pmp1 negatively regulates Pmk1 MAP kinase by direct dephosphorylation. Consistently, the deletion of pmk1(+) suppresses the Cl--sensitive growth defect of ppb1 null. Thus, calcineurin and the Pmk1 MAP kinase pathway may play antagonistic functional roles in the Cl- homeostasis.

Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.

We recently found, using cultured mouse cell systems, that newly synthesized catalytic (C) subunits of cyclic AMP-dependent protein kinase undergo a posttranslational modification that reduces their electrophoretic mobilities in sodium dodecyl sulfate (SDS)-polyacrylamide gels and activates them for binding to a Sepharoseconjugated inhibitor peptide. Using an Escherichia coli expression system, we now show that recombinant murine Ca subunit undergoes a similar modification and that the modification results in a large increase in protein kinase activity. Threonine phosphorylation appears to be responsible for both the enzymatic activation and the electrophoretic mobility shift. The phosphothreonine-deficient form of C subunit had reduced affinities for the ATP analogs p-fluorosulfonyl-[14CJbenzoyl 5'-adenosine and adenosine 5'-O-(3-thiotriphosphate) as well as for the Sepharose-conjugated inhibitor peptide; it also had markedly elevated Kms for both ATP and peptide substrates. Autophosphorylation of C-subunit preparations enriched for this phosphothreonine-deficient form reproduced the changes in enzyme activity and SDS-gel mobility that occur in intact cells. A mutant form of the recombinant C subunit with Ala substituted for Thr-197 (the only C-subunit threonine residue known to be phosphorylated in mammalian cells) was similar in SDS-polyacrylamide gel electrophoresis mobility and activity to the phosphothreonine-deficient form of wild-type C subunit. In contrast to the wild-type subunit, however, the Ala-197 mutant form could not be shifted or activated by incubation with the phosphothreoninecontaining wild-type form. We conclude that posttranslational autophosphorylation of Thr-197 is a critical step in intracellular expression of active C subunit.It has been known for some time that catalytic (C) subunit of cyclic AMP (cAMP)-dependent protein kinase purified from animal tissues is phosphorylated at two sites, 22); these sites are also apparently phosphorylated in Escherichia coli (results cited in reference 32). A third site (Ser-10) can be phosphorylated by C subunit in vitro (29). Dephosphorylation of native C subunit has been difficult to achieve (22), and as a result, the importance of phosphorylation to C subunit function has remained unknown. Because of its specific labeling by a peptide-based affinity reagent, Thr-197 has been localized to a region in or near the active site of C subunit (17); recent crystallographic data substantiate this localization and suggest that the phosphate on Thr-197 might contribute to stabilization of the active conformation of C subunit (11,12

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.

The ppb1؉ gene encodes a fission yeast homologue of the mammalian calcineurin. We have recently shown that Ppb1 is essential for chloride ion homeostasis, and acts antagonistically with Pmk1 mitogen-activated protein kinase pathway. In an attempt to identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature, and isolated a mutant, its3-1. its3؉ was shown to be an essential gene encoding a functional homologue of phosphatidylinositol-4-phosphate 5-kinase (PI(4)P5K). The temperature upshift or addition of FK506 induced marked disorganization of actin patches and dramatic increase in the frequency of septation in the its3-1 mutants but not in the wild-type cells. Expression of a green fluorescent protein-tagged Its3 and the phospholipase C␦ pleckstrin homology domain indicated plasma membrane localization of PI(4)P5K and phosphatidylinositol 4,5-bisphosphate. These green fluorescent proteintagged proteins were concentrated at the septum of dividing cells, and the mutant Its3 was no longer localized to the plasma membrane. These data suggest that fission yeast PI(4)P5K Its3 functions coordinately with calcineurin and plays a key role in cytokinesis, and that the plasma membrane localization of Its3 is the crucial event in cytokinesis.Calcineurin, Ca 2ϩ /calmodulin-dependent protein phosphatase, is a molecular target for the specific immunosuppressive drugs, such as cyclosporin A or tacrolimus (FK506), 1 used in organ transplantation. These drugs induce their biological effects by forming an initial complex with cytosolic proteins termed immunophilins. These drug-immunophilin complexes then bind to and inhibit calcineurin (1). Calcineurin is widely distributed in mammalian tissues, and may have additional functions other than its well characterized role in lymphocytes. The inhibition of these functions may contribute to the side effects of these drugs. A better understanding of the biological roles of calcineurin in different cell types should promote the development of improved strategies for immunosuppression.Calcineurin is conserved from yeast to man (2-4). We have been studying calcineurin signal transduction pathways in fission yeast Schizosaccharomyces pombe because this system is amenable to genetics and has many advantages in terms of relevance to higher systems. S. pombe has a single gene encoding the catalytic subunit of calcineurin, ppb1 ϩ , that is essential for cytokinesis (4, 5). We have previously shown that ppb1 ϩ plays an essential role in maintaining chloride ion homeostasis, and acts antagonistically with Pmk1 mitogen-activated protein kinase pathway (6 -8). To identify new components in the calcineurin signaling pathway, we have developed a simple genetic screen using the immunosuppressive drug FK506 for mutants that depend on calcineurin for growth, and have identified eight complementation groups (its1-8 for immunosuppressant-and temperature-sensitive, to be described elsewhere in det...