Takayoshi Kuno

Decreased Serotonin S2 and Increased Dopamine D2 Receptors in Chronic Schizophrenics

Serotonin S2 and dopamine D2 receptors in the prefrontal cortex and caudate nucleus of postmortem brains of chronic schizophrenics were studied using 3H-ketanserin and 3H-spiperone, respectively. In the prefrontal cortex of schizophrenics, we found a significant decrease in the maximum number of 3H-ketanserin binding sites (Bmax), with no change in the dissociation constant (Kd). Conversely, both Bmax and Kd of 3H-spiperone binding to the caudate nucleus were significantly increased in the schizophrenic patients. There were no differences in receptor indices between patients who were taking neuroleptics until their death and those who had taken none for 2 months or more prior to death. These findings suggest that alterations in S2 receptors in the prefrontal cortex may reflect the disease process, per se, and that the increase in the number of D2 receptors in the caudate nucleus of schizophrenics is not due solely to neuroleptic medication.

The fission yeast pmk1+ gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway.

We have isolated a gene, pmk1+, a third mitogen-activated protein kinase (MAPK) gene homolog from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence shows the most homology (63 to 65% identity) to those of budding yeast Saccharomyces Mpk1 and Candida Mkc1. The Pmk1 protein contains phosphorylated tyrosines, and the level of tyrosine phosphorylation was increased in the dsp1 mutant which lacks an attenuating phosphatase for Pmk1. The level of tyrosine phosphorylation appears constant during hypotonic or heat shock treatment. The cells with pmk1 deleted (delta pmk1) are viable but show various defective phenotypes, including cell wall weakness, abnormal cell shape, a cytokinesis defect, and altered sensitivities to cations, such as hypersensitivity to potassium and resistance to sodium. Consistent with a high degree of conservation of amino acid sequence, multicopy plasmids containing the MPK1 gene rescued the defective phenotypes of the delta pmk1 mutant. The frog MAPK gene also suppressed the pmk1 disruptant. The results of genetic analysis indicated that Pmk1 lies on a novel MAPK pathway which does not overlap functionally with the other two MAPK pathways, the Spk1-dependent mating signal pathway and Sty1/Spc1/Phh1-dependent stress-sensing pathway. In Saccharomyces cerevisiae, Mpk1 is involved in cell wall integrity and functions downstream of the protein kinase C homolog. In contrast, in S. pombe, Pmk1 may not act in a linear manner with respect to fission yeast protein kinase C homologs. Interestingly, however, these two pathways are not independent; instead, they regulate cell integrity in a coordinate manner.

Distinct Cellular Expression of Calcineurin Aα and Aβ in Rat Brain

Abstract: Specific polyclonal antibodies that distinguish the two distinct isoforms of the catalytic subunit of calmodulin‐dependent protein phosphatase, calcineurin Aα and Aβ, were prepared, and the distribution of calcineurin Aα and Aβ in rat brain was studied using immunochemical and immunocytochemical techniques. Immunochemical measurement revealed that the regional distributions of the two isoforms differed and that Aα was more abundant than Aβ in the rat brain. The subcellular distribution patterns of both isoforms were similar. Both isoforms were highly enriched in cytosolic fractions, including the synaptosomal cytosol. Immunocytochemical analysis revealed that both Aα and Aβ immunoreactivities differed in regional and cellular localizations. These different patterns of expression suggest that the two isoforms of calcineurin A may each have specific functions in modulating neuronal activity in particular cell types.

pmp1+, a suppressor of calcineurin deficiency, encodes a novel MAP kinase phosphatase in fission yeast.

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.

The complete primary structure of calcineurin A, a calmodulin binding protein homologous with protein phosphatases 1 and 2A

A complementary DNA (cDNA) clone encoding the catalytic subunit of calcineurin (calcineurin A) has been isolated from a rat brain cDNA library. The primary structure of the cDNA consists of 2,337 nucleotides including the entire coding region for 521 amino acids, and the calculated molecular mass is 58,643 Da. The calcineurin A is strikingly homologous to protein phosphatases 1 and 2A, approximately 50% of the amino acids over an internal 250-residue region between residues 78 and 329 being identical. Twenty four amino acid-residue region between residues 391 and 414 shows the consensus structural features for a calmodulin-binding domain. These data suggest that the allosteric character of this chimeric enzyme is generated by gene fusion of two separate protein families.

Phosphatidylinositol-4-phosphate 5-kinase Its3 and calcineurin Ppb1 coordinately regulate cytokinesis in fission yeast

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 theits3-1 mutants but not in the wild-type cells. Expression of a green fluorescent protein-tagged Its3 and the phospholipase Cδ pleckstrin homology domain indicated plasma membrane localization of PI(4)P5K and phosphatidylinositol 4,5-bisphosphate. These green fluorescent protein-tagged 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.

Molecular Cloning of Two Additional Members of the Neural Visinin‐Like Ca2+‐Binding Protein Gene Family

Abstract: We have isolated a rat cDNA clone encoding a neural visinin‐like Ca2+‐binding protein (NVP), which we designate NVP‐1. To identify additional molecular forms of NVP, a rat brain cDNA library was screened for their presence using an NVP‐1 cDNA probe under low‐stringency hybridization conditions. Two types of cDNA clones encoding structurally related proteins, designated NVP‐2 and NVP‐3, have been isolated. The deduced amino acid sequences of NVP‐2 and NVP‐3 are 89.0% and 68.6% identical to that of NVP‐1, respectively, and contain consensus sequences for EF‐hand Ca2+‐binding sites. Northern blot analysis shows that NVP‐1, NVP‐2, and NVP‐3 mRNAs are most highly expressed in brain and are differentially expressed in various regions of rat brain. These results suggest that NVP‐2 and NVP‐3 are additional members of the NVP gene family.

cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae

We have isolated a cDNA clone encoding a homolog of mammalian calcineurin B (the regulatory subunit of calmodulin-dependent protein phosphatase) by screening a cDNA expression library of Saccharomyces cerevisiae with antiserum against bovine calcineurin B. The yeast calcineurin B homolog (YCNB) is composed of 175 amino acids with a calculated molecular mass of 19,639 daltons and contains four putative Ca(2+)-binding domains. The amino-acid alignment of YCNB with human calcineurin B demonstrates 53% sequence identity and 82% homology. Southern blot analysis indicates that the gene for YCNB is a single-copy gene. Thus, yeast calmodulin-dependent protein phosphatase apparently has a heterodimeric structure similar to that of the enzyme in mammalians.

Evidence for a second isoform of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A)

We have used a previously characterized rat cDNA clone for the catalytic (A) subunit of calmodulin-dependent protein phosphatase (calcineurin), which we designated A alpha, to isolate cDNA clones coding for a second isoform of the A subunit, A beta. The A beta cDNA encodes a protein of 525 amino acids that is 81% identical with A alpha. The N-terminal region is dissimilar and contains a characteristic proline-rich sequence. The region homologous to protein phosphatases 1 and 2A (region between residues 87 and 338, 91% identical) and the calmodulin binding domain (region between residues 401 and 424, 96% identical) are highly conserved. The presence of two genes coding for calcineurin A suggests the possibility of important functional differences in the two enzymes.

The MAPK kinase Pek1 acts as a phosphorylation-dependent molecular switch

The mitogen-activated protein kinase (MAPK) pathway is a highlyconserved eukaryotic signalling cascade that converts extracellular signals into various outputs, such as cell growth and differentiation. MAPK is phosphorylated and activated by a specific MAPK kinase (MAPKK): MAPKK is therefore considered to be an activating regulator of MAPK. Pmk1 is a MAPK that regulates cell integrity and which, with calcineurin phosphatase, antagonizes chloride homeostasis in fission yeast. We have now identified Pek1, a MAPKK for Pmk1 MAPK. We show here that Pek1, in its unphosphorylated form, acts as a potent negative regulator of Pmk1 MAPK signalling. Mkh1, an upstream MAPKK kinase (MAPKKK), converts Pek1 from being an inhibitor to an activator. Our results indicate that Pek1 has a dual stimulatory and inhibitory function which depends on its phosphorylation state. This switch-like mechanism could contribute to the all-or-none physiological response mediated by the MAPK signalling pathway.