Hideo Michibata

Cloning and characterization of a novel human phosphodiesterase that hydrolyzes both cAMP and cGMP (PDE10A).

cDNA encoding a novel phosphodiesterase (PDE) was isolated from a human fetal lung cDNA library and designated PDE10A. The deduced amino acid sequence contains 779 amino acids, including a putative cGMP binding sequence in the amino-terminal portion of the molecule and a catalytic domain that is 16–47% identical in amino acid sequence to those of other PDE families. Recombinant PDE10A transfected and expressed in COS-7 cells hydrolyzed cAMP and cGMP with K m values of 0.26 and 7.2 μm, respectively, and V max with cGMP was almost twice that with cAMP. Of the PDE inhibitors tested, dipyridamole was most effective, with IC50 values of 1.2 and 0.45 μm for inhibition of cAMP and cGMP hydrolysis, respectively. cGMP inhibited hydrolysis of cAMP, and cAMP inhibited cGMP hydrolysis with IC50 values of 14 and 0.39 μm, respectively. Thus, PDE10A exhibited properties of a cAMP PDE and a cAMP-inhibited cGMP PDE. PDE10A transcripts were particularly abundant in the putamen and caudate nucleus regions of brain and in thyroid and testis, and in much lower amounts in other tissues. The PDE10A gene was located on chromosome 6q26 by fluorescentin situ hybridization analysis. PDE10A represents a new member of the PDE superfamily, exhibiting unique kinetic properties and inhibitor sensitivity.

Isolation and Characterization of Two Novel Phosphodiesterase PDE11A Variants Showing Unique Structure and Tissue-specific Expression

cDNAs encoding a novel phosphodiesterase, phosphodiesterase 11A (PDE11A), were isolated by a combination of reverse transcriptase-polymerase chain reaction using degenerate oligonucleotide primers and rapid amplification of cDNA ends. Their catalytic domain was identical to that of PDE11A1 (490 amino acids) reported during the course of this study. However, the cDNAs we isolated had N termini distinct from PDE11A1, indicating two novel N-terminal variants of PDE11A. PDE11A3 cDNA encoded a 684-amino acid protein including one complete and one incomplete GAF domain in the N-terminal region. PDE11A4 was composed of 934 amino acids including two complete GAF domains and shared 630 C-terminal amino acids with PDE11A3 but had a distinct N terminus containing the putative phosphorylation sites for cAMP- and cGMP-dependent protein kinases. PDE11A3 transcripts were specifically expressed in testis, whereas PDE11A4 transcripts were particularly abundant in prostate. Recombinant PDE11A4 expressed in COS-7 cells hydrolyzed cAMP and cGMP with K m values of 3.0 and 1.4 μm, respectively, and the V maxvalue with cAMP was almost twice that with cGMP. Although PDE11A3 showed the same K m values as PDE11A4, the relativeV max values of PDE11A3 were approximately one-sixth of those of PDE11A4. PDE11A4, but not PDE11A3, was phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro. Thus, the PDE11A gene undergoes tissue-specific alternative splicing that generates structurally and functionally distinct gene products.

A Novel Interaction of cGMP-dependent Protein Kinase I with Troponin T

cGMP-dependent protein kinase (cGK) is a major intracellular receptor of cGMP and is implicated in several signal transduction pathways. To identify proteins that participate in the cGMP/cGK signaling pathway, we employed the yeast two-hybrid system with cGK Iα as bait. cDNAs encoding slow skeletal troponin T (skTnT) were isolated from both mouse embryo and human skeletal muscle cDNA libraries. The skTnT protein interacted with cGK Iβ but not with cGK II nor cAMP-dependent protein kinase. The yeast two-hybrid and in vitro binding assays revealed that the N-terminal region of cGK Iα, containing the leucine zipper motif, is sufficient for the association with skTnT. In vivoanalysis, mutations in cGK Iα, which disrupted the leucine zipper motif, were shown to completely abolish the binding to skTnT. Furthermore, cGK I also interacted with cardiac TnT (cTnT) but not with cardiac troponin I (cTnI). Together with the observations that cTnI is a good substrate for cGK I and is effectively phosphorylated in the presence of cTnT in vitro, these findings suggest that TnT functions as an anchoring protein for cGK I and that cGK I may participate in the regulation of muscle contraction through phosphorylation of TnI.

Cloning of a Gene for a Novel Epithelium-specific Cytosolic Phospholipase A2, cPLA2δ, Induced in Psoriatic Skin

Psoriasis is a common skin disease characterized by hyperplastic regenerative epidermal growth and infiltration of immunocytes. The etiology of psoriasis is unknown, although several genetic and cellular factors have been elucidated. To find new psoriasis-related genes, we have cloned cDNAs that are differentially expressed between normal and psoriatic skins. Among these clones, we have identified a new gene that codes for a new member of the type IV cytosolic phospholipase A2 (cPLA2) family. We refer to this gene as cPLA2δ. It encodes a polypeptide of 818 amino acids that has significant homology with known cPLA2 proteins in the C2 and catalytic domains. The cPLA2δ gene was mapped to the 15q13-14 chromosomal locus, near to the locus of the cPLA2β gene, from which it is separated by a physical distance of about 220 kb. To identify the phospholipase A2 activity of cPLA2δ, we transfected COS-7 cells with His-tagged cPLA2δ. The cell lysate from these cells had calcium-dependent phospholipase A2 activity. Northern blot analysis revealed that a cPLA2δ transcript of about 4 kb is expressed in stratified squamous epithelia, such as those in skin and cervix, but not in other tissues. In situ hybridization and immunohistochemistry revealed that cPLA2δ is expressed strongly in the upper spinous layer of the psoriatic epidermis, expressed weakly and discontinuously in atopic dermatitis and mycosis fungoides, and not detected in the epidermis of normal skin; cPLA2α is not detected in either normal or psoriatic skin. These results suggest that cPLA2δ exhibits a unique distribution pattern compared with that of known cPLA2 subtypes, and it may play a critical role in inflammation in psoriatic lesions.

Characterization and effects of methyl-2- (4-aminophenyl)-1,2-dihydro-1-oxo-7- (2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel potent inhibitor of cGMP-binding cGMP-specific phosphodiesterase (PDE5)

An isoquinolone derivative, methyl-2-(4-aminophenyl)-1, 2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), was found to be a novel potent inhibitor of cyclic GMP (cGMP)-binding cGMP-specific phosphodiesterase (PDE5). We investigated the inhibitory effects of T-1032 on six PDE isozymes isolated from canine tissues. T-1032 specifically inhibited the hydrolysis of cGMP by PDE5 partially purified from canine lung, at a low concentration (IC(50) = 1.0 nM, K(i) = 1.2 nM), in a competitive manner. In contrast, the IC(50) values of T-1032 for PDE1, PDE2, PDE3, and PDE4 were more than 1 microM. T-1032 also inhibited PDE6 from canine retina with an IC(50) of 28 nM, which is of the same order of magnitude as the IC(50) of sildenafil. cGMP hydrolytic activities of two alternative splice variants of canine PDE5 expressed in COS-7 cells were inhibited by this compound to a similar extent. T-1032 increased the intracellular concentration of cGMP in cultured rat vascular smooth muscle cells in the presence and absence of C-type natriuretic peptide, an activator of membrane-bound guanylate cyclase, whereas the compound did not change cyclic AMP levels. These data indicated that T-1032, which belongs to a new structural class of PDE5 inhibitors, is a potent and selective PDE5 inhibitor. This compound may be useful in pharmacological studies to examine the role of a cGMP/PDE5 pathway in tissues.

Identification of a conserved residue responsible for the autoinhibition of cGMP-dependent protein kinase Iα and β

We isolated a constitutively active form of cGMP‐dependent protein kinase Iα (cGK Iα) by PCR‐driven random mutagenesis. The replacement of Ile‐63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cGMP. The hydrophobicity at position 63 is essential for the inactive state of cGK Iα, and Ile‐78 of cGK Iβ is also required for the autoinhibitory property. Furthermore, cGK Iα (Ile‐63‐Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is.