Identification of a surface on the beta-propeller protein RACK1 that interacts with the cAMP-specific phosphodiesterase PDE4D5.

A strategy of mutagenesis followed by yeast two-hybrid assay was used to determine the sites on the WD-repeat protein Receptor for Activated C Kinase 1 (RACK1) necessary for it to interact with the cAMP-specific phosphodiesterase isoform PDE4D5. Analysis of deletion mutations demonstrated that WD-repeats 5-7, inclusively, of RACK1 contained the major site for interaction with PDE4D5. A reverse two-hybrid screen focusing on WD-repeats 5-7 of RACK1 isolated 11 single amino acid mutations from within this region that blocked the interaction. The ability of these mutations to block the interaction was confirmed by "pull-down" assays using bacterially expressed glutathione-S-transferase (GST)-RACK1 and mammalian cell-expressed PDE4D5. A model of RACK1 structure, based on the structural similarity of RACK1 to other beta-propeller WD-repeat proteins, indicated that the majority of the amino acids identified by mutagenesis are clustered in a discrete surface of RACK1. We propose that this surface of RACK1 is the major site for its interaction with the unique amino-terminal region of PDE4D5.

Surgically induced cryptorchidism-related degenerative changes in spermatogonia are associated with loss of cyclic adenosine monophosphate-dependent phosphodiesterases type 4 in abdominal testes of rats.

The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.

ERK2 mitogen-activated protein kinase binding, phosphorylation, and regulation of the PDE4D cAMP-specific phosphodiesterases. The involvement of COOH-terminal docking sites and NH2-terminal UCR regions.

The cAMP-specific phosphodiesterase family 4, subfamily D, isoform 3 (PDE4D3) is shown to have FQF and KIM docking sites for extracellular signal-regulated kinase 2 (ERK2) (p42(MAPK)). These straddle the target residue, Ser(579), for ERK2 phosphorylation of PDE4D3. Mutation of either or both of these docking sites prevented ERK2 from being co-immunoprecipitated with PDE4D3, ablated the ability of epidermal growth factor to inhibit PDE4D3 through ERK2 action in transfected COS cells, and attenuated the ability of ERK2 to phosphorylate PDE4D3 in vitro. The two conserved NH(2)-terminal blocks of sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2), that characterize PDE4 long isoforms, are proposed to amplify the small, inherent inhibitory effect that ERK2 phosphorylation exerts on the PDE4D catalytic unit. In contrast to this, the lone intact UCR2 region found in PDE4D1 directs COOH-terminal ERK2 phosphorylation to cause the activation of this short isoform. From the analysis of PDE4D3 truncates, it is suggested that UCR1 and UCR2 provide a regulatory signal integration module that serves to orchestrate the functional consequences of ERK2 phosphorylation. The PDE4D gene thus encodes a series of isoenzymes that are either inhibited or activated by ERK2 phosphorylation and thereby offers the potential for ERK2 activation either to increase or decrease cAMP levels in cellular compartments.

UCR1 and UCR2 domains unique to the cAMP-specific phosphodiesterase family form a discrete module via electrostatic interactions.

The cAMP-specific phosphodiesterases (PDE4) enzymes contain unique "signature" regions of amino acid sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2). UCR1 and UCR2 are located between the extreme amino-terminal region and the catalytic region of the PDE4 enzymes. The UCR1 of the PDE4D3 isoform was used as a "bait" in a two-hybrid screen, which identified a PDE4D cDNA clone containing UCR2 and the catalytic region but not UCR1. Two-hybrid and "pull down" analysis of constructs incorporating various regions of the PDE4D3 cDNA demonstrated that the carboxyl-terminal region of UCR1 interacted specifically with the amino-terminal region of UCR2. The interaction was blocked by mutations of two positively charged amino acids (Arg-98 and Arg-101 to alanine) located within an otherwise largely hydrophobic region of UCR1. Mutation of three negatively charged amino acids in UCR2 (Glu-146, Glu-147, and Asp-149, all to alanine) also blocked the interaction. The phosphorylation of UCR1 by cAMP-dependent protein kinase (PKA) in vitro attenuated the ability of UCR1 to interact with UCR2. Mutation of the PKA substrate site in UCR1 (Ser-54) to aspartic acid, which mimics the activation of PDE4D3 by PKA, profoundly reduced the interaction between UCR1 and UCR2. Our data are consistent with a model in which UCR1 and UCR2 act as independent domains whose interaction is determined by electrostatic interactions and which may be disrupted by PKA phosphorylation. We suggest that the UCR1 and UCR2 domains may form a module that interacts with and regulates the PDE4 catalytic region.

Physical mapping and promoter structure of the murine cAMP-specific phosphodiesterase pde4a gene.

The Pde4a gene is a mammalian homolog of the dunce learning and memory gene of Drosophila melanogaster and encodes cAMP-specific phosphodiesterases, targets for drugs with antidepressant and anti-inflammatory actions in humans. We have analyzed the intron/exon and promoter structure of the murine Pde4a gene. Pde4a encodes at least two different transcripts, each generated by alternative mRNA splicing and the use of alternative promoters. The majority of Pde4a exons are tightly clustered at the 3' end of the gene. The 5' region of the gene contains at least one widely separated exon, which encodes the 5' end of a distinct mRNA transcript and contains a separate promoter and transcriptional start site. Analysis of YAC clones determined that the Pde4a gene maps to the 4-cM region of Chromosome (Chr) 9, close to Ldlr and Epor, in a region syntenic to human PDE4A.

The unique N-terminal domain of the cAMP phosphodiesterase PDE4D4 allows for interaction with specific SH3 domains.

Of the five PDE4D isoenzymes, only the PDE4D4 cAMP specific phosphodiesterase was able to bind to SH3 domains. Only PDE4D4 and PDE4A5, but not any other PDE4A, B, C and D isoforms expressed in rat brain, bound to src, lyn and fyn kinase SH3 domains. Purified PDE4D4 could bind to purified lyn SH3. PDE4D4 and PDE4A5 both exhibited selectivity for binding the SH3 domains of certain proteins. PDE4D4 did not bind to WW domains. We suggest that an important function of the unique N-terminal region of PDE4D4 may be to allow for association with certain SH3 domain-containing proteins.

The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform.

The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, beta'-coatomer protein and Gsbeta, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (Ka approximately 7 nM) [corrected] when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3-4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.

Stimulation of p70S6 kinase via a growth hormone-controlled phosphatidylinositol 3-kinase pathway leads to the activation of a PDE4A cyclic AMP-specific phosphodiesterase in 3T3-F442A preadipocytes.

The challenge of 3T3-F442A fibroblasts with growth hormone led to both a decrease in the mobility on SDS/PAGE and activation of the PDE4A cyclic AMP-specific phosphodiesterase isoform PDE4A5. Activation was mediated by a JAK-2-dependent pathway coupled to the activation of phosphatidylinositol 3-kinase and p70S6 kinase. Activation was not dependent on the ability of growth hormone to stimulate ERK2 or protein kinase C or any effect on transcription. Blockade of activation of murine PDE4A5 ablated the ability of growth hormone to decrease intracellular cAMP levels. Antisense depletion of murine PDE4A5 mimicked the ability of rolipram to enhance the growth hormone-stimulated differentiation of 3T3-F442A cells to adipocytes. It is suggested that activation of PDE4A5 by growth hormone serves as a brake on the differentiation processes.

Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene.

We have isolated and characterized complete cDNAs for two isoforms (HSPDE4D4 and HSPDE4A5) encoded by the human PDE4D gene, one of four genes that encode cAMP-specific rolipram-inhibited 3',5'-cyclic nucleotide phosphodiesterases (type IVPDEs; PDE4 family). The HSPDE4D4 and HSPDE4D5 cDNAs encode proteins of 810 and 746 amino acids respectively. A comparison of the nucleotide sequences of these two cDNAs with those encoding the three other human PDE4D proteins (HSPDE4D1, HSPDE4D2 and HSPDE4D3) demonstrates that each corresponding mRNA transcript has a unique region of sequence at or near its 5'-end, consistent with alternative mRNA splicing. Transient expression of the five cDNAs in monkey COS-7 cells produced proteins of apparent molecular mass under denaturing conditions of 68, 68, 95, 119 and 105 kDa for isoforms HSPDE4D1-5 respectively. Immunoblotting of human cell lines and rat brain demonstrated the presence of species that co-migrated with the proteins produced in COS-7 cells. COS-cell-expressed and native HSPDE4D1 and HSPDE4D2 were found to exist only in the cytosol, whereas HSPDE4D3, HSPDE4D4 and HSPDE4D5 were found in both cytosolic and particulate fractions. The IC50 values for the selective PDE4 inhibitor rolipram for the cytosolic forms of the five enzymes were similar (0.05-0.14 microM), whereas they were 2-7-fold higher for the particulate forms of HSPDE4D3 and HSPDE4D5 (0.32 and 0.59 microM respectively), than for the corresponding cytosolic forms. Our data indicate that the N-terminal regions of the HSPDE4D3, HSPDE4D4 and HSPDE4D5 proteins, which are derived from alternatively spliced regions of their mRNAs, are important in determining their subcellular localization, activity and differential sensitivity to inhibitors.

Molecular cloning and transient expression in COS7 cells of a novel human PDE4B cAMP-specific phosphodiesterase, HSPDE4B3.

5'-Rapid amplification of cDNA ends, done on poly(A)+ RNA from human U87 cells, was used to identify 420 bp of novel 5' sequence of a PDE4B cAMP-specific phosphodiesterase (PDE). This identified an open reading frame encoding a putative 721-residue 'long-form' PDE4B splice variant, which we term HSPDE4B3. HSPDE4B3 differs from the two known PDE4B forms by virtue of its unique 79-residue N-terminal region, compared with the unique N-terminal regions of 94 and 39 residues found in HSPDE4B1 and HSPDE4B2 respectively. In transfected COS7 cells the two long forms, HSPDE4B1 and HSPDE4B3, had molecular masses of approx. 104 and approx. 103 kDa respectively. Expressed in COS-7 cells, the three HSPDE4B isoforms were found in the high-speed supernatant (cytosol) fraction as well as both the high-speed pellet (P2) and low-speed pellet (P1) fractions. All isoforms showed similar Km values for cAMP hydrolysis (1.5-2.6 microM). The maximal activities of the soluble cytosolic activity of the two long forms were very similar, whereas that of the short form, HSPDE4B2, was approx. 4-fold higher. Particulate-associated HSPDE4B1 and HSPDE4B2 were less active (approx. 40%) than their cytosol forms, whereas particulate HSPDE4B3 was similar in activity to its cytosolic form. Particulate and cytosolic forms of HSPDE4B1 and HSPDE4B3 were similarly inhibited by rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone}, the selective inhibitor of PDE4 (IC50 0.05-0.1 microM), whereas particulate-associated HSPDE4B2 was profoundly (approx. 10-fold) more sensitive (IC50 0.02 microM) to rolipram inhibition than its cytosolic form (IC50 0.2 microM). The various particulate-associated HSPDE4B isoforms showed very different susceptibilities to solubilization with the detergent Triton X-100 and high NaCl concentration. A novel cDNA, called pRPDE74, was obtained by screening a rat olfactory lobe cDNA library. This contained an open reading frame encoding a 721-residue protein that showed approx. 96% amino acid identity with HSPDE4B3 and is proposed to reflect the rat homologue of this human enzyme and is thus called RNPDE4B3. Alternative splicing of mRNA generated from both the human and rat PDE4B genes produces long and short splice variants that have unique N-terminal splice regions. It is suggested that these alternatively spliced regions determine changes in the maximal catalytic activity of the isoforms, their susceptibility to inhibition by rolipram and mode of interaction with particulate fractions.

Alternative splicing of cAMP-specific phosphodiesterase mRNA transcripts. Characterization of a novel tissue-specific isoform, RNPDE4A8.

In order to characterize the structure and regulation of members of the cAMP-specific phosphodiesterase (PDE) family (Type IV PDEs; PDE4 family), we have cloned from the rat a cDNA, pRPDE39, encoding a novel member of this family, which we call RNPDE4A8. Sequencing of the pRPDE39 cDNA shows it to be encoded by the rat PDE4A gene, but to differ from two other PDE4A transcripts, RD1 (pRPDE8; RNPDE4A1) and pRPDE6 (RNPDE4A5), by the presence of a unique region at its 5' end, consistent with alternative mRNA splicing. The pRPDE39 cDNA encodes a predicted protein of 763 amino acids, of which all but 21, located at the extreme amino terminus, are found in the pRPDE6 protein. Expression of pRPDE39 in COS cells produced a protein of 98 +/- 1.4 kDa, as determined by immunoblotting with an antiserum specific to the carboxyl-terminal regions of all PDE4A proteins, compared to a predicted value of 87.5 kDa. RNase protection analysis detected pRPDE39 mRNA only in testis. Immunoblotting of testis extracts demonstrated two bands of 97 +/- 2 and 87 +/- 3 kDa, the larger of which co-migrated with the band seen in COS cells expressing pRPDE39. COS cell expressed pRPDE39 partitioned between a high speed pellet (particulate) fraction (15% of protein; 8% of activity) and a cytosolic fraction. The particulate fraction had a Km for cAMP of 3.3 +/- 0.6 microM, and the cytosolic fraction a Km of 5.4 +/- 2.8 microM. The Vmax values for the pRPDE39 protein, relative to the RD1 protein, were 0.16 +/- 0.06 and 0.29 +/- 0.05 for the particulate and cytosolic forms, respectively. The pRPDE39-encoded PDE activity could not be removed from the particulate fraction by high salt concentrations, or by nonionic detergents. The pRPDE39-encoded enzyme was inhibited by rolipram at an IC50 of 0.5 +/- 0.2 microM for the particulate form and 1.0 +/- 0.2 microM for the cytosolic form, which are values typical of PDE4 family members. The highly tissue-specific distribution of the pRPDE39 mRNA suggest that the pRPDE39 protein functions to modulate a cAMP signaling pathway that is present largely, if not exclusively, in the testis.

Differential CNS expression of alternative mRNA isoforms of the mammalian genes encoding cAMP-specific phosphodiesterases.

To study alternative splicing and tissue-specific expression of the mammalian genes encoding type-IV cAMP-specific phosphodiesterases, which are homologs of the dnc learning and memory gene of Drosophila melanogaster, we cloned seven cDNAs from four rat loci (PDE1, PDE2, PDE3 and PDE4) homologous to dnc. The deduced amino-acid sequences of the proteins encoded by the rat loci were shown to have a 1:1 correspondence with those encoded by the four human dnc homologs. The proteins encoded by at least one cDNA from each of the four rat loci contained novel N-terminal upstream conserved regions (UCR1 and UCR2), described previously in proteins encoded by the human dnc homologs and by dnc. cDNAs from three of the rat loci (PDE2, PDE3 and PDE4) had a structure consistent with alternative splicing of the 5' coding regions of their respective mRNAs. UCR1, and in one case a portion of UCR2, were absent in one of the alternatively spliced transcripts from these three loci. RNase protection analysis showed that the rat PDE3 and PDE4 loci were each expressed at relatively constant levels in multiple regions of the brain, while PDE2 transcripts were more abundant in temporal cortex and brainstem. One of the alternatively spliced mRNAs from the PDE4 locus was relatively more abundant in temporal cortex and cerebellum. One alternatively spliced transcript from the PDE3 locus was expressed more abundantly in parietal cortex. Both of the alternatively spliced transcripts from the human DPDE4 locus (the homolog of rat PDE4) were expressed in temporal cortex.

Myasthenia gravis after allogeneic bone marrow transplantation: relationship to chronic graft-versus-host disease.

Three patients with chronic graft-versus-host disease (GVHD) developed myasthenia gravis (MG) 762 to 1,180 days after allogeneic bone marrow transplantation. Symptoms of MG were observed after taper or discontinuation of immunosuppressive treatment of chronic GVHD. All patients developed antibodies to acetylcholine receptor, and one had antibody formation to striated muscle. One patient died of complications of treatment of MG. The severity of disease underscores the importance of the differential diagnosis and the need for prompt therapy of this late complication after human bone marrow transplantation.

Second marrow infusion for poor graft function after allogeneic marrow transplantation.

Fifty-seven patients undergoing allogeneic marrow transplantation had persistent poor graft function after transplant and received a second marrow infusion from the original donor. Eight donors were HLA-non-identical family members and 49 were HLA-identical siblings. Poor function of the initial graft occurred in the absence of demonstrable rejection or persistent malignancy. Preparative reconditioning was not given before the second marrow infusion. Thirty-four of the 57 patients survived more than 1 month after the boost and were evaluated for haematopoietic recovery. All 34 subsequently achieved a neutrophil count of more than 500 X 10(6)/l and 20 became independent of platelet transfusions. Forty-nine (86%) of the 57 patients demonstrated acute graft-versus-host disease (GVHD). In 24 of the 49 patients GVHD increased in severity (10 patients) or first appeared (14 patients) after the boost. Chronic GVHD developed in all of the 20 patients who survived more than 150 days after the second infusion. Currently, 10 patients survive (median follow-up of more than 5 years) and all have normal haematologic function. Although haematologic reconstitution and increased GVHD occurred in some patients receiving second marrow infusions, the relation of these outcomes to the marrow boost is unclear in the absence of a control group.