Refolding and kinetic characterization of the phosphodiesterase-8A catalytic domain

Cyclic nucleotide phosphodiesterase-8 (PDE8) hydrolyzes the second messenger cAMP and is involved in many biological processes such as testosterone production. Although the bacterial and mammalian expression systems have been extensively tried, production of large quantity of soluble and active PDE8 remains to be a major hurdle for pharmacological and structural studies. Reported here is a detailed protocol of refolding and purification of large quantity of the PDE8A1 catalytic domain (residues 480-820) and kinetic characterization of the refolded protein. This protocol yielded about 8 mg of the PDE8A catalytic domain from 2l Escherichia coli culture, which has at least 40-fold higher activity than those reported in literature. The PDE8A1 catalytic domain has k(cat) of 4.0 s(-1) for Mn(2+) and 2.9s(-1) for Mg(2+), and the K(M) values of 1-1.8 microM. In addition, the PDE8A1 (205-820) fragment that contains both PAS and catalytic domains was expressed in E. coli and refolded. This PDE8A1 (205-820) fragment has k(cat) of 1.1 s(-1) and K(M) of 0.28 microM, but aggregated at high concentration. The K(M) of PDE8A1 (205-820) is 2- to 7-fold higher than the K(M) values of 40-150 nM for the full-length PDE8s in literature, but about 6-fold lower than that of the catalytic domain. Thus, the K(M) difference likely implies an allosteric regulation of the PDE8A activity by its PAS domain.     

TbPDE1, a novel class I phosphodiesterase of Trypanosoma brucei.

Cyclic nucleotide specific phosphodiesterases (PDEs) are important components of all cAMP signalling networks. In humans, 11 different PDE families have been identified to date, all of which belong to the class I PDEs. Pharmacologically, they have become of great interest as targets for the development of drugs for a large variety of clinical conditions. PDEs in parasitic protozoa have not yet been extensively investigated, despite their potential as antiparasitic drug targets. The current study presents the identification and characterization of a novel class I PDE from the parasitic protozoon Trypanosoma brucei, the causative agent of human sleeping sickness. This enzyme, TbPDE1, is encoded by a single-copy gene located on chromosome 10, and it functionally complements PDE-deficient strains of Saccharomyces cerevisiae. Its C-terminal catalytic domain shares about 30% amino acid identity, including all functionally important residues, with the catalytic domains of human PDEs. A fragment of TbPDE1 containing the catalytic domain could be expressed in active form in Escherichia coli. The recombinant enzyme is specific for cAMP, but exhibits a remarkably high Km of > 600 microm for this substrate.     

Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.     

The gamma subunit of the rod photoreceptor cGMP phosphodiesterase can modulate the proteolysis of two cGMP binding cGMP-specific phosphodiesterases (PDE6 and PDE5) by caspase-3

We have investigated whether the proteolysis of members of the cGMP binding phosphodiesterases (PDE6, PDE5A1, and PDE10A2) by caspase-3 is modulated by the gamma inhibitor subunit of PDE6. We show here that purified caspase-3 proteolyses PDE6, an enzyme composed of two nonidentical catalytic subunits (termed alpha and beta) with molecular mass of 88 and 84 kDa. The proteolysis of PDE6 produced a single fragment with a molecular mass of 78 kDa. This corresponds to the possible cleavage of the caspase-3 consensus DFVD site (amino acids: 164-168) in the alpha subunit and leads to a 50% decrease in the cGMP hydrolysing activity of the enzyme. The addition of rod PDEgamma to the incubation completely blocked the cleavage of PDE6 by caspase-3. In contrast, rod PDEgamma converted PDE5A1 (molecular mass of 98 kDa) to a better substrate for caspase-3. This resulted in the formation of four major fragments with molecular mass of 82-83, 67, 43, and 34 kDa. In addition, caspase-3 induced an approximately 80% reduction in the activity of a partially purified preparation of PDE5A1 in the presence of rod PDEgamma. Caspase-3 also cleaved PDE10A2 (molecular mass of 95 kDa) to a single 48-kDa fragment. This was consistent with cleavage of the DLFD site (amino acids: 312-315) in PDE10A2. In contrast with both PDE6 and PDE5A1, rod PDEgamma was without effect on this enzyme. These data show that rod PDEgamma interacts with at least two members of the cGMP binding PDE family (PDE5A1 and PDE6) and can exert differential effects on the cleavage of these enzymes by caspase-3.     

Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis

Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.     

Identification of the gamma subunit-interacting residues on photoreceptor cGMP phosphodiesterase, PDE6alpha '

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory gamma subunit (Pgamma). Chimeric proteins between cone PDE6alpha' and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by Pgamma. Substitution of the segment PDE5-(773-820) by the corresponding PDE6alpha'-(737-784) sequence in the wild-type PDE5 or in a PDE5/PDE6alpha' chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pgamma. The catalytic properties of the chimeric PDEs remained similar to those of PDE5. Ala-scanning mutational analysis of the Pgamma-binding region, PDE6alpha'-(750-760), revealed PDE6alpha' residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired the inhibition of chimeric PDE by Pgamma. The analysis of the catalytic properties of mutant PDEs and a model of the PDE6 catalytic domain suggest that residues Met(758) and Gln(752) directly bind Pgamma. A model of the PDE6 catalytic site shows that PDE6alpha'-(750-760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pgamma to Met(758) would effectively block access of cGMP to the catalytic cavity, providing a structural basis for the mechanism of PDE6 inhibition.     

Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity

Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain). The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate. A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied. The rate, cGMP dependence, and stoichiometry of phosphorylation of the PDE5 R domain by the catalytic subunit of cAMP-dependent protein kinase are comparable with that of the holoenzyme. Migration in native polyacrylamide gels suggests that either cGMP binding or phosphorylation produces distinct conformers of the R domain. Phosphorylation of the R domain increases affinity for cGMP approximately 10-fold (K(D) values 97.8 +/- 17 and 10.0 +/- 0.5 nm for unphospho- and phospho-R domains, respectively). [(3)H]cGMP dissociates from the phospho-R domain with a single rate (t(12) = 339 +/- 30 min) compared with the biphasic pattern of the unphospho-R domain (t(12) = 39.0 +/- 4.8 and 265 +/- 28 min, for the fast and slow components, respectively). Thus, cGMP-directed regulation of PDE5 phosphorylation and the resulting increase in cGMP binding affinity occur largely within the R domain. Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis.     

The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization

Retinal rod cGMP phosphodiesterase (PDE6 family) is the effector enzyme in the vertebrate visual transduction cascade. Unlike other known PDEs that form catalytic homodimers, the rod PDE6 catalytic core is a heterodimer composed of alpha and beta subunits. A system for efficient expression of rod PDE6 is not available. Therefore, to elucidate the structural basis for specific dimerization of rod PDE6, we constructed a series of chimeric proteins between PDE6alphabeta and PDE5, which contain the N-terminal GAFa/GAFb domains, or portions thereof, of the rod enzyme. These chimeras were co-expressed in Sf9 cells in various combinations as His-, myc-, or FLAG-tagged proteins. Dimerization of chimeric PDEs was assessed using gel filtration and sucrose gradient centrifugation. The composition of formed dimeric enzymes was analyzed with Western blotting and immunoprecipitation. Consistent with the selectivity of PDE6 dimerization in vivo, efficient heterodimerization was observed between the GAF regions of PDE6alpha and PDE6beta with no significant homodimerization. In addition, PDE6alpha was able to form dimers with the cone PDE6alpha' subunit. Furthermore, our analysis indicated that the PDE6 GAFa domains contain major structural determinants for the affinity and selectivity of dimerization of PDE6 catalytic subunits. The key dimerization selectivity module of PDE6 has been localized to a small segment within the GAFa domains, PDE6alpha-59-74/PDE6beta-57-72. This study provides tools for the generation of the homodimeric alphaalpha and betabeta enzymes that will allow us to address the question of functional significance of the unique heterodimerization of rod PDE6.     

Isolation and characterization of PDE10A, a novel human 3', 5'-cyclic nucleotide phosphodiesterase.

A gene encoding a novel human 3', 5'-cyclic nucleotide phosphodiesterase (PDE) was identified and characterized. PDE10A1 encodes a protein that is 779 amino acids in length. An incomplete cDNA for a second 5'-splice variant, PDE10A2, was isolated. The proteins encoded by the two variants share 766 amino acids in common. This common region includes an amino-terminal domain with partial homology to the cGMP-binding domains of PDE2, PDE5 and PDE6 as well as a carboxy-terminal region with homology to the catalytic regions of mammalian PDEs. Northern analysis revealed that PDE10A is widely expressed. The PDE10A gene was mapped to three yeast artificial chromosomes (YACs) that contain human DNA from chromosome 6q26-27. A recombinant protein corresponding to the 766 amino acid region common to PDE10A1 and PDE10A2 was expressed in yeast. It hydrolyzed both cAMP and cGMP. Inhibitors that are selective for other PDE families are poor inhibitors of PDE10A; however, PDE10A is inhibited by the non-specific PDE inhibitor, IBMX.     

Isolation and characteristics of functionally active proteolytically modified forms of tyrosyl-tRNA synthetase from bovine liver

Functionally active proteolytic modified form of tyrosyl-tRNA-synthetase has been isolated in a homogeneous form from the bovine liver under incomplete blocking of endogenous proteolysis. The isolation scheme is described. From the data of gel electrophoresis under denaturing conditions the molecular weight of this form is 39 +/- 1.5 kDa and from the data of gel filtration under native conditions -84 kDa. Thus, this form as well as the native enzyme is a dimer of the alpha 2-type. As compared to the native enzyme (Mm 2 x 59 kDa) a proteolytically modified form has a fragment of the polypeptide chain about 20 kDa long split out (this fragment is not essential for catalytic activity). The values of catalytic characteristics of the modified form in tRNA(Tyr) aminoacylation reaction (Km = 1.19 microM and kcat = 2.99 min-1) are close to those obtained for the main form of the enzyme (0.69 microM and 2.97 min-1, respectively). Amino acid composition of the low-molecular form of tyrosyl-tRNA-synthetase has been determined. It was found that the fragment split out in limited proteolysis was characterized by very high content of positively charged lysine residues (46 residues). A proteolytically modified form of tyrosyl-tRNA-synthetase possesses, like the main form, the affinity to high-molecular rRNA but it is eluted from the column filled with rRNA-sepharose at lower salt concentration (50 mM KCl) as compared to the main form of the enzyme (100 mM KCl).     

Refolding and purification of recombinant human PDE7A expressed in Escherichia coli as inclusion bodies

We have investigated the refolding and purification of the catalytic domain of human 3',5'-cyclic nucleotide phosphodiesterase 7A1 (PDE7A1) expressed in Escherichia coli. A cDNA encoding an N-terminal-truncated PDE7A1(147-482-His) was amplified by RT-PCR from human peripheral blood cells and inserted into the vector pET21-C for bacterial expression of the enzyme fused to a C-terminal His-tag. The PDE was found to be expressed in the form of inclusion bodies which could be refolded to an active enzyme in buffer containing high concentrations of arginine hydrochloride, ethylene glycol, and magnesium chloride at pH 8.5. The PDE7A1(147-482-His) construct could be purified after dialysis and concentration steps by either Zn2+-IDA-Sepharose chromatography or ResourceQ ion-exchange chromatography to homogeneity. In comparison to the metal-chelate column, the ResourceQ purification resulted in a distinctly better yield and enrichment of the protein. Both the Vmax (0.46 micromol. min(-1). mg(-1) ) and the K(m) (0.1 microM) of the purified enzyme were found to be comparable with published data for native or recombinant catalytically active expressed PDE7A1. Using SDS/PAGE, a molecular mass of 39 kDa was determined (theoretical value 38.783 kDa). As known from several other mammalian PDEs, size-exclusion chromatography using refolded PDE7A1(147-482-His) indicated the formation of dimers. The purified enzyme was soluble at concentrations up to 100 microg/ml. A further increase of protein concentration resulted, however, in precipitation of the enzyme.     

Comparison of enzymatic characterization and gene organization of cyclic nucleotide phosphodiesterase 8 family in humans

Full-length cDNAs of human cyclic nucleotide phosphodiesterase 8B (PDE8B) were isolated. Enzymatic characteristics of a dominant variant encoding a protein of 885 residues (PDE8B1) were compared with those of PDE8A1. The recombinant PDE8A1 and PDE8B1 proteins of an entire form were produced in both cytosolic and membrane fractions of the transfected COS cells. The human PDE8B1 was a high-affinity cAMP-PDE with K(m) value of 101+/-12 nM for cAMP, which is greater than that of PDE8A1 (40+/-1 nM). Relative V(max) value of PDE8A1 was 57+/-8% compared with that of PDE8B1 (100+/-12%). Although PDE8A1 was moderately inhibited by dipyridamole with IC(50) value of 8+/-2 microM, the compound antagonized the PDE8B1 activity at three-fold higher concentration (IC(50)=23+/-2 microM). The human PDE8B gene was composed of 22 exons, spanning over 217 kb. Although overall sequence identity between PDE8A1 and PDE8B1 was 68%, positions of junctions of each exon between the PDE8A1 and PDE8B1 sequences were well matched, indicating evolutionary relatedness of both genes.     

Coexpression of human cAMP-specific phosphodiesterase activity and high affinity rolipram binding in yeast.

Studies by various investigators have demonstrated that the low Km, cAMP-specific phosphodiesterase (PDE IV) is selectively inhibited by a group of compounds typified by rolipram and Ro 20-1724. In addition to inhibiting the catalytic activity of PDE IV, rolipram binds to a high affinity binding site present in brain homogenates. Although it has been assumed that the high affinity rolipram-binding site is PDE IV, no direct evidence has been produced to support this assumption. The present studies were undertaken to determine whether the rolipram-binding site is coexpressed with PDE IV catalytic activity in Saccharomyces cerevisiae genetically engineered to express human recombinant monocytic PDE IV (hPDE IV). Expressing hPDE IV cDNA in yeast resulted in a 20-fold increase in PDE activity that was evident within 1 h of induction and reached a maximum by 3-6 h. The recombinant protein represented hPDE IV as judged by its immunoreactivity, molecular mass (approximately 88 kDa), kinetic characteristics (cAMP Km = 3.1 microM; cGMP Km greater than 100 microM), sensitivity to rolipram (Ki = 0.06 microM), and insensitivity to siguazodan (PDE III inhibitor) and zaprinast (PDE V inhibitor). Saturable, high affinity [3H] (R)-rolipram-binding sites (Kd = 1.0 nM) were coexpressed with PDE activity, indicating that both binding activity and catalytic activity are properties of the same protein. A limited number of compounds were tested for their ability to inhibit hPDE IV catalytic activity and compete for [3H](R)-rolipram binding. Analysis of the data revealed little correlation (r2 = 0.35) in the structure-activity relationships for hPDE IV inhibition versus competition for [3H] (R)-rolipram binding. In fact, certain compounds (e.g. (R)-rolipram Ro 20-1724) possessed a 10-100-fold selectivity for inhibition of [3H] (R)-rolipram binding over hPDE IV inhibition, whereas others (e.g. dipyridamole, trequinsin) possessed a 10-fold selectivity for PDE inhibition. Thus, although the results of these studies demonstrate that hPDE IV activity and high affinity [3H](R)-rolipram binding are properties of the same protein, they do not provide clear cut evidence linking the binding site with the PDE inhibitory activity of rolipram and related compounds.     

Crystal structures of phosphodiesterases 4 and 5 in complex with inhibitor 3-isobutyl-1-methylxanthine suggest a conformation determinant of inhibitor selectivity

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. Crystal structures of the catalytic domains of cGMP-specific PDE5A1 and cAMP-specific PDE4D2 in complex with the nonselective inhibitor 3-isobutyl-1-methylxanthine have been determined at medium resolution. The catalytic domain of PDE5A1 has the same topological folding as that of PDE4D2, but three regions show different tertiary structures, including residues 79-113, 208-224 (H-loop), and 341-364 (M-loop) in PDE4D2 or 535-566, 661-676, and 787-812 in PDE5A1, respectively. Because H- and M-loops are involved in binding of the selective inhibitors, the different conformations of the loops, thus the distinct shapes of the active sites, will be a determinant of inhibitor selectivity in PDEs. IBMX binds to a subpocket that comprises key residues Ile-336, Phe-340, Gln-369, and Phe-372 of PDE4D2 or Val-782, Phe-786, Gln-817, and Phe-820 of PDE5A1. This subpocket may be a common site for binding nonselective inhibitors of PDEs.     

Type III phosphodiesterase plays a necessary role in the growth-promoting actions of insulin, insulin-like growth factor-I, and Ha p21ras in Xenopus laevis oocytes.

Three phosphodiesterase (PDE) type III inhibitors were tested and found to inhibit Xenopus oocyte maturation induced by insulin with apparent IC50 values of 2.2 +/- 0.2 microM Cl-930, 25 +/- 3 microM imazodan (Cl-914), and 786 +/- 237 microM piroximone (MDL 19,205). The same rank order of potencies was observed for inhibition of insulin-like growth factor-I (IGF-I)-induced oocyte maturation, with IC50 values of 5.5 +/- 0.9 microM Cl-930, 54 +/- 4 microM imazodan, and 1190 +/- 395 microM piroximone. Oocyte maturation induced by microinjection of Ha p21ras was also inhibited by pretreatment of oocytes with Cl-930 or imazodan, with IC50 values of 4.3 +/- 1.2 and 59 +/- 4 microM, respectively. Progesterone-induced maturation was not affected by PDE III inhibitor action; and, neither type IV PDE inhibitors (Ro 20, 1724 or rolipram) nor dipyridamole (a type V PDE inhibitor) inhibited cell division induced by IGF-I or microinjected Ha p21ras. In addition, while insulin-stimulated oocyte PDE activity measured in vivo after microinjection of 200 microM [3H] cAMP was inhibited by nonselective and type III-specific drugs (with IC50 values of 4.2 +/- 1.8 microM Cl-930 and 26 +/- 6 microM imazodan), type IV and type V inhibitors did not inhibit hormone-stimulated enzyme activity. This pharmacological evidence demonstrates a necessary role for PDE III in insulin-, IGF-I-, and p21ras-induced meiotic cell division in Xenopus laevis oocytes.     

Three-dimensional structures of PDE4D in complex with roliprams and implication on inhibitor selectivity

Selective inhibitors against the 11 families of cyclic nucleotide phosphodiesterases (PDEs) are used to treat various human diseases. How the inhibitors selectively bind the conserved PDE catalytic domains is unknown. The crystal structures of the PDE4D2 catalytic domain in complex with (R)- or (R,S)-rolipram suggest that inhibitor selectivity is determined by the chemical nature of amino acids and subtle conformational changes of the binding pockets. The conformational states of Gln369 in PDE4D2 may play a key role in inhibitor recognition. The corresponding Y329S mutation in PDE7 may lead to loss of the hydrogen bonds between rolipram and Gln369 and is thus a possible reason explaining PDE7's insensitivity to rolipram inhibition. Docking of the PDE5 inhibitor sildenafil into the PDE4 catalytic pocket further helps understand inhibitor selectivity.     

Inhibitors of phosphodiesterase III block stimulation of Xenopus laevis oocyte ribosomal S6 kinase activity by insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) stimulated Xenopus laevis oocyte ribosomal S6 kinase activity 5- to 10-fold, with an apparent EC50 of 0.8 +/- 0.1 nM after 90 min of hormone treatment. IGF-I-stimulated enzyme activity was inhibited by treatment of oocytes with nonselective phosphodiesterase (PDE) inhibitors, with apparent IC50 values of 2 +/- 1 microM papaverine, 20 +/- 2 microM isobutylmethylxanthine, and 128 +/- 16 microM theophylline. Type III PDE inhibitors also inhibited IGF-I-stimulated S6 kinase activity with IC50 values of 9.7 +/- 0.3 microM Cl-930 and 84 +/- 23 microM imazodan (Cl-914). These drugs apparently affected an intracellular molecular event leading to activation of S6 kinase, since Cl-930 prevented IGF-I-stimulation of S6 kinase, but had no direct inhibitory effect when added to the S6 kinase enzyme assay mixture. While hormone-stimulated S6 kinase activity was inhibited by isobutylmethylxanthine (nonselective PDE inhibitor) and Cl-930 (PDE III inhibitor), Ro 20, 1724 and rolipram (PDE IV inhibitors) and dipyridamole (PDE V inhibitor) had no significant effect on activated enzyme levels. The time course for IGF-I stimulation of oocyte S6 kinase displayed a small early peak of activity approximately 0.15-0.4 time required for 50% of cell population to display white spots (GVBD50) and a second major increase in activity at 0.6-0.7 GVBD50 that was sustained until meiotic maturation was complete. The second wave of enzyme activation was inhibited by Cl-930, but the early increase was not.(ABSTRACT TRUNCATED AT 250 WORDS)