The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.

The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid regulation of progesterone receptor expression in cultured rat gonadotropes.

During the preovulatory period, the pituitary action of progesterone is biphasic, moving from a severalfold augmentation of the gonadotropin release action of GnRH to a suppression of GnRH efficacy, which occurs in rats over a period of about 12 h, but the extent to which these biphasic effects are dependent on alterations in progesterone receptor (PR) expression is not known. To address this, as well as the localization of PR in cultured rat pituitary cells, we used cells from ovariectomized rats cultured +/- 0.2 nM E2 with acute progesterone treatment on day 3. Northern blot of poly(A+) RNA extracts showed multiple PR messenger RNA (mRNA) transcripts between 4.8-10.2 kb; E2 treatment led to a 5- to 6-fold increase in the predominant PR mRNA transcripts (5.1 and 10.1 kb). In the presence of E2, 200 nM progesterone resulted in a decrease in steady-state PR mRNA levels by 3 h of exposure, with the greatest decrease around 6 h (50% of E2 control) and recovery by 12 h. Similarly treated pituitary cultures were subjected to dual immunofluorescence staining for LH and PR. In the absence of E2, PR was undetectable. In the presence of E2, essentially all LH-positive cells were positive for PR and only 1-2% of PR-immunopositive cells were negative for LH, possibly reflecting FSH-exclusive gonadotropes. PR staining was predominantly nuclear, but 20 nM progesterone led to a gradual increase in cytoplasmic staining, with the nuclear-to-cytoplasmic ratio decreasing to near unity by 9-12 h of exposure. In summary, we show for the first time, that PR colocalizes with LH in cultured female rat pituitary cells and that E2 induces expression of PR mRNA, as well as PR protein, in rat gonadotropes. In the presence of E2, progesterone causes a rapid but transient down-regulation of PR message; recovery of PR mRNA is accompanied by an increase in cytoplasmic PR, suggestive of an increase in synthesis. These dynamic changes implicate the gonadotrope PR as having a significant role within the temporal context of the rat preovulatory period.

Multiple pathway signal transduction by the cAMP-dependent protein kinase.

Since its discovery a quarter century ago, the cAMP-dependent protein kinase has been a central model for study of the mode of transduction of second messenger signals; more than 300 protein kinases are now known to play keys roles in cellular control. Multiple cellular events are initiated by the activation of the cAMP-dependent protein kinase and correlated with these events has been the identification of a broad spectrum of protein substrates. From model substrates and inhibitors an excellent understanding has been obtained of the "optimum" sequence for protein phosphorylation by the cAMP-dependent protein kinases, and now, from pioneering crystal structure studies, we are beginning to understand exactly how an optimum substrate can interact with and be efficiently phosphorylated by the kinase. The next important step is for us to understand the full sequence of events that occurs within the cell upon activation of the protein kinase, and it is abundantly evident that this is indeed a complex process. It is not sufficient to simply know which proteins are phosphorylated but it is critical that we understand the dynamics of the events surrounding the phosphorylation of multiple proteins, what factors dictate those dynamics, and what might happen when the sequence of events is perturbed. This review focuses on the first simple question that must be addressed, namely, how might proteins vary as substrates for the cAMP-dependent protein kinase and what ramifications might such variations have for the consequential events within the cell?

Multiplicity of the beta form of the cAMP-dependent protein kinase inhibitor protein generated by post-translational modification and alternate translational initiation.

Two distinct species of the thermostable inhibitor of the cAMP-dependent protein kinase, PKIalpha and PKIbeta, exist that are the products of separate genes. The PKIbeta form, as first isolated from rat testis, is a 70-amino acid protein, but the genomic sequence suggested that an alternate form might exist, arising as a consequence of alternate translational initiation. This species, now termed PKIbeta-78, has been synthesized by bacterial expression, demonstrated to be equipotent with PKIbeta-70, and also now demonstrated to occur in vivo. By Western blot analyses, six additional species of PKIbeta are also evident in tissues. Two of these represent the phospho forms of PKIbeta-78 and PKIbeta-70. The other four represent phospho and dephospho forms of two higher molecular mass PKIbeta species. These latter forms are currently termed PKIbeta-X and PKIbeta-Y, awaiting the full elucidation of their molecular identity. In adult rat testis and cerebellum, PKIbeta-70, PKIbeta-X, and PKIbeta-Y constitute 39, 23, and 32% and 15, 29, and 54% of the total tissue levels, respectively. In adult rat testis, 35-42% of each of these three species is present as a monophospho form, whereas no phosphorylation of them is evident in cerebellum. PKIbeta-78 is present at much lower levels in both rat testis and cerebellum (approximately 6 and 2% of the total, respectively) and almost entirely as a monophospho species. PKIbeta-78, like PKIbeta-70, is a high affinity and specific inhibitor of the cAMP-dependent protein kinase. PKIbeta-Y and PKIbeta-X, in contrast, also significantly inhibit the cGMP-dependent protein kinase.

The inhibitor protein of the cAMP-dependent protein kinase-catalytic subunit interaction. Parameters of complex formation.

The formation of a complex between the catalytic subunit of the cAMP-dependent protein kinase and the Inhibitor Protein of this enzyme has been examined by means of nondenaturing gel electrophoresis. Two forms of complex were identified, both containing a 1:1 molar ratio of the component proteins. The formation of the major of the two forms is markedly enhanced by the presence of nucleotide triphosphate and divalent cation. Either Mg2+ or Mn2+ serves to promote complex formation. With Mg2+, only ATP is effective for enhancing complex formation, whereas with Mn2+ complex formation occurs to an equal extent with ATP, GTP, ITP, and adenyl-5'-yl imidodiphosphate. The formation of the two complexes is only minimally dependent upon nucleotide triphosphate. It is suggested that the two types of complex are a result of different species of catalytic subunit. Two principal forms of the complex have been detected occurring maximally in approximately a 2.5:1 ratio. In the accompanying paper (Fletcher, W.H., Van Patten, S.M., Cheng, H-C., and Walsh, D.A. (1986) J. Biol. Chem. 261, 5504-5513), we have described the use of a fluoresceinated derivative of catalytic subunit as a cytochemical probe to localize the Inhibitor Protein and the regulatory subunit of the protein kinase. The integrity of this fluorophore has been further characterized using the method of examining catalytic subunit-Inhibitor Protein interaction delineated here.

Cytochemical identification of the regulatory subunit of the cAMP-dependent protein kinase by use of fluorescently labeled catalytic subunit. Examination of protein kinase dissociation in hepatoma cells responding to 8-Br-cAMP stimulation.

Homogeneous catalytic subunit from the cAMP-dependent protein kinase, when derivatized with a fluorophore, was used as a cytochemical probe to locate intracellular sites of the protein kinase regulatory subunit. After conjugation, the fluoresceinated catalytic subunit (F:C), derivatized to a stoichiometry of approximately 1 mol/mol, retained near full activity as judged by specific activity and by titration against either regulatory subunit or Inhibitor Protein of the protein kinase. With this molecular probe the dissociated regulatory subunit was localized by direct cytochemistry in Reuber H-35 hepatoma cells that had been exposed, while intact, for 0-120 min to 10(-4) M 8-Br-cAMP. After stimulation, cultures were fixed and washed and then incubated for 16 h with F:C. Following 8-Br-cAMP stimulation, extensive binding of the probe to both cytoplasmic and nucleolar sites was observed. This binding was diminished but not eliminated when 50 microM cAMP was present during the incubation of the fixed cells with F:C that was eliminated by a 40-fold molar excess of underivatized catalytic subunit but not by heat-denatured catalytic subunit, and was not reduced by a 20-fold molar excess of cGMP-dependent protein kinase, examined plus or minus cGMP. Collectively, the results allow the conclusion that the F:C probe binds free regulatory subunit. The time course of its change with 8-Br-cAMP (measured as the difference between binding in the presence or absence of cAMP during the postfixation treatment) mirrors that previously reported for changes in the catalytic subunit in these cells, also identified cytochemically (Byus, C. V., and Fletcher, W.H. (1982) J. Cell Biol. 93, 727-734). The binding of the F:C probe, detected when cAMP is present during postfixation treatment, may possibly represent binding to free Inhibitor Protein of the cAMP-dependent protein kinase. If so, it was at a level of approximately 20% of the maximal level of detectable regulatory subunit, and it also showed cytosolic and nucleolar localization.

The inhibitor protein of the cyclic AMP-dependent protein kinase-catalytic subunit interaction. Composition of multiple complexes.

It has been previously demonstrated that the combination of pure preparations of the inhibitor protein of the cyclic AMP-dependent protein kinase and the catalytic subunit of this enzyme resulted in the formation of multiple complexes [Van Patten, Fletcher & Walsh (1986) J. Biol. Chem. 261, 5514-5523]. In the present study it is demonstrated that these multiple species occur because the bovine heart protein kinase preparation contains multiple forms of catalytic subunit [Kinzel, Hotz, König, Gagelmann, Pyerin, Reed, Köbler, Hofmann, Obst, Gensheimer, Goldblatt & Shaltiel (1987) Arch. Biochem. Biophys. 253, 341-349].

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase contains two stable phosphorylation sites, Thr-197 and Ser-338 (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). Thr-197 is very resistant to dephosphorylation and thus cannot typically be autophosphorylated in vitro once the stable subunit is formed. Ser-338 is slowly dephosphorylated and can be rephosphorylated autocatalytically. In addition to these two stable phosphorylation sites, a new site of autophosphorylation, Ser-10, was identified. Phosphorylation at Ser-10 does not have a major effect on activity, and phosphates from Ser-10 or Ser-338 are not transferred to physiological substrates such as the type II regulatory subunit. Autophosphorylation at Ser-10 is associated with one of the two major isoelectric variants of the catalytic subunit. The form having the more acidic pI can be autophosphorylated at Ser-10 while the more basic form of the catalytic subunit cannot. Phosphorylation at Ser-10 does not account for the two isoenzyme forms. Since the reason for two isoelectric variants of the catalytic subunit is still unknown, it is not possible to provide a structural basis for the difference in accessibility of Ser-10 to phosphorylation. Either Ser-10 is not accessible in the more basic form of the catalytic subunit or some other type of post- or cotranslational modification causes Ser-10 to be a poor substrate. Whether the myristoyl group at the amino-terminal Gly is important for Ser-10 autophosphorylation remains to be established. The isoenzyme forms of the catalytic subunit do not correspond to the gene products coded for by the C alpha and C beta genes.

Tyrosine kinase catalyzed phosphorylation and inactivation of the inhibitor protein of the cAMP-dependent protein kinase.

The inhibitor protein of the cAMP-dependent protein kinase is a potential high affinity regulator of cAMP function. We now show that it is phosphorylated in Tyr7 by the intrinsic tyrosine kinase activity of epidermal growth factor receptor. The phosphorylated form can be readily separated from the unphosphorylated protein by high pressure liquid chromatography which has permitted the isolation of stoichiometrically phosphorylated protein. Using this method, it has been demonstrated that this phosphorylation, which occurs within the inhibitor protein's active domain, results in a 6 to 9-fold decrease in inhibitory potency. Possibly, a component of growth control could be the coupling of tyrosine kinase activity to cAMP-mediated cellular proliferation via the regulation of the efficacy of the inhibitor protein.

An active twenty-amino-acid-residue peptide derived from the inhibitor protein of the cyclic AMP-dependent protein kinase.

Digestion with Staphylococcus aureus V8 proteinase of the inhibitor protein of the cyclic AMP-dependent protein kinase results in the sequential formation of three active inhibitory peptides. The smallest active peptide has the sequence Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp . This 20-amino-acid-residue peptide has 20-40% of the activity of the native molecule and a Ki of 0.2 nM. Inhibition, as a minimum, appears to be based upon the inhibitor protein containing the recognition sequences that dictate protein-substrate-specificity. This inhibitory peptide also has sequence homology with the phosphorylation site for a protein kinase other than the cyclic AMP-dependent enzyme.

Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase.

The form of inhibitor protein of the cAMP-dependent protein kinase (PKI) that has been most thoroughly studied is a protein purified from rabbit skeletal muscle. Beale et al. previously isolated a species of PKI from rat testis that appeared from its amino acid composition to be quite distinct from the rabbit skeletal muscle protein [Beale, E. G., Dedman, J. R. & Means, A. R. (1977) J. Biol. Chem. 252, 6322-6327]. The amino acid sequence of a form of rat testis PKI has now been determined both by sequencing overlapping peptide fragments for 95% of the protein and by the isolation of a cDNA clone containing the coding region for the 70-amino acid protein. The sequence of the 70-amino acid testis PKI displays a maximum of only 41% sequence identity with the previously sequenced 75-amino acid rabbit skeletal muscle PKI. However, the two forms have identical potency as inhibitors and the key amino acids of the pseudosubstrate site, shown to be critical for maximal inhibition with the rabbit skeletal muscle PKI, have been conserved in the testis protein. The rabbit skeletal muscle and rat testis PKIs most likely represent distinct isoforms. The nucleotide sequence of the rat testis PKI cDNA suggests that a second form of testis PKI, longer by 8 additional amino-terminal amino acids, might also be produced.

A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase.

As an important new reagent for studying the cAMP-dependent protein kinase, a 20-residue peptide has been synthesized that corresponds to the active site of the skeletal muscle inhibitor protein. This synthetic peptide inhibits the protein kinase competitively with a Ki = 2.3 nM; its sequence, Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr- Gly-Arg-Arg-Asn-Ala-Ile-His-Asp, is that of a peptide previously reported by us which was derived from the native inhibitor protein by V8 protease digestion (Cheng, H. C., Van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem. J. 231, 655-661). Studies with analogues of this peptide show that its high affinity binding to the protein kinase (as also of the inhibitor protein) appears to be due to it mimicking the protein substrate by binding to the catalytic site via the arginine-cluster basic subsite (Formula: see text), and also to a critical contribution from one or more of the 6 N-terminal residues (Formula: see text). The availability of this high affinity synthetic peptide should open up a variety of avenues to probe the cellular actions of cAMP.

Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase.

Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine. Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI. Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures.

Specific testicular cellular localization and hormonal regulation of the PKIalpha and PKIbeta isoforms of the inhibitor protein of the cAMP-dependent protein kinase.

We have previously demonstrated that there exist two distinct genes for the thermostable inhibitor protein of the cAMP-dependent protein kinase, PKIalpha and PKIbeta (Van Patten, S. M., Howard, P., Walsh, D. A., and Maurer, R. A. (1992) Mol. Endocrinol. 6, 2114-2122). We have also shown that in the testis, at least eight forms of PKIbeta exist, differing as a result of at least post-translational modification and alternate translational initiation (Kumar, P., Van Patten, S. M., and Walsh, D. A. (1997) J. Biol. Chem. 272, 20011-20020). We now report that in the testis, there is a unique cellular distribution of protein kinase inhibitor forms, with PKIbeta being essentially (if not exclusively) a germ cell protein and PKIalpha being expressed primarily in Sertoli cells. Furthermore, there is a progressive change in the forms of PKIbeta that are present within germ cells with development that is initiated in testis tubules and continues as the germ cells migrate through the epididymis. These conclusions are derived from studies with isolated cell populations and with the at/at germ cell-deficient mouse line, by in situ hybridization, and by following the developmental expression of these proteins in both testis and epididymis. We have also shown that follicle-stimulating hormone (FSH) can increase the expression of both PKIalpha and PKIbeta. The FSH-regulated expression of PKIalpha in the Sertoli cell likely occurs via the normal route of second messenger signal transduction. In contrast, the FSH-dependent PKIbeta expression must arise by some form of Sertoli cell-germ cell intercommunication.

Oxidation of methionine residues in antithrombin. Effects on biological activity and heparin binding.

Commercially available human plasma-derived preparations of the serine protease inhibitor antithrombin (AT) were shown to contain low levels of oxidation, and we sought to determine whether oxidation might be a means of regulating the protein's inhibitory activity. A recombinant form of AT, with similarly low levels of oxidation as purified, was treated with hydrogen peroxide in order to study the effect of oxidation, specifically methionine oxidation, on the biochemical properties of this protein. AT contains two adjacent methionine residues near the reactive site loop cleaved by thrombin (Met314 and Met315) and two exposed methionines that border on the heparin binding region of AT (Met17 and Met20). In forced oxidations with hydrogen peroxide, the methionines at 314 and 315 were found to be the most susceptible to oxidation, but their oxidation did not affect either thrombin-inhibitory activity or heparin binding. Methionines at positions 17 and 20 were significantly oxidized only at higher concentrations of peroxide, at which point heparin affinity was decreased. However at saturating heparin concentrations, activity was only marginally decreased for these highly oxidized samples of AT. Structural studies indicate that highly oxidized AT is less able to undergo the complete conformational change induced by heparin, most probably due to oxidation of Met17. Since this does not occur in less oxidized, and presumably more physiologically relevant, forms of AT such as those found in plasma preparations, oxidation does not appear to be a means of controlling AT activity.

Transgenically produced human antithrombin: structural and functional comparison to human plasma-derived antithrombin.

Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma-derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.