AQP2 is a substrate for endogenous PP2B activity within an inner medullary AKAP-signaling complex.

We have demonstrated that inner medullary collecting duct (IMCD) heavy endosomes purified from rat kidney IMCD contain the type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, PKCzeta, and an RII-binding protein (relative molecular mass ~90 kDa) representing a putative A kinase anchoring protein (AKAP). Affinity chromatography of detergent-solubilized endosomes on cAMP-agarose permits recovery of a protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKCzeta. With the use of small-particle flow cytometry, RII and PKCzeta were localized to an identical population of endosomes, suggesting that these proteins are components of an endosomal multiprotein complex. (32)P-labeled aquaporin-2 (AQP2) present in these PKA-phosphorylated endosomes was dephosphorylated in vitro by either addition of exogenous PP2B or by an endogenous endosomal phosphatase that was inhibited by the PP2B inhibitors EDTA and the cyclophilin-cyclosporin A complex. We conclude that IMCD heavy endosomes possess an AKAP multiprotein-signaling complex similar to that described previously in hippocampal neurons. This signaling complex potentially mediates the phosphorylation of AQP2 to regulate its trafficking into the IMCD apical membrane. In addition, the PP2B component of the AKAP-signaling complex could also dephosphorylate AQP2 in vivo.

A-kinase-anchoring protein AKAP95 is targeted to the nuclear matrix and associates with p68 RNA helicase.

The cell nucleus is structurally and functionally organized by the nuclear matrix. We have examined whether the nuclear cAMP-dependent protein kinase-anchoring protein AKAP95 contains specific signals for targeting to the subnuclear compartment and for interaction with other proteins. AKAP95 was expressed in mammalian cells and found to localize exclusively to the nuclear matrix. Mutational analysis was used to identify determinants for nuclear localization and nuclear matrix targeting of AKAP95. These sites were found to be distinct from previously identified DNA and protein kinase A binding domains. The nuclear matrix-targeting site is unique but conserved among members of the AKAP95 family. Direct binding of AKAP95 to isolated nuclear matrix was demonstrated in situ and found to be dependent on the nuclear matrix-targeting site. Moreover, Far Western blot analysis identified at least three AKAP95-binding proteins in nuclear matrix isolated from rat brain. Yeast two-hybrid cloning identified one binding partner as p68 RNA helicase. The helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells, associated in vitro, and were precipitated as a complex from solubilized cell extracts. The results define novel protein-protein interactions among nuclear matrix proteins and suggest a potential role of AKAP95 as a scaffold for coordinating assembly of hormonally responsive transcription complexes.

The molecular basis for protein kinase A anchoring revealed by solution NMR.

Compartmentalization of signal transduction enzymes into signaling complexes is an important mechanism to ensure the specificity of intracellular events. Formation of these complexes is mediated by specialized protein motifs that participate in protein-protein interactions. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) is localized through interaction of the regulatory (R) subunit dimer with A-kinase-anchoring proteins (AKAPs). We now report the solution structure of the type II PKA R-subunit fragment RIIalpha(1-44), which encompasses both the AKAP-binding and dimerization interfaces. This structure incorporates an X-type four-helix bundle dimerization motif with an extended hydrophobic face that is necessary for high-affinity AKAP binding. NMR data on the complex between RIIalpha(1-44) and an AKAP fragment reveals extensive contacts between the two proteins. Interestingly, this same dimerization motif is present in other signaling molecules, the S100 family. Therefore, the X-type four-helix bundle may represent a conserved fold for protein-protein interactions in signal transduction.

Identification of cGMP-dependent protein kinase anchoring proteins (GKAPs).

To promote both efficiency and selectivity, many protein kinases and phosphatases are maintained in specific subcellular microenvironments through their association with anchoring proteins. In this study, we describe a new class of proteins, called GKAPS, that specifically bind the Type II cGMP-dependent protein kinase (PKG). GKAPs were detected in rat aorta, brain, and intestine using a protein overlay technique. The PKG binding proteins were distinct from AKAPs, proteins known to bind the cAMP-dependent protein kinase (PKA). Furthermore, a synthetic peptide that blocks association of PKA with AKAPs did not affect the PKG-GKAP interaction. Deletion mutagenesis was used to map the GKAP binding determinants within PKG to the N-terminal regulatory region. While most GKAPs were tissue-specific, a ubiquitous PKG-binding protein was detected and identified as myosin. Analysis of myosin fragments revealed that PKG binds within Subfragment 2. The results define a novel class of anchoring proteins that may target PKG for specific functional roles.

Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95.

The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N. J. Lamb, and J. D. Scott (1994) J. Biol. Chem. 269, 7658-7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414-692 of human AKAP95 was expressed in Escherichia coli and shown to bind RIIalpha. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIalpha binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIalpha was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RIIalpha overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RIIalpha was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIalpha may be cell cycle-dependent.

Mutational analysis of the A-kinase anchoring protein (AKAP)-binding site on RII. Classification Of side chain determinants for anchoring and isoform selective association with AKAPs.

Compartmentalization of the type II cAMP-dependent protein kinase is conferred by interaction of the regulatory subunit (RII) with A-Kinase Anchoring Proteins (AKAPs). The AKAP-binding site involves amino-terminal residues on each RII protomer and is formed through dimerization. A site-directed mutagenesis strategy was utilized to assess the contribution of individual residues in either RII isoform, RIIalpha or RIIbeta, for interaction with various anchoring proteins. Substitution of long-chain or bulky hydrophobic groups (leucines or phenylalanines) for isoleucines at positions 3 and 5 in RIIalpha decreased AKAP-binding up to 24 +/- 3 (n = 8)-fold, whereas introduction of valines had minimal effects. Replacement with hydrophilic residues (serine or asparigine) at both positions abolished AKAP binding. Mutation of proline 6 in RIIalpha reduced binding for four AKAPs (Ht31, MAP2, AKAP79, and AKAP95) from 2.3 to 20-fold (n = 4) whereas introduction of an additional proline at position 6 in RIIbeta increased or conferred binding toward these anchoring proteins. Therefore, we conclude that beta-branched side chains at positions 3 and 5 are favored determinants for AKAP-binding and prolines at positions 6 and 7 increase or stabilize RIIalpha interaction with selected anchoring proteins.

Cloning and characterization of a novel A-kinase anchoring protein. AKAP 220, association with testicular peroxisomes.

Compartmentalization of the type II cyclic AMP-dependent kinase (PKA) is achieved through association of the regulatory subunit (RII) with A-kinase anchoring proteins (AKAPs). Using an interaction cloning strategy with RIIalpha as a probe, we have isolated cDNAs encoding a novel 1129-amino acid protein that contains both a PKA binding region and a peroxisome targeting motif. Northern analysis detected mRNAs of 9.7 and 7.3 kb in several rat tissues with the highest levels present in the brain and testis. Western analysis and RII overlay experiments showed that the protein is approximately 220 kDa and was, therefore, named AKAP 220. Immunoprecipitation of AKAP 220 from rat testis extracts resulted in co-purification of the type II PKA holoenzyme. The specific activity of PKA increased 458-fold from 7.2 pmol/min/mg in the cell lysate to 3.3 nmol/min/mg in the immunoprecipitate. Immunohistochemical analysis of rat testicular TM4 cells showed that AKAP 220 and a proportion of RII were co-localized in microbodies that appear to be a subset of peroxisomes. Collectively, these results suggest that AKAP 220 may play a role in targeting type II PKA for cAMP-responsive peroxisomal events.

Multinuclear magnetic resonance and mutagenesis studies of the histidine residues of human mitochondrial ferredoxin.

Human mitochondrial ferredoxin is a [2Fe-2S] protein that functions to transfer electrons from NADPH-dependent ferredoxin reductase to cytochrome P450 enzymes. Two of the three histidines of human ferredoxin are strictly conserved in the sequences of all known vertebrate ferredoxins, and one of these (His56) is adjacent to Cys55, which serves as one of the ligands to the iron-sulfur cluster. All but 16 of its residues show sequence identity with those of bovine ferredoxin. It has been proposed for bovine ferredoxin that His56 hydrogen bonds with a labile sulfur and that the reduction of the iron-sulfur center is accompanied by the uptake of a proton by this histidine [Lambeth, J. D., Seybert, D. W., Lancaster, J. R., Jr., Salerno, J. C., & Kamin, H. (1982) Mol. Cell. Biochem. 45, 13-31]. In this paper, we report procedures for labeling human ferredoxin uniformly with 15N using 15NH4Cl and selectively with 13C by the incorporation of [U-13C]histidine. Most of the imidazole 1H, 13C, and 15N resonances of the three histidines have been assigned by heteronuclear two-dimensional single- and multiple-bond correlation spectroscopy. Site-directed mutagenesis was used in assigning the NMR signals from His56. The pKa values of His10 (6.5) and His62 (5.8) in oxidized human ferredoxin were found to be similar to those reported previously for the corresponding residues of bovine ferredoxin [Greenfield, N. J., Wu, X., & Jordan, F. (1989) Biochim. Biophys. Acta 995, 246-254; Miura, S., Tamita, S., & Ichikawa, Y. (1991) J. Biol. Chem. 266, 19212-19216].(ABSTRACT TRUNCATED AT 250 WORDS)

Association of protein kinase A and protein phosphatase 2B with a common anchoring protein.

Specificity of protein kinases and phosphatases may be achieved through compartmentalization with preferred substrates. In neurons, adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase (PKA) is localized at postsynaptic densities by association of its regulatory subunit with an A kinase anchor protein, AKAP79. Interaction cloning experiments demonstrated that AKAP79 also binds protein phosphatase 2B, or calcineurin (CaN). A ternary complex of PKA, AKAP, and CaN was isolated from bovine brain, and colocalization of the kinase and the phosphatase was established in neurites of cultured hippocampal neurons. The putative CaN-binding domain of AKAP79 is similar to that of the immunophilin FKBP-12, and AKAP79 inhibited CaN phosphatase activity. These results suggest that both PKA and CaN are targeted to subcellular sites by association with a common anchor protein and thereby regulate the phosphorylation state of key neuronal substrates.

Human regulatory subunit RI beta of cAMP-dependent protein kinases: expression, holoenzyme formation and microinjection into living cells.

The human regulatory subunit RI beta of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin. The human recombinant RI beta protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RI beta antiserum. Furthermore, the purified recombinant RI beta protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit C alpha. The rate of RI beta 2C alpha 2 holoenzyme formation was faster in the presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active C alpha-subunit, due to cAMP binding to RI beta. Compared to a RI alpha 2C alpha 2 holoenzyme, the RI beta 2C alpha 2 holoenzyme exhibited a more than twofold higher sensitivity to cAMP. The subcellular localization of RI beta was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm. A cytoplasmic distribution was observed when free RI beta was injected, whereas free C alpha injected into the cytoplasm appeared in the nucleus. When holoenzymes with labeled RI beta and unlabeled C alpha, or unlabeled RI beta and labeled C alpha, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RI beta was the labeled subunit of the in vivo dissociated holoenzyme. In contrast, nuclear fluorescence was evident from the release and translocation of labeled C alpha from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.

Type II regulatory subunit (RII) of the cAMP-dependent protein kinase interaction with A-kinase anchor proteins requires isoleucines 3 and 5.

Compartmentalization of the type II cAMP-dependent protein kinase is maintained by association of the regulatory subunit (RII) with A-Kinase Anchor Proteins (AKAPs). In previous studies (Scott, J. D., Stofko, R. E., McDonald, J. R., Comer, J. D., Vitalis, E. A., and Mangili J. (1990) J. Biol. Chem. 265, 21561-21566) we have shown that dimerization of RII alpha was required for interaction with the cytoskeletal component microtubule-associated protein 2. In this report we show that the localization and dimerization domains of RII alpha are contained within the first thirty residues of each RII protomer. RII des-5 (an amino-terminal deletion mutant lacking residues 1-5) was unable to bind AKAPs but retained the ability to dimerize. RII alpha I3A,I5A (a mutant where isoleucines 3 and 5 were replaced with alanine) was unable to bind a variety of AKAPs. Mutation of both isoleucines decreased AKAP binding without affecting dimerization, cAMP binding, or the overall secondary structure of the protein. Measurement of RII alpha I3A,I5A interaction with the human thyroid AKAP, Ht 31, by two independent methods suggests that mutation of isoleucines 3 and 5 decreases affinity by at least 6-fold. Therefore, we propose that two isoleucine side chains on each RII protomer are principle sites of contact with the conserved amphipathic helix binding domain on AKAPs.

Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase.

The subcellular location of the type II cAMP-dependent protein kinase is dictated by the interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Using an interaction cloning strategy with RII alpha as a probe, we have isolated cDNAs encoding a novel 761-amino acid protein (named AKAP 95) that contains both RII- and DNA-binding domains. Deletion analysis and peptide studies revealed that the RII-binding domain of AKAP 95 is located between residues 642 and 659 and includes a predicted amphipathic helix. Zinc overlay and DNA binding studies suggest that the DNA-binding domain is composed of two CC/HH-type zinc fingers between residues 464 and 486 and residues 553 and 576. The AKAP was detected in a nuclear matrix fraction, and immunofluorescence using purified anti-AKAP 95 antibodies revealed a distinct nuclear staining in a variety of cell types. Direct overlay of fluorescein isothiocyanate-labeled RII alpha onto fixed rat embryo fibroblasts showed that high-affinity binding sites for RII exist in the nucleus and that these sites are blocked by an anchoring inhibitor peptide. Furthermore, AKAP 95 was detected in preparations of RII that were purified from cellular extracts using cAMP-agarose. The results suggest that AKAP 95 could play a role in targeting type II cAMP-dependent protein kinase for cAMP-responsive nuclear events.

A-kinase anchoring proteins: a key to selective activation of cAMP-responsive events?

The cAMP-dependent protein kinase (PKA) regulates a variety of diverse biochemical events through the phosphorylation of target proteins. Because PKA is a multifunctional enzyme with a broad substrate specificity, its compartmentalization may be a key regulatory event in controlling which particular target substrates are phosphorylated. In recent years it has been demonstrated that differential localization of the type II holoenzyme is directed through interaction of the regulatory subunit (RII) with a family of A-Kinase Anchoring Proteins (AKAPs). In this report, we review evidence for PKA compartmentalization and discuss the structural and functional properties of AKAPs.

Electrostatic interactions stabilizing ferredoxin electron transfer complexes. Disruption by "conservative" mutations.

Mitochondrial ferredoxins mediate electron transfer from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes. Previous studies on human ferredoxin, in which acidic residues were replaced with neutral amino acids, established that Asp-76 and Asp-79 are are important for binding to both reductase and P450 (Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612). Here we report that replacement of Asp----Glu at position 76 or 79, whereas maintaining negative charge at these positions also results in dramatic decreases in binding affinity for both electron transfer partners (5-100-fold, delta(delta G) approximately 1.0-2.8 kcal/mol). These results imply that the active electron transfer complexes in these systems are dominated by a stable form which requires specific pairwise electrostatic interactions of fixed geometry for recognition and binding. This mechanism contrasts with that proposed for other electron transfer systems (as exemplified by cytochrome c) in which electrostatic interactions are believed to function primarily in precollisional orientation leading to "encounter complexes" having multiple geometries of similar free energy.

Site-specific mutations in human ferredoxin that affect binding to ferredoxin reductase and cytochrome P450scc.

Ferredoxins found in animal mitochondria function in electron transfer from NADPH-dependent ferredoxin reductase (Fd-reductase) to cytochrome P450 enzymes. To identify residues involved in binding of human ferredoxin to its electron transfer partners, neutral amino acids were introduced in a highly conserved acidic region (positions 68-86) by site-directed mutagenesis of the cDNA. Mutant ferredoxins were produced in Escherichia coli, and separate assays were used to determine the effect of substitutions on the capacity of each mutant to bind to Fd-reductase and cytochrome P450scc and to participate in the cholesterol side chain cleavage reaction. Replacements at several positions (mutants D68A, E74Q, and D86A) did not significantly affect activity, suggesting that acidic residues at these positions are not required for binding or electron transfer interactions. In contrast, substitutions at positions 76 and 79 (D76N and D79A) caused dramatic decreases in activity and in the affinity of ferredoxin for both Fd-reductase and P450scc; this suggests that the binding sites on ferredoxin for its redox partners overlap. Other substitutions (mutants D72A, D72N, E73A, E73Q, and D79N), however, caused differential effects on binding to Fd-reductase and P450scc, suggesting that the interaction sites are not identical. We propose a model in which Fd-reductase and P450scc share a requirement for ferredoxin residues Asp-76 and Asp-79 but have other determinants that differ and play an important role in binding. This model is consistent with the hypothesis that ferredoxin functions as a mobile shuttle in steroidogenic electron transfer, and it is considered unlikely that a functional ternary complex is formed.

1H NMR spectra of vertebrate [2Fe-2S] ferredoxins. Hyperfine resonances suggest different electron delocalization patterns from plant ferredoxins.

We report the observation of paramagnetically shifted (hyperfine) proton resonances from vertebrate mitochondrial [2Fe-2S] ferredoxins. The hyperfine signals of human, bovine, and chick [2Fe-2S] ferredoxins are described and compared with those of Anabaena 7120 vegetative ferredoxin, a plant-type [2Fe-2S] ferredoxin studied previously [Skjeldal, L., Westler, W. M., & Markley, J. L. (1990) Arch. Biochem. Biophys. 278, 482-485]. The hyperfine resonances of the three vertebrate ferredoxins were very similar to one another both in the oxidized state and in the reduced state, and slow (on the NMR scale) electron self-exchange was observed in partially reduced samples. For the oxidized vertebrate ferredoxins, hyperfine signals were observed downfield of the diamagnetic envelope from +13 to +50 ppm, and the general pattern of peaks and their anti-Curie temperature dependence are similar to those observed for the oxidized plant-type ferredoxins. For the reduced vertebrate ferredoxins, hyperfine signals were observed both upfield (-2 to -18 ppm) and downfield (+15 to +45 ppm), and all were found to exhibit Curie-type temperature dependence. This pattern and temperature dependence are distinctly different from those found with reduced plant-type ferredoxins which have signal centered around +120 ppm with Curie-type temperature dependence, assigned to cysteines which interact with Fe(III), and signals centered around +20 ppm with anti-Curie temperature dependence, assigned to cysteines which interact with Fe(II) [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271].(ABSTRACT TRUNCATED AT 250 WORDS)

Hybribox: a device for processing numbers of radioactively labeled hybridization filters with a minimum of personal exposure.

The device described permits the rapid and efficient processing of large numbers of filter hybridizations while virtually eliminating exposure to radioactive emission from the labeled probe. The filters are annealed and washed without being transferred from the holder. Throughout the process, the Plexiglas composition of the device absorbs the beta particles and exposure to the hands and arms is essentially zero. Since the hybridization solution can be reused several times, the cost involved approximates that of the conventional methods for hybridization.

Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli.

A cDNA fragment encoding human ferredoxin, a mitochondrial [2Fe-2S] protein, was introduced into Escherichia coli by using an expression vector based on the approach of Nagai and Thøgersen [Nagai, K. & Thøgersen, M. C. (1984) Nature (London) 309, 810-812]. Expression was under control of the lambda PL promoter and resulted in production of ferredoxin as a cleavable fusion protein with an amino-terminal fragment derived from bacteriophage lambda cII protein. The fusion protein was isolated from the soluble fraction of induced cells and was specifically cleaved to yield mature recombinant ferredoxin. The recombinant protein was shown to be identical in size to ferredoxin isolated from human placenta (13,546 Da) by NaDodSO4/PAGE and partial amino acid sequencing. E. coli cells expressing human ferredoxin were brown in color, and absorbance and electron paramagnetic resonance spectra of the purified recombinant protein established that the [2Fe-2S] center was assembled and incorporated into ferredoxin in vivo. Recombinant ferredoxin was active in steroid hydroxylations when reconstituted with cytochromes P-450scc and P-450(11) beta and exhibited rates comparable to those observed for ferredoxin isolated from human placenta. This expression system should be useful in production of native and structurally altered forms of human ferredoxin for studies of ferredoxin structure and function.