A-kinase anchoring proteins as the basis for cAMP signaling.

Common challenges to any cell are the processing of the extracellular stimuli it receives into intracellular signaling cascades that initiate a multitude of diverse biological functions. However, many of these stimuli act via a common signaling pathway, suggesting the cell must somehow discriminate between different stimuli and respond accordingly. Subcellular targeting through the association with adaptor and scaffolding proteins has emerged as a key mechanism by which cells maintain signaling specificity. Compartmentation of cAMP signaling is maintained by the clustering of cAMP signaling enzymes in discrete units by the scaffolding protein A-kinase anchoring proteins (AKAP). In doing so, AKAPs provide the molecular architecture for the cAMP micordomains that underlie the spacial-temporal control of cAMP signaling.

mAKAP and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope.

The physical association of regulatory enzymes and ion channels at relevant intracellular sites contributes to the diversity and specificity of second messenger-mediated signal transduction in cells. mAKAP is a scaffolding protein that targets the cAMP-dependent protein kinase A and phosphodiesterase type 4D3 to the nuclear envelope of differentiated cardiac myocytes. Here we present data that the mAKAP signaling complex also includes nuclear envelope-resident ryanodine receptors and protein phosphatase 2A. The ryanodine receptor is the major cardiac ion channel responsible for calcium-induced calcium release from intracellular calcium ion stores. As demonstrated by a combination of immunohistochemistry and tissue fractionation, mAKAP is targeted specifically to the nuclear envelope, whereas the ryanodine receptor is present at both the sarcoplasmic reticulum and nuclear envelope intracellular membrane compartments. At the nuclear envelope, a subset of cardiac ryanodine receptor is bound to mAKAP and via the association with mAKAP may be regulated by protein kinase A-mediated phosphorylation. By binding protein kinase A and ryanodine receptor, mAKAP may serve as the scaffold for a cAMP- and calcium ion-sensitive signaling complex.

mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module.

Spatiotemporal regulation of protein kinase A (PKA) activity involves the manipulation of compartmentalized cAMP pools. Now we demonstrate that the muscle-selective A-kinase anchoring protein, mAKAP, maintains a cAMP signaling module, including PKA and the rolipram-inhibited cAMP-specific phosphodiesterase (PDE4D3) in heart tissues. Functional analyses indicate that tonic PDE4D3 activity reduces the activity of the anchored PKA holoenzyme, whereas kinase activation stimulates mAKAP-associated phosphodiesterase activity. Disruption of PKA- mAKAP interaction prevents this enhancement of PDE4D3 activity, suggesting that the proximity of both enzymes in the mAKAP signaling complex forms a negative feedback loop to restore basal cAMP levels.

mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes.

The compartmentalization of second messenger-activated protein kinases contributes to the fidelity of hormone-mediated signal transduction events. For example, the cAMP-dependent protein kinase is tethered at specific intracellular locations through association with A-kinase anchoring proteins (AKAPs). We now report the cloning of mAKAP, an anchoring protein found predominantly in heart, skeletal muscle and brain, and whose expression is induced in neonatal ventriculocytes by treatment with hypertrophic stimuli. mAKAP is targeted to the nuclear membrane of differentiated myocytes. Analysis of mAKAP-green fluorescent protein (GFP) fusion constructs revealed that nuclear membrane targeting is conferred by two regions of the protein, between residues 772-915 and 915-1065, which contain spectrin-like repeat sequences. Heterologous expression of the mAKAP targeting sequences displaced the endogenous anchoring protein from the nuclear membrane, demonstrating that mAKAP targeting is saturable. Collectively, these data suggest that a domain containing spectrin-like repeats mediates targeting of the anchoring protein mAKAP and the cAMP-dependent protein kinase holoenzyme to the nuclear membrane in response to differentiation signals.

Regulation of intrasteric inhibition of the multifunctional calcium/calmodulin-dependent protein kinase.

A regulatory region involved in both autoinhibition and calmodulin (CaM) binding has previously been identified in the multifunctional Ca2+/CaM-dependent protein kinase (CaM kinase II). We have tested the role of various segments of the regulatory region in autoinhibition by the analysis of a series of truncation, substitution, and deletion mutants of the CaM kinase II alpha subunit (CaM kinase II alpha). Unexpectedly, the sequence Lys-Lys-Phe-Asn at positions 291-294, adjacent to the CaM binding domain, was found to be sufficient to maintain an inhibited state in a truncated form of the kinase. However, these residues are not essential in the context of the full-length protein, indicating the importance of additional residues from the overlapping CaM binding domain. We propose here a molecular model for CaM kinase II alpha based on the three-dimensional structure of the cAPK-PKI-(5-24) (protein kinase inhibitor fragment) complex. It is predicted from this model that autoinhibition is of the pseudosubstrate variety and that autophosphorylation of Thr-286 could occur by an intersubunit reaction in the holoenzyme complex.

Variable effects of phosphorylation of Pit-1 dictated by the DNA response elements.

Pit-1, a tissue-specific POU domain transcription factor, is required for the activation of the prolactin, growth hormone, and Pit-1 promoters that confer regulation by epidermal growth factor, adenosine 3',5'-monophosphate (cAMP), and phorbol esters. Pit-1 is phosphorylated in pituitary cells at two distinct sites in response to phorbol esters and cAMP. Phosphorylation of Pit-1 modifies its conformation on DNA recognition elements and results in increased binding at certain sites and decreased binding at other sites, dependent on DNA sequences adjacent to the core Pit-1 binding motif. One residue (Thr220), located in the POU homeodomain within a sequence conserved throughout the POU-domain family, confers these responses.

Calcium/calmodulin-dependent protein kinase mediates a pathway for transcriptional regulation.

Calcium influx in response to extracellular signals can modulate gene transcription. A constitutive, calcium/calmodulin-independent mutant of type II calcium/calmodulin-dependent protein kinase was capable of increasing the transcription rate of specific genes independently of protein kinase C activation. This increase was mediated by transferable cis-active elements capable of binding the transcription factor CAAT/enhancer binding protein. Therefore, the activation of type II calcium/calmodulin-dependent protein kinase in response to stimuli that increase intracellular calcium is proposed to represent a distinct second messenger pathway in calcium-mediated regulation of gene transcription.

The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions.

Pit-1 is a member of a family of transcription factors sharing two regions of homology: a highly conserved POU-specific (POUS) domain and a more divergent homeodomain (POUHD). Analysis of mutant Pit-1 proteins suggests that, while the POUHD is required and sufficient for low affinity DNA binding, the POUS domain is necessary for high affinity binding and accurate recognition of natural Pit-1 response elements. Pit-1 is monomeric in solution but associates as a dimer on its DNA response element, exhibiting DNA-dependent protein-protein interactions requiring the POUS domain. Analysis of alpha-helical domains and conserved structures in Pit-1 suggests that POU domain proteins interact with their DNA recognition sites differently than classic homeodomain proteins, with both the POUHD and the POUS domain contacting DNA. Transcriptional activity of Pit-1 on enhancer elements is conferred primarily by a Ser- and Thr-rich N-terminal region unrelated to other known transcription-activating motifs.

A family of POU-domain and Pit-1 tissue-specific transcription factors in pituitary and neuroendocrine development.

The anterior pituitary gland provides a model for investigating the molecular basis for the appearance of phenotypically distinct cell types, within an organ, a central question in development. The rat prolactin and growth hormone genes are selectively expressed in distinct cell types (lactotrophs and somatotrophs) of the anterior pituitary gland, which reflect differential mechanisms of gene activation or restriction because of interactions of multiple factors binding to these genes. We find that the pituitary-specific 33,000 dalton transcription factor, Pit-1, normally expressed in somatotrophs, lactotrophs, and thyrotrophs, can bind to and activate both growth hormone and prolactin promoters in vitro at levels even tenfold lower than those normally present in pituitary cells. In the case of the prolactin gene, high levels of expression in transgenic animals required two cis-active regions; a distal enhancer (-1.8 to -1.5 kb) and a proximal region (-422 to +33 bp). Each of these regions alone can direct low levels of fusion gene expression to prolactin-producing cell types in transgenic mice, but a synergistic interaction between these regions is necessary for high levels of expression. The initial appearance of the prolactin transgene expression closely follows the appearance of high levels of Pit-1, but later increases in expression coincident with appearance of mature lactotrophs suggest the operation of additional, critical positive factor(s). Unexpectedly, transgenes containing the distal enhancer removed from its normal context are expressed in both the prolactin-producing lactotrophs and the TSH-producing thyrotrophs, thereby suggesting that sequences flanking this enhancer are necessary to restrict expression to the correct cell type within the pituitary. These data indicate that distinct processes of gene activation and restriction are necessary for the fidelity of cell-type specific expression within an organ. Consistent with this model, we find that lactotroph cell lines that cannot express the growth hormone gene contain high levels of functional Pit-1. We suggest a large, highly related POU-domain gene family, potentially exceeding 100 members, has been conserved and expanded in evolution to meet the increasing requirements for more intricate patterns of cell phenotypes. The POU-domain subgroup of the homeodomain gene family, in concert with other homeodomain proteins and with other classes of transcription factors, is likely to contribute to the establishment of the mammalian neuroendocrine system.

Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation.

Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase converts it from a Ca2(+)-dependent to a Ca2(+)-independent or autonomous kinase, a process that may underlie some long-term enhancement of transient Ca2+ signals. We demonstrate that the neuronal alpha subunit clone expressed in COS-7 cells (alpha-CaM kinase) is sufficient to encode the regulatory phenomena characteristic of the multisubunit kinase isolated from brain. Activity of alpha-CaM kinase is highly dependent on Ca2+/calmodulin. It is converted by autophosphorylation to an enzyme capable of Ca2(+)-independent (autonomous) substrate phosphorylation and autophosphorylation. Using site-directed mutagenesis, we separately eliminate five putative autophosphorylation sites within the regulatory domain and directly examine their individual roles. Ca2+/calmodulin-dependent kinase activity is fully retained by each mutant, but Thr286 is unique among the sites in being indispensable for generation of an autonomous kinase.

Molecular cloning of a brain-specific calcium/calmodulin-dependent protein kinase.

A calcium/calmodulin-dependent protein kinase type II (CaM-K) alpha-subunit cDNA has been cloned from rat brain. This enzyme is encoded by a 5.1-kilobase mRNA expressed exclusively in the brain. Hybridization histochemistry reveals that the CaM-K mRNA expression corresponds to the distribution of the immunoreactive alpha-subunit protein, suggesting that the high enzyme levels in specific brain areas reflect regional differences in gene expression. The sequence of CaM-K alpha-subunit cDNA indicates a 478-amino acid (54-kDa) protein with three functional domains. The domain organization suggests a structural model for calcium/calmodulin-dependent and independent states that might subserve short- and long-term responses to transient stimuli.

A rat brain isozyme of angiotensin-converting enzyme. Unique specificity for amidated peptide substrates.

We have purified angiotensin-converting enzyme (ACE, EC 3.4.15.1) from rat brain corpus striatum and rat lung. The brain enzyme has Mr 165,000 by sodium dodecyl sulfate gel electrophoresis, whereas the lung enzyme is 175,000. This difference is not an artifact of preparation since mixture of the two tissues prior to purification results in isolation of two proteins with Mr 165,000 and 175,000. Separation of tryptic fragments of 125I-labeled lung and brain ACE by reverse-phase chromatography yields distinct but similar patterns. No differences between the native enzymes are detected in dansyl-tripeptide cleavage specificity, inhibitor profile, immunological properties, sucrose gradient sedimentation, or gel filtration of ACE from the two tissues. However, lung and brain ACE can be differentiated in their ability to cleave amidated peptides. Both lung and brain ACE cleave Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substance P) via two pathways. In one pathway, ACE first releases Gly-Leu-Met-NH2 and then dipeptides sequentially from the carboxyl terminus. The other first produces Leu-Met-NH2, and then releases dipeptides to leave substance P 1-5. Lung ACE favors initial tripeptide release 3:1, while the striatal enzyme acts via the two pathways to a similar extent. Lung and striatal ACE also differ in their ability to degrade other amidated peptides. His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 (substance K) and bombesin are degraded by striatal but not lung ACE. Physalaemin and luteinizing hormone-releasing hormone are cleaved by both enzymes, while eledoisin, kassinin, thyrotropin-releasing hormone, and substance P 5-11 are not cleaved by either enzyme. Physalaemin is degraded more rapidly by the lung enzyme. The coincidence of an ACE isozyme with substance P and substance K in the descending striatonigral pathway and the unique ability of this isozyme to cleave substance P and substance K suggest that one or both of these peptides is a physiological substrate for striatonigral ACE.

A fluorometric assay for angiotensin-converting enzyme activity.

A simple and sensitive assay for angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity has been developed which employs fluorescently labeled tripeptides. ACE hydrolyzes dansylphenylalanyl-arginyl-tryptophan or dansyl-phenylalanyl-arginyl-phenylalanine, liberating dansyl-phenylalanine and a dipeptide. Dansyl-phenylalanine partitions quantitatively into chloroform, whereas the substrates are virtually insoluble in chloroform. This allows rapid measurement of ACE activity with high signal-to-noise ratios even when microliter aliquots of human serum are assayed. Inhibition studies of the dansyl-tripeptide cleaving activity of human serum and rat lung, the identity of the products of enzyme action, and the regional distribution of enzyme activity among rat tissues demonstrate that only ACE cleaves these substrates under the conditions employed here. This assay may be useful for the clinical measurement of human serum ACE activity and for research investigations of ACE from a variety of tissues.

[3H]Captopril binding to membrane associated angiotensin converting enzyme.

[3H]Captopril binding to membrane fractions of rat tissues is saturable and reversible with a KD of 2.4 nM. [3H]Captopril binding and angiotensin converting enzyme measured with hippuryl-L-histidine-L-leucine are distributed in parallel between different tissues and brain regions, with highest levels in the choroid plexus, lung and corpus striatum. Captopril, N-(1(S)-carboxy-3-phenyl-propyl)-L-alanyl-L-proline, N-(1(S)-carboxy-3-phenyl-propyl)-L-lysyl-L-proline, teprotide, thiorphan and S-acetylcaptopril each have similar potencies for inhibition of [3H]captopril binding and of angiotensin converting enzyme. These data strongly indicate that [3H]captopril binds selectively to angiotensin converting enzyme. [3H]Captopril binding evaluation should help clarify the localization and function of angiotensin converting enzyme and assist in defining pharmacologic actions of captopril.