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Protein kinase A (PKA) signaling exemplifies phosphorylation-based signaling as we understand it today. Its catalytic-subunit structure and dynamics continue to advance our understanding of kinase mechanics as the first protein kinase catalytic domain to be identified, sequenced, cloned, and structurally detailed. The PKA holoenzyme elaborates on the role of its regulatory subunits and maintains our understanding of cAMP-dependent cellular signaling. The activation of PKA holoenzymes by cAMP is an example of specialized protein allostery, emphasizing the relevance of protein binding interfaces, unstructured regions, isoform diversity, and dynamics-based allostery. This review provides the most up-to-date overview of PKA structure and function, including a description of the catalytic and regulatory subunits' structures. In addition, the structure, activation, and allostery of holoenzymes are covered. [ABSTRACT FROM AUTHOR]

It is difficult to imagine where the signaling community would be today without the Protein Data Bank. This visionary resource, established in the 1970s, has been an essential partner for sharing information between academics and industry for over 3 decades. We describe here the history of our journey with the protein kinases using cAMP-dependent protein kinase as a prototype. We summarize what we have learned since the first structure, published in 1991, why our journey is still ongoing, and why it has been essential to share our structural information. For regulation of kinase activity, we focus on the cAMP-binding protein kinase regulatory subunits. By exploring full-length macromolecular complexes, we discovered not only allostery but also an essential motif originally attributed to crystal packing. Massive genomic data on disease mutations allows us to now revisit crystal packing as a treasure chest of possible protein:protein interfaces where the biological significance and disease relevance can be validated. It provides a new window into exploring dynamic intrinsically disordered regions that previously were deleted, ignored, or attributed to crystal packing. Merging of crystallography with cryo-electron microscopy, cryo-electron tomography, NMR, and millisecond molecular dynamics simulations is opening a new world for the signaling community where those structure coordinates, deposited in the Protein Data Bank, are just a starting point!

Background: The Saccharomyces cerevisiae Snf1 complex is a member of the AMP-activated protein kinase family and plays an important role in response to environmental stress. The α catalytic subunit Snf1 regulates the activity of the protein kinase, while the β regulatory subunits Sip1/Sip2/Gal83 specify substrate preferences and stress response capacities of Snf1. In this study, we aim to investigate the effects of SNF1 overexpression on the cell tolerance and glucose consumption of S. cerevisiae in high glucose, ethanol, and heat stresses and to explore the valid Snf1 form in the light of β subunits in these stresses. Results: The results suggest that overexpression of SNF1 is effective to improve cell resistance and glucose consumption of S. cerevisiae in high glucose, ethanol, and heat stresses, which might be related to the changed accumulation of fatty acids and amino acids and altered expression levels of genes involved in glucose transport and glycolysis. However, different form of β regulatory subunits dominated in stresses with regard to cell tolerance and glucose utilization. The Sip1 isoform was more necessary to the growth and glucose consumption in ethanol stress. The glucose uptake largely depended on the Sip2 isoform in high sugar and ethanol stresses. The Gal83 isoform only contributed inferior effect on the growth in ethanol stress. Therefore, redundancy and synergistic effect of β subunits might occur in high glucose, ethanol, and heat stresses, but each subunit showed specificity under various stresses. Conclusions: This study enriches the understanding of the function of Snf1 protein kinase and provides an insight to breed multi-stress tolerant yeast strains. [ABSTRACT FROM AUTHOR]

Assembly of multi enzyme complexes at subcellular localizations by anchoring- and scaffolding proteins represents a pivotal mechanism for achieving spatiotemporal regulation of cellular signaling after hormone receptor targeting [for review, see (1)]. In the 3' 5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) signaling pathway it is generally accepted that specificity is secured at several levels. This includes at the first level stimulation of receptors coupled to heterotrimeric G proteins which through stimulation of adenylyl cyclase (AC) forms the second messenger cAMP. Cyclic AMP has several receptors including PKA. PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. The R subunit is the receptor for cAMP and compartmentalizes cAMP signals through binding to cell and tissue-specifically expressed A kinase anchoring proteins (AKAPs). The current dogma tells that in the presence of cAMP, PKA dissociates into an R subunit dimer and two C subunits which are free to phosphorylate relevant substrates in the cytosol and nucleus. The release of the C subunit has raised the question how specificity of the cAMP and PKA signaling pathway is maintained when the C subunit no longer is attached to the R subunit-AKAP complex. An increasing body of evidence points toward a regulatory role of the cAMP and PKA signaling pathway by targeting the C subunits to various C subunit binding proteins in the cytosol and nucleus. Moreover, recent identification of isoform specific amino acid sequences, motifs and three dimensional structures have together provided new insight into how PKA at the level of the C subunit may act in a highly isoform-specific fashion. Here we discuss recent understanding of specificity of the cAMP and PKA signaling pathway based on C subunit subcellular targeting as well as evolution of the C subunit structure that may contribute to the dynamic regulation of C subunit activity. [ABSTRACT FROM AUTHOR]

Fluorescence spectroscopy was used to differentiate between different states of acrylodan-labeled cAMP-dependent protein kinase catalytic subunits in urea, guanidine hydrochloride and 3-( N-morpholino)propanesulfonic acid solutions, by measuring changes in the emission spectrum of the protein-coupled dye, which is very sensitive to its microenvironment. Decomposition of the observed fluorescence spectra by a parameterized log-normal distribution function allowed the resolution of overlapping spectral bands and revealed the formation of three distinct protein states, denominated as native, denatured and unfolded structures. At low denaturant concentrations the formation of the denatured form from the native protein was observed, and this process was characterized by a blue-shift of the fluorescence spectrum of acrylodan, indicating that the dye was transferred into some water-deficit hydrophobic environment inside the protein molecule. Therefore, formation of a 'dry molten globule' structure could be suggested in state. At high denaturant concentrations a red-shift of the emission spectrum of the protein-coupled probe was observed indicating significant extrusion of the dye molecule into water environment as a result of the unfolding of the protein structure. [ABSTRACT FROM AUTHOR]

A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSC5) exists and is under intense investigation. Mice heterozygous for a null allele of prkar1a (Prkar1a+/-), the primary receptor for cyclic adenosine monophosphate (cAMP) and regulator of protein kinase A (PKA) activity, developed bone lesions that were derived from cAMP-responsive osteogenic cells and resembled fibrous dysplasia (FD). Prkar1a+/- mice were crossed with mice that were heterozygous for catalytic subunit Cα (Prkaca+/-), the main PKA activity-mediating molecule, to generate a mouse model with double heterozygosity for prkar1a and prkaca (Prkar1a+/-Prkaca+/-). Unexpectedly, Prkar1a+/-Prkaca+/- mice developed a greater number of osseous lesions starting at 3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions originated from an area proximal to the growth plate, expressed osteogenic cell markers, and showed higher PKA activity that was mostly type II (PKA-ll) mediated by an alternate pattern of catalytic subunit expression. Gene expression profiling confirmed a preosteo-blastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial transition and increased Wnt signaling. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by alternate PKA and catalytic sub-unit activity; abnormal proliferation of these cells leads to skeletal lesions that have similarities to human FD and bone tumors. [ABSTRACT FROM AUTHOR]

Abstract: There are two major genes encoding the catalytic subunits of protein kinase A, Cα and Cβ. The functional significance of these isoforms is enigmatic. Lymphoid cells of the immune system express both Cα and Cβ. In this study we tested the role of Cα and Cβ in regulating immune cell reactivity to antigens using mice carrying a targeted disruption of the Cα and Cβ gene respectively. Cα and Cβ ablation both resulted in a 50% reduction in PKA-specific kinase activity and the level of PKA type I but not PKA type II. Moreover, despite that C subunit ablation did not affect immune cell development and homeostasis, Cα but not Cβ ablation augmented expression of the activation marker CD69 on lymphocytes. CD69 induction coincided with immune cell hyperresponsiveness and was associated with reduced sensitivity to cAMP-mediated inhibition of anti-CD3 induced T cell proliferation. Our results imply that Cα is required for normal immune cell reactivity and demonstrates isoform-specific effects and non-redundant functions of C subunit isoforms expressed in the same cell. [Copyright &y& Elsevier]

Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here, we report the crystal structures of rat CK2α mutant (rCK2α-ΔC, 1-335) and CK2β (rCK2β). The overall topology of rCK2α-AC and rCK2β are very similar to the human enzyme, although large structural differences could be observed in the N-terminal domain of rCK2α-AC. Our reported structure of rCK2α-AC is in the close conformation state while the counterpart hCK2α is in the open conformation state, indicating the conformation of CK2α molecule has high plasticity. The structure of rCK2β represents the conformation of free CK2β. Upon CK2β binding, the C-terminal region undergoes a drastic conformational change. The major region of interaction within the interface of CK2α/CK2β may serve as a bridge to transmit the conformational change and thus regulate the activity of CK2α. [ABSTRACT FROM AUTHOR]

The Drosophila Swiss Cheese (SWS) protein and its vertebrate ortholog Neuropathy Target Esterase (NTE) are required for neuronal survival and glial integrity. In humans, NTE is the target of organophosphorous compounds which cause a paralyzing axonal degeneration and recently mutations in NTE have been shown to cause a Hereditary Spastic Paraplegia called NTE-related Motor-Neuron Disorder. SWS and NTE are concentrated in the endoplasmic reticulum and both have been shown to have an esterase function against an artificial substrate. However, the functional mechanisms and the pathways in which SWS/NTE are involved in are still widely unknown. Here, we show that SWS interacts specifically with the C3 catalytic subunit of cAMP activated protein kinase (PKA-C3), which together with orthologs in mouse (Pkare) and human (PrKX) forms a novel class of catalytic subunits of unknown function. This interaction requires a domain of SWS which shows homology to regulatory subunits of PKA and, like conventional regulatory subunits, the binding of SWS to the PKA-C3 inhibits its function. Consistent with this result, expression of additional PKA-C3 induces degeneration and enhances the neurodegenerative phenotype in sws mutants. We also show that the complex formation with the membrane-bound SWS tethers PKA-C3 to membranes. We therefore propose a model in which SWS acts as a noncanonical subunit for PKA-C3, whereby the complex formation regulates the localization and kinase activity of PKA-C3, and that disruption of this regulation can induce neurodegeneration. [ABSTRACT FROM AUTHOR]

Several isoforms of the cAMP-dependent protein kinase catalytic subunit (C-subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue-specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C-subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C-subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Cα-isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C-subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C-subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract. [ABSTRACT FROM AUTHOR]

Abstract: Neuroendocrine (NE) cells may play a role in prostate cancer progression. Both androgen deprivation and cAMP are well known inducers of NE differentiation (NED) in the prostate. Gene-expression profiling of LNCaP cells, incubated in androgen stripped medium, showed that the Cβ isoform of PKA is up-regulated during NE differentiation. Furthermore, by using semi-quantitative RT-PCR and immunoblotting analysis, we observed that the Cβ splice variants are differentially regulated during this process. Whereas the Cβ2 splice variant is down-regulated in growth arrested LNCaP cells, the Cβ1, Cβ3 and Cβ4 variants, as well as the RIIβ subunit of PKA, are induced in NE-like LNCaP cells. The opposite effect of Cβ expression could be mimicked by androgen stimulation, implying the Cβ gene of PKA as a putative new target gene for the androgen receptor in prostate cancer. Moreover, to investigate expression of PKA subunits during prostate cancer progression, we did immunoblotting of several prostatic cell lines and normal and tumor tissue from prostate cancer patients. Interestingly, multiple Cβ subunits were also observed in human prostate specimens, and the Cβ2 variant was up-regulated in tumor cells. In conclusion, it seems that the Cβ isoforms play different roles in proliferation and differentiation and could therefore be potential markers for prostate cancer progression. [Copyright &y& Elsevier]

Abstract: cAMP-dependent protein kinase (PKA) plays a crucial role in the release of the catch state of molluskan muscles, but the nature of the enzyme in such tissues is unknown. In this paper, we report the purification of the catalytic (C) subunit of PKA from the posterior adductor muscle (PAM) of the sea mussel Mytilus galloprovincialis. It is a monomeric protein with an apparent molecular mass of 40.0±2.0kDa and Stoke’s radius 25.1±0.3Å. The protein kinase activity of the purified enzyme was inhibited by both isoforms of the PKA regulatory (R) subunit that we had previously characterized in the mollusk, and also by the inhibitor peptide PKI5–24. On the other hand, the main proteins of the contractile apparatus of PAM were partially purified and their ability to be phosphorylated in vitro by purified PKA C subunit was analyzed. The results showed that twitchin, a high molecular mass protein associated with thick filaments, was the better substrate for endogenous PKA. It was rapidly phosphorylated with a stoichiometry of 3.47±0.24mol Pmol−1 protein. Also, catchin, paramyosin, and actin were phosphorylated, although more slowly and to a lesser extent. On the contrary, myosin heavy chain (MHC) and tropomyosin were not phosphorylated under the conditions used. [Copyright &y& Elsevier]

Melanoma is one of the fastest rising malignancies in the United States. When detected early, primary melanomas are curable through surgery. However, despite significant improvements in diagnosis and surgical, local and systemic therapy, mortality rate in metastatic melanoma remains high. Furthermore, genetic alterations associated with the development and stepwise progression of melanoma, are still unclear. Previous reports show that the catalytic kinase subunit of the cAMP-dependent protein kinase is secreted by tumor cells and can be detected in the serum of cancer patients. We examine in this report the clinical significance of this secreted C subunit kinase termed extracellular protein kinase (ECPKA) in melanoma patients. Our results showed the presence of ECPKA activity in the serum of melanoma patients and correlate with the appearance and size of the tumor. Most importantly, surgical removal of melanoma causes a precipitous decrease in ECPKA activity in the sera of patients, suggesting that ECPKA may be a novel predictive marker in melanoma. [Copyright &y& Elsevier]

Increased cAMP levels inhibit sclerotial development and raise oxalate production in Sclerotinia sclerotiorum. These effects were hypothesized to be mediated by protein kinase A (PKA). To test this hypothesis, a pka1 catalytic subunit gene was cloned and disrupted. Southern hybridization confirmed disruption, and northern analysis revealed loss of a wild-type pka1 transcript. Mutant strains were cAMP-responsive, pathogenic, and had wild-type levels of PKA activity. Phylogenetic analyses of pka1 with other fungal pka genes and encoded peptides suggest that filamentous fungi possess two pka paralogs. Multiple lines of evidence presented in this work suggest that a second pka catalytic subunit gene, pka2, exists and contributes the majority of PKA activity in S. sclerotiorum. [Copyright &y& Elsevier]

The cyclic AMP-dependent phosphorylation of proteins is essential for the initiation of sperm motility in salmonid fishes. This study isolated cDNA for the catalytic subunit of a cAMP-dependent protein kinase (PKA-C) from rainbow trout testis. The deduced amino acid sequence shows 75–80% identity to sequences previously reported in other organisms. However, the N-terminal regions of PKA-C from the testis as well as ovary in the trout appear slightly shorter than those from other tissues, suggesting that small PKA-C might be specific to germ cells. An immunofluorescence study using polyclonal antibody against trout testis PKA-C shows that it localizes along sperm flagellum. Furthermore, immunoelectron microscopy revealed that PKA-C is anchored to the outer arm dynein of flagellar axonemes. These results suggest that PKA-C is involved in regulating the flagellar motility of sperm via phosphorylation of a subunit of the outer arm dynein. [Copyright &y& Elsevier]

The protein kinase CK2 holoenzyme is composed of two regulatory β subunits and two catalytic α or α' subunits. Although experimental evidence for involvement of the enzyme in the regulation of cell proliferation is accumulating, the exact mechanism of its action is still unclear. The subcellular localization of the enzyme may be a key to its function. We have recently shown that the CK2 holoenzyme is tightly associated with the Golgi complex and the endoplasmic reticulum. Centrosomes, which organize spindle formation during the cell cycle and microtubule cytoskeleton formation and, thereby, the location and orientation of different organelles in the cell, are in close vicinity to the Golgi complex. Because several kinases and phosphatases have been described to regulate the functions of the centrosome, we analysed the association of CK2 with these organelles. Using biochemical cell fractionation and coimmunoprecipitation, we never found the holoenzyme but only the catalytic asubunits associated with the centrosome. These data were confirmed by immunoelectron microscopy. Thus, the present data point to a particular role of the catalytic α and α' subunit of protein kinase CK2, which may be different from their roles in the holoenzyme. [ABSTRACT FROM AUTHOR]

Cyclic adenosine monophosphate-dependent protein kinase (PKA) is involved in various biological functions in neurons. To investigate the subcellular localization of PKA, we stained cultured hippocampal neurons with anti-PKA catalytic subunit antisera. PKA catalytic subunit colocalized with microtubules (MTs) in dendrites as well as with the actin filaments (F-actin) in growth cones. After treatment with cytochalasin B, the colocalization of PKA catalytic subunits with MTs was enhanced, whereas the colocalization with F-actin was suppressed. This result indicates that PKA is anchored to the actin and MT cytoskeletons, and disruption of F-actin releases PKA to the cytoplasm, which then leads to an increase in the amount of PKA in MT domains in the neuron. [Copyright &y& Elsevier]

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation. [ABSTRACT FROM AUTHOR]

Protein kinase A (PKA) are tetramers of two catalytic and two regulatory subunits, docked at precise intracellular sites to provide localized phosphorylating activity, triggered by cAMP binding to regulatory subunits and subsequent dissociation of catalytic subunits. It is unclear whether in the brain PKA dissociated subunits may also be found. PKA catalytic subunit was examined in various mouse brain areas using immunofluorescence, equilibrium binding and western blot, to reveal its location in comparison to regulatory subunits type RI and RII. In the cerebral cortex, catalytic subunits colocalized with clusters of RI, yet not all RI clusters were bound to catalytic subunits. In stria terminalis, catalytic subunits were in proximity to RI but separated from them. Catalytic subunits clusters were also present in the corpus striatum, where RII clusters were detected, whereas RI clusters were absent. Upon cAMP addition, the distribution of regulatory subunits did not change, while catalytic subunits were completely released from regulatory subunits. Unpredictably, catalytic subunits were not solubilized; instead, they re-targeted to other binding sites within the tissue, suggesting local macromolecular reorganization. Hence, the interactions between catalytic and regulatory subunits of protein kinase A consistently vary in different brain areas, supporting the idea of multiple interaction patterns. [ABSTRACT FROM AUTHOR]