Subunit Of cAMP-dependent Protein Kinase Research Articles

The cAMP signaling module regulates sperm motility in the liverwort Marchantia polymorpha

Adenosine 3',5'-cyclic monophosphate (cAMP) is a universal signaling molecule that acts as a second messenger in various organisms. It is well established that cAMP plays essential roles across the tree of life, although the function of cAMP in land plants has long been debated. We previously identified the enzyme with both adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) activity as the cAMP-synthesis/hydrolysis enzyme COMBINED AC with PDE (CAPE) in the liverwort Marchantia polymorpha. CAPE is conserved in streptophytes that reproduce with motile sperm; however, the precise function of CAPE is not yet known. In this study, we demonstrate that the loss of function of CAPE in M. polymorpha led to male infertility due to impaired sperm flagellar motility. We also found that two genes encoding the regulatory subunits of cAMP-dependent protein kinase (PKA-R) were also involved in sperm motility. Based on these findings, it is evident that CAPE and PKA-Rs act as a cAMP signaling module that regulates sperm motility in M. polymorpha. Therefore, our results have shed light on the function of cAMP signaling and sperm motility regulators in land plants. This study suggests that cAMP signaling plays a common role in plant and animal sperm motility.

Stimulus-Induced Activation of the Glycoprotein Hormone α-Subunit Promoter in Human Placental Choriocarcinoma Cells: Major Role of a tandem cAMP Response Element.

The glycoprotein hormones LH, FSH, TSH and chorionic gonadotropin consist of a common α-subunit and a hormone-specific β-subunit. The α-subunit is expressed in the pituitary and the placental cells, and its expression is regulated by extracellular signal molecules. Much is known about the regulation of the α-subunit gene in the pituitary, but few studies have addressed the regulation of this gene in trophoblasts. The aim of this study was to characterize the molecular mechanism of stimulus-induced α-subunit gene transcription in JEG-3 cells, a cellular model for human trophoblasts, using chromatin-embedded reporter genes under the control of the α-subunit promoter. The results show that increasing the concentration of the second messengers cAMP or Ca2+, or expressing the catalytic subunit of cAMP-dependent protein kinase in the nucleus activated the α-subunit promoter. Similarly, the stimulation of p38 protein kinase activated the α-subunit promoter, linking α-subunit expression to stress response. The stimulation of a Gαq-coupled designer receptor activated the α-subunit promoter, involving the transcription factor CREB, linking α-subunit expression to hormonal stimulation and an increase in intracellular Ca2+. Deletion mutagenesis underscores the importance of a tandem cAMP response element within the glycoprotein hormone α-subunit promoter, which acts as a point of convergence for a multiple signaling pathway.

Functional Study of cAMP-Dependent Protein Kinase A in Penicillium oxalicum.

Signaling pathways play a crucial role in regulating cellulase production. The pathway mediated by signaling proteins plays a crucial role in understanding how cellulase expression is regulated. In this study, using affinity purification of ClrB, we have identified sixteen proteins that potentially interact with ClrB. One of the proteins, the catalytic subunit of cAMP-dependent protein kinase A (PoPKA-C), is an important component of the cAMP/PKA signaling pathway. Knocking out PoPKA-C resulted in significant decreases in the growth, glucose utilization, and cellulose hydrolysis ability of the mutant strain. Furthermore, the cellulase activity and gene transcription levels were significantly reduced in the ΔPoPKA-C mutant, while the expression activity of CreA, a transcriptional regulator of carbon metabolism repression, was notably increased. Additionally, deletion of PoPKA-C also led to earlier timing of conidia production. The expression levels of key transcription factor genes stuA and brlA, which are involved in the production of the conidia, showed significant enhancement in the ΔPoPKA-C mutant. These findings highlight the involvement of PoPKA-C in mycelial development, conidiation, and the regulation of cellulase expression. The functional analysis of PoPKA-C provides insights into the mechanism of the cAMP/PKA signaling pathway in cellulase expression in filamentous fungi and has significant implications for the development of high-yielding cellulase strains.

Abstract 5658: MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies

Abstract The generation of bispecific molecules, in which two target-specific moieties are engineered into a single entity, offers an attractive approach to optimize biological activity which may improve clinical outcomes. Among various formats of bispecific molecules, the heterodimeric Fc currently is a favorable scaffold for the design of bispecific antibodies because of its structural similarity to the natural antibody. However, development of molecules based on this concept is challenged by the presence of potential homodimer contamination, reduced yield, and stability loss relative to the natural Fc. Here, we report the development of the MG-Ig platform by grafting a single chain fragment variable (scFv) onto the hinge region of a full-size immunoglobulin with a different specificity to produce a trivalent IgG-like bispecific antibody that retains natural homodimer Fc. Whereas the MG-Ig platform is evolved from the Dock-and-Lock (DNL) technology which exploits the interaction between the anchoring domain (AD) of A kinase anchor proteins and the dimerization and docking domain (DDD) of regulatory subunit of cAMP-dependent protein kinase, the main distinctions include 1) the AD-fused scFv monomer is judiciously designed to graft onto the DDD dimer inserted into the hinge region of IgG, forming a bispecific antibody with three (1+2) binding arms and 2) all modular subunits are expressed in a single host cell and self-assemble to generate the bioactive conjugate in situ. To validate the method, bispecific T-cell engagers, designated 3scFv × hL0125C (CD3 × Trop2) and 3scFv × hACD20C (CD3 × CD20), which redirect T cells to Trop2 and CD20, respectively, have been produced with high yield and purity. Flow cytometry analysis demonstrated the bispecific binding of 3scFv × hL0125C to both CD3 on Jurkat and Trop2 on ME-180 with unaltered affinities. These T-cell redirecting bispecific antibodies mediated dose-dependent killing of target cancer cells when co-incubated with human PBMCs. Based on the 60-h viability assays using MTS, the IC50 of 3scFv × hL0125C was about 6 pM for MDA-MB-468, 13 pM for MDA-MB-231, 20 pM for BT-474, and 18 pM for HCT116. In 3D culture of HCT116 cells, the formation of multicellular tumor spheroids was impeded by 3scFv × hL0125C at 19.5 pM or higher, which is consistent with the cytotoxicity assays for tumor cells grown in monolayer. In addition, 3scFv × hL0125C was stable in human plasma for 7 days at 37°C, retaining bispecific integrity and binding activity. These data are promising and merit further development of MG-Ig bispecific antibodies for preclinical and clinical studies. Citation Format: Donglin Liu, Li Zeng, Berenice E. Liu, Chien-Hsing Chang. MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5658.

RIIβ-PKA in GABAergic Neurons of Dorsal Median Hypothalamus Governs White Adipose Browning.

The RIIβ subunit of cAMP-dependent protein kinase A (PKA) is expressed in the brain and adipose tissue. RIIβ-knockout mice show leanness and increased UCP1 in brown adipose tissue. The authors have previously reported that RIIβ reexpression in hypothalamic GABAergic neurons rescues the leanness. However, whether white adipose tissue (WAT) browning contributes to the leanness and whether RIIβ-PKA in these neurons governs WAT browning are unknown. Here, this work reports that RIIβ-KO mice exhibit a robust WAT browning. RIIβ reexpression in dorsal median hypothalamic GABAergic neurons (DMH GABAergic neurons) abrogates WAT browning. Single-cell sequencing, transcriptome sequencing, and electrophysiological studies show increased GABAergic activity in DMH GABAergic neurons of RIIβ-KO mice. Activation of DMH GABAergic neurons or inhibition of PKA in these neurons elicits WAT browning and thus lowers body weight. These findings reveal that RIIβ-PKA in DMH GABAergic neurons regulates WAT browning. Targeting RIIβ-PKA in DMH GABAergic neurons may offer a clinically useful way to promote WAT browning for treating obesity and other metabolic disorders.

The Forgotten PKAs: New Insights Revealed by the First Crystal Structure of a PKA C Beta Splice Variant, Cβ4ab

A major regulatory mechanism of enzyme function in cells is protein phosphorylation that is carried out by the large protein kinase family. While the catalytic (C) subunit of cAMP‐dependent protein kinase A (PKA) has served as a prototype for this family, our enormous knowledge base is skewed towards Cα1, only one of the many tissue specific C subunit isoforms and splice variants expressed in cells. Accordingly, all published crystal structures are of Cα1. The other PKA splice variants, especially the Cβ splice variant gene products of PRKACB, have been obscure. In an effort to re‐balance our PKA perspective we have solved a 2.6 A crystal structure of the PKA splice variant Cβ4ab, which not only crystallized in a new condition but also grew in a thus far for PKA not seen space group P21 with four molecules per asymmetric unit. Compared to Cα1, the isoform with the most deposited structures in the protein data bank, the protein sequence of Cβ4ab differs by 10% or 35 out of 350 amino acids. A majority of these amino acid differences cluster in the N‐terminal and C‐terminal tails. The rest are located mostly in the N‐lobe. Some hot spots of amino acid variation nucleate two sets of C subunit dimers that interact in a novel way to create polymers within the crystal. In our structure, Cβ4ab is in a closed conformation with the inhibitor peptide IP20 bound but surprisingly no electron density is observed for either of the two Mg ions usually present with IP20 and nucleotide. SPR data for the protein kinase inhibitor provides further insights into the differences between Cβ4ab and Cα. Our structure and binding studies suggest that in addition to C beta being expressed in a tissue specific manner, they are also functionally non‐redundant. The newly revealed inter‐molecular interaction sites within the dimers highlight regions of the C subunit that have been less studied so far and are likely to have important regulatory functions.

FgSfl1 and Its Conserved PKA Phosphorylation Sites Are Important for Conidiation, Sexual Reproduction, and Pathogenesis in Fusarium graminearum

The fungal plant pathogen, Fusarium graminearum, contains two genes, FgCPK1 and FgCPK2, encoding the catalytic subunits of cAMP-dependent protein kinase A. FgCPK1 and FgCPK2 are responsible for most of the PKA activities and have overlapping functions in various cellular processes in F. graminearum. The cpk1 cpk2 double mutant was significantly reduced in growth, rarely produced conidia, and was non-pathogenic. In this study, we found that the cpk1 cpk2 double mutant was unstable and produced fast-growing spontaneous sectors that were defective in plant infection. All spontaneous suppressor strains had mutations in FgSFL1, a transcription factor gene orthologous to SFL1 in yeast. Thirteen suppressor strains had non-sense mutations at Q501, three suppressor strains had frameshift mutations at W198, and five suppressor strains had mutations in the HSF binding domain of FgSfl1. Only one suppressor strain had both a non-synonymous mutation at H225 and a non-sense mutation at R490. We generated the SFL1 deletion mutant and found that it produced less than 2% of conidia than that of the wild-type strain PH-1. The sfl1 mutant was significantly reduced in the number of perithecia on carrot agar plates at 7 days post-fertilization (dpf). When incubated for more than 12 days, ascospore cirrhi were observed on the sfl1 mutant perithecia. The infection ability of the sfl1 deletion mutant was also obviously defective. Furthermore, we found that in addition to the S223 and S559 phosphorylation sites, FgSFL1 had another predicted phosphorylation site: T452. Interestingly, the S223 phosphorylation site was responsible for sexual reproduction, and the T452 phosphorylation site was responsible for growth and sexual reproduction. Only the S559 phosphorylation site was found to play an important role in conidiation, sexual reproduction, and infection. Overall, our results indicate that FgSFL1 and its conserved PKA phosphorylation sites are important for vegetative growth, conidiation, sexual reproduction, and pathogenesis in F. graminearum.

PKA Cβ: a forgotten catalytic subunit of cAMP-dependent protein kinase opens new windows for PKA signaling and disease pathologies.

3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits are encoded by the two major genes PRKACA and PRKACB, respectively. The PRKACA gene encodes two known splice variants, the ubiquitously expressed Cα1 and the sperm-specifically expressed Cα2. In contrast, the PRKACB gene encodes several splice variants expressed in a highly cell and tissue-specific manner. The Cβ proteins are called Cβ1, Cβ2, Cβ3, Cβ4 and so-called abc variants of Cβ3 and Cβ4. Whereas Cβ1 is ubiquitously expressed, Cβ2 is enriched in immune cells and the Cβ3, Cβ4 and their abc variants are solely expressed in neuronal cells. All Cα and Cβ splice variants share a kinase-conserved catalytic core and a C-terminal tail encoded by exons 2 through 10 in the PRKACA and PRKACB genes, respectively. All Cα and Cβ splice variants with the exception of Cα1 and Cβ1 are hyper-variable at the N-terminus. Here, we will discuss how the PRKACA and PRKACB genes have developed as paralogs that encode distinct and functionally non-redundant proteins. The fact that Cα and Cβ splice variant mutations are associated with numerous diseases further opens new windows for PKA-induced disease pathologies.

Molecular Determinants of PKA RIα Driven Liquid‐Liquid Phase Separation

We recently discovered a regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS), which is critical for effective cAMP compartmentation. PKA RIα LLPS is enhanced by cAMP, which results in phase separated bodies that dynamically sequester cAMP. Interestingly, although cAMP-dependent dissociation of the canonical PKA complex, which consists of regulatory (R) and catalytic (C) subunits, allows RIα to undergo LLPS, the PKA C subunit is co-recruited to RIα phase separated bodies, resulting in high PKA activity. In this study, we performed systematic mutations to the dimerization/docking (D/D) domain, inhibitory sequence (IS), and cyclic nucleotide binding (CNB) domains to elucidate the molecular drivers of RIα LLPS. Mutations of key residues within the CNB domains and within the D/D domain and N3A motif of the CNB-A domain disrupt PKA RIα LLPS, indicating cAMP binding and RIα dimerization are required for LLPS. Conversely, mutations within the IS pseudo-substrate increase RIα LLPS, indicating a disrupted R:C complex enhances LLPS. The IS mutations also prevented recruitment of the catalytic subunit to the RIα LLPS, providing opportunities to further probe this non-canonical recruitment mechanism. Importantly, the loss of RIα LLPS leads to disruption of cAMP compartmentation, and increased cell proliferation, DNA synthesis, and anchorage independent growth, suggesting that dysregulated cAMP signaling plays a role in tumorigenesis.

Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A

An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera’s allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

Deficiency of the RI\u03b2 subunit of protein kinase A causes body tremor and impaired fear conditioning memory in rats

The RIβ subunit of cAMP-dependent protein kinase (PKA), encoded by Prkar1b, is a neuronal isoform of the type I regulatory subunit of PKA. Mice lacking the RIβ subunit exhibit normal long-term potentiation (LTP) in the Schaffer collateral pathway of the hippocampus and normal behavior in the open-field and fear conditioning tests. Here, we combined genetic, electrophysiological, and behavioral approaches to demonstrate that the RIβ subunit was involved in body tremor, LTP in the Schaffer collateral pathway, and fear conditioning memory in rats. Genetic analysis of WTC-furue, a mutant strain with spontaneous tremors, revealed a deletion in the Prkar1b gene of the WTC-furue genome. Prkar1b-deficient rats created by the CRISPR/Cas9 system exhibited body tremor. Hippocampal slices from mutant rats showed deficient LTP in the Schaffer collateral–CA1 synapse. Mutant rats also exhibited decreased freezing time following contextual and cued fear conditioning, as well as increased exploratory behavior in the open field. These findings indicate the roles of the RIβ subunit in tremor pathogenesis and contextual and cued fear memory, and suggest that the hippocampal and amygdala roles of this subunit differ between mice and rats and that rats are therefore beneficial for exploring RIβ function.

Reversible phosphorylation of a protein from Trypanosoma equiperdum that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases

Homologous proteins of the cAMP-dependent protein kinase (PKA) regulatory and catalytic subunits have been identified in Trypanosoma equiperdum (TeqR-like and TeqC-like, respectively). Partially purified TeqR-like from parasites isolated in the presence of glucose migrated as an apparent 55 kDa/57 kDa polypeptide doublet when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a single polypeptide of 57 kDa was obtained when parasites were deprived of glucose, a condition that has been shown to activate a TeqC-like enzyme. As revealed by immunoblots using anti-phospothreonine antibodies, the 57 kDa band corresponded to a form of TeqR-like that was phosphorylated in threonine residues. TeqR-like phosphorylation was reversible since the level of phospho-TeqR-like decreased once glucose was readded to glucose starved-parasites. Dephospho- and phospho-TeqR-like proteins are monomers with native molecular masses of 54.93–57.41 kDa, Stokes radii of 3.42–3.37 nm, and slightly asymmetric shapes (frictional ratio f/fo = 1.36–1.32). A protein kinase of ∼40 kDa was also partially purified from glucose deprived-trypanosomes, which corresponded to the TeqC-like enzyme by its ability to phosphorylate kemptide, its inhibition by PKA-specific inhibitors, and its immunorecognition by anti-PKA catalytic subunit antibodies. TeqR-like and TeqC-like did not coelute following anion-exchange chromatography, revealing that these proteins are not associated forming a complex in T. equiperdum. Yet, when TeqR-like was incubated in vitro with TeqC-like in the presence of Mg2+ and ATP, the 55 kDa dephospho form of the 55kDa/57 kDa polypeptide doublet of TeqR-like was converted into the 57 kDa phospho form, demonstrating that TeqR-like is a substrate for TeqC-like.

Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

Germline and Mosaic Variants in PRKACA and PRKACB Cause a Multiple Congenital Malformation Syndrome

PRKACA and PRKACB code for two catalytic subunits (Cα and Cβ) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cβ subunits of PKA during human development.

Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

The heterotrimeric G-protein beta subunit Gpb1 controls hyphal growth under low oxygen conditions through the protein kinase A pathway and is essential for virulence in the fungus Mucor circinelloides.

Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G-protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1-encoding gene is up-regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild-type strain. Mutation in a regulatory subunit of cAMP-dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild-type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.

Gene expression studies of Saccharum spontaneum, a wild relative of sugarcane in response to salinity stress.

The study is aimed to assess the morphological, physiological, and molecular responses of seven Saccharum spontaneum clones for salinity stress. These clones (IND-07-1462, IND-07-1465, IND-07-1470, IND-07-1471, IND 16-1761, IND 16-1762, and IND 16-1763) were subjected to salinity stress at two different concentrations of electrical conductivity 6 and 8 ds/m after 60 days of planting. All seven genotypes showed a decrease in relative water content and nitrate reductase activity with an increase in severity of salt stress. The effect was more pronounced in IND-07-1471, while IND-16-1762 exhibited only a minimum drop. Similarly we observed an increase in proline content and lipid peroxidation activity for the genotype IND-07-1471, while IND-16-1762 showed minimum increase. Molecular profiling of genes/transcription factors like salt overly sensitive, responsive to abscissic acid, dirigent, myeloblastosis, ethylene responsive factor associated with salinity stress tolerance showed 19-, 18-, 17-, 10-, and 9-fold increased expression at 8 ds/m of salinity stress, respectively, in IND-16-1762 showed. Based on the evidences obtained from expression profiling, we have cloned the conserved regions of RAB and SOS1 genes. The domain of SOS and RAB was identified as a regulatory subunit of cAMP-dependent protein kinases which is involved in a signaling pathway.

Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A.

In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI's most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme's binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.

Physico-chemical properties of two point mutants of small heat shock protein HspB6 (Hsp20) with abrogated cardioprotection

Physico-chemical properties of HspB6 S10F and P20L mutants with abrogated cardioprotective activity and associated with different forms of cardiomyopathy were analyzed. Under normal conditions both the wild-type HspB6 and its mutants formed small size oligomers (dimers) with apparent molecular weight of 50–60 kDa. Under crowding conditions (0.5 M trimethylamine N-oxide, TMAO) the wild-type HspB6 remained predominantly dimeric or formed small molecular weight complexes, whereas both mutants tended to form high molecular weight complexes. Catalytic subunit of cAMP-dependent protein kinase phosphorylated the wild-type HspB6 and its S10F mutant with comparable rate. The rate of P20L mutant phosphorylation was higher than that of the wild-type HspB6. S10F and P20L mutations did not affect interaction of phosphorylated HspB6 with universal adapter proteins 14-3-3. The wild-type HspB6 was resistant to heat-induced denaturation and aggregation, whereas both its mutants were denatured and started to aggregate at temperature much lower than its wild-type counterpart. Titration with fluorescent probe bis-ANS was accompanied by larger increase of fluorescence in the case of both mutants than in the case of the wild-type HspB6. Both mutants possessed higher chaperone-like activity than the wild-type protein. It is concluded that both S10F and P20L mutations are accompanied by increase of hydrophobicity of the very N-terminal region of HspB6 leading to increased aggregation at elevated temperature, formation of large complexes under crowding conditions and increased chaperone-like activity measured in vitro. Increased hydrophobicity and self-association can affect substrate specificity and interaction with certain target proteins thus leading to decrease or complete abrogation of cardioprotective activity.

Comprehensive Characterization of the Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase by Top-Down Mass Spectrometry.

Reversible phosphorylation plays critical roles in cell growth, division, and signal transduction. Kinases which catalyze the transfer of γ-phosphate groups of nucleotide triphosphates to their substrates are central to the regulation of protein phosphorylation and are therefore important therapeutic targets. Top-down mass spectrometry (MS) presents unique opportunities to study protein kinases owing to its capabilities in comprehensive characterization of proteoforms that arise from alternative splicing, sequence variations, and post-translational modifications. Here, for the first time, we developed a top-down MS method to characterize the catalytic subunit (C-subunit) of an important kinase, cAMP-dependent protein kinase (PKA). The recombinant PKA C-subunit was expressed in Escherichia coli and successfully purified via his-tag affinity purification. By intact mass analysis with high resolution and high accuracy, four different proteoforms of the affinity-purified PKA C-subunit were detected, and the most abundant proteoform was found containing seven phosphorylations with the removal of N-terminal methionine. Subsequently, the seven phosphorylation sites of the most abundant PKA C-subunit proteoform were characterized simultaneously using tandem MS methods. Four sites were unambiguously identified as Ser10, Ser11, Ser18, and Ser30, and the remaining phosphorylation sites were localized to Ser2/Ser3, Ser358/Thr368, and Thr[215-224]Tyr in the PKA C-subunit sequence with a 20mer 6xHis-tag added at the N-terminus. Interestingly, four of these seven phosphorylation sites were located at the 6xHis-tag. Furthermore, we have performed dephosphorylation reaction by Lambda protein phosphatase and showed that all phosphorylations of the recombinant PKA C-subunit phosphoproteoforms were removed by this phosphatase.