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Worldwide, tuberculosis is the second leading infectious killer and multidrug resistance severely hampers disease control. Mycolic acids are a unique category of lipids that are essential for viability, virulence, and persistence of the causative agent, Mycobacterium tuberculosis (Mtb). Therefore, enzymes involved in mycolic acid biosynthesis represent an important class of drug targets. We previously showed that the (3R)‐hydroxyacyl‐ACP dehydratase (HAD) protein HadD is dedicated mainly to the production of ketomycolic acids and plays a determinant role in Mtb biofilm formation and virulence. Here, we discovered that HAD activity requires the formation of a tight heterotetramer between HadD and HadB, a HAD unit encoded by a distinct chromosomal region. Using biochemical, structural, and cell‐based analyses, we showed that HadB is the catalytic subunit, whereas HadD is involved in substrate binding. Based on HadBDMtb crystal structure and substrate‐bound models, we identified determinants of the ultra‐long‐chain lipid substrate specificity and revealed details of structure–function relationship. HadBDMtb unique function is partly due to a wider opening and a higher flexibility of the substrate‐binding crevice in HadD, as well as the drastically truncated central α‐helix of HadD hotdog fold, a feature described for the first time in a HAD enzyme. Taken together, our study shows that HadBDMtb, and not HadD alone, is the biologically relevant functional unit. These results have important implications for designing innovative antivirulence molecules to fight tuberculosis, as they suggest that the target to consider is not an isolated subunit, but the whole HadBD complex. [ABSTRACT FROM AUTHOR]

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!

Abstract 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.

Worldwide, tuberculosis is the second leading infectious killer and multidrug resistance severely hampers disease control. Mycolic acids are a unique category of lipids that are essential for viability, virulence, and persistence of the causative agent, Mycobacterium tuberculosis (Mtb). Therefore, enzymes involved in mycolic acid biosynthesis represent an important class of drug targets. We previously showed that the (3R)-hydroxyacyl-ACP dehydratase (HAD) protein HadD is dedicated mainly to the production of ketomycolic acids and plays a determinant role in Mtb biofilm formation and virulence. Here, we discovered that HAD activity requires the formation of a tight heterotetramer between HadD and HadB, a HAD unit encoded by a distinct chromosomal region. Using biochemical, structural, and cell-based analyses, we showed that HadB is the catalytic subunit, whereas HadD is involved in substrate binding. Based on HadBD Mtb crystal structure and substrate-bound models, we identified determinants of the ultra-long-chain lipid substrate specificity and revealed details of structure-function relationship. HadBD Mtb unique function is partly due to a wider opening and a higher flexibility of the substrate-binding crevice in HadD, as well as the drastically truncated central α-helix of HadD hotdog fold, a feature described for the first time in a HAD enzyme. Taken together, our study shows that HadBD Mtb , and not HadD alone, is the biologically relevant functional unit. These results have important implications for designing innovative antivirulence molecules to fight tuberculosis, as they suggest that the target to consider is not an isolated subunit, but the whole HadBD complex., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

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]

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases known to regulate important cellular functions by phosphorylating the inositol ring of inositol-phospholipids (PtdIns) at 3' position. The PI3Kα is a heterodimer and the activation of the catalytic subunit (p110α) is regulated by its regulatory subunit (p85α). The current work deals with studying the activation mechanism of the PI3Kα using multi micro-second molecular dynamic simulations. Structural changes involved in activation mechanism is studied by gradually releasing the inhibitory effects of different domains of regulatory subunit namely, n-terminal SH2 (nSH2) and inter SH2 (iSH2). The observation shows that even in the presence of n-terminal and inter SH2 domain (niSH2) of regulatory subunit, the catalytic domain has some intrinsic activation activity and the presence of c-terminal SH2 (cSH2) domain may be required for complete inhibition. The release of nSH2 domain leads to loss of interactions between iSH2 domain (regulatory subunit) and C2 and kinase domain (catalytic subunit). The study shows that early events in the activation mechanism involve the movement of the ABD domain of the catalytic subunit along with the linker region between ABD and RBD region which may lead to movement of ABD closer to the CLobe of the kinase domain. This movement is essentially as it triggers the rearrangement of CLobe especially the catalytic loop and activation loop which bring catalytic important residues closer to ATP and PIP2(phosphatidylinositol-4,5-bisphosphate). Water mediated interaction analysis reveal that water may be playing an important role in the transfer of phosphate from ATP to PIP2. The study shows that initial signal for release of inhibitory effect of the regulatory subunit might be propagated through the linker region between ABD and RBD through allosteric effect to different regions of the protein. These understanding of early events during the activation mechanism may help in the design of better therapeutic targeting PI3K. [Display omitted] • The catalytic domain of PI3Kα has intrinsic activation activity and the presence of n-terminal SH2, inter SH2 and c-terminal SH2 (cSH2) domains may be required for its complete inhibition. • Early activation events involve the movement of the ABD domain, linker region between ABD and RBD domain. • Movement of ABD closer to the CLobe of the kinase domain, leads to rearrangement in kinase domain essential for substrate phosphorylation. • Linker region between ABD and RBD domain may act as hinge for the movement of ABD domain. • Water may be playing an important role in the transfer of phosphate from ATP to PIP2. • The LYS776, HIS917, SER919, HIS936 and LYS944 may play important role in transfer of phosphate from ATP to PIP2. [ABSTRACT FROM AUTHOR]

Abstract Protein kinase A (PKA), also known as cAMP dependent protein kinase, is an essential component of many signaling pathways, many of which regulate key developmental processes. Inactive PKA is a tetrameric holoenzyme, comprised of two catalytic (PRKAC), and two regulatory subunits. Upon cAMP binding, the catalytic subunits are released and thereby activated. There are multiple isoforms of PKA catalytic subunits, but their individual roles are not well understood. In order to begin studying their roles in zebrafish development, it is first necessary to identify the spatial and temporal expression profiles for each prkac subunit. Here we evaluate the expression profiles for the four zebrafish prkacs : prkacαa , αb, βa, and βb , at key developmental time points: 24, 48 and 72 h post fertilization. We show that zebrafish prkacs are expressed throughout the developing nervous system, each showing unique expression patterns. This body of work will inform future functional studies into the roles of PKA during development. Highlights • prkac subunits are expressed throughout the developing zebrafish nervous system. • Zebrafish have 4 protein kinase A catalytic subunit genes; prkcαa, αb, βa, and βb. • Each prkac subunit shows unique expression patterns in zebrafish development. [ABSTRACT FROM AUTHOR]

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.

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.

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.

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]

The catalytic subunits of protein kinase CK2 are classified into two subtypes: CK2α1 and CK2α2. CK2α1 is an attractive drug‐discovery target for various diseases such as cancers and nephritis. CK2α2 is defined as an off‐target of CK2α1 and is a potential target in the development of male contraceptive drugs. High‐resolution crystal structures of both isozymes are likely to provide crucial clues for the design of selective inhibitors of CK2α1 and/or CK2α2. To date, several crystal structures of CK2α1 have been solved at high resolutions of beyond 1.5 Å. However, crystal structures of CK2α2 have barely achieved a low resolution of around 3 Å because of the formation of needle‐shaped crystals. In this study, new crystal forms were exploited and one provided a crystal structure of CK2α2 at 1.89 Å resolution. This result, together with the structure of CK2α1, will assist in the development of highly selective inhibitors for both isozymes. [ABSTRACT FROM AUTHOR]

The cAMP-Protein Kinase A signaling, anchored on CpkA, is necessary for appressorium development and host penetration, but indispensable for infectious growth in Magnaporthe oryzae. In this study, we identified and characterized the gene encoding the second catalytic subunit, CPK2, whose expression was found to be lower compared to CPKA at various stages of pathogenic growth in M. oryzae. Deletion of CPK2 caused no alterations in vegetative growth, conidiation, appressorium formation, or pathogenicity. Surprisingly, the cpkAΔcpk2Δ double deletion strain displayed significant reduction in growth rate and conidiation compared to the single deletion mutants. Interestingly, loss of CPKA and CPK2 resulted in morphogenetic defects in germ tubes (with curled/wavy and serpentine growth pattern) on hydrophobic surfaces, and a complete failure to produce appressoria therein, thus suggesting an important role for CPK2-mediated cAMP-PKA in surface sensing and response pathway. CPKA promoter-driven expression of CPK2 partially suppressed the defects in host penetration and pathogenicity in the cpkAΔ. Such ectopic CPK2 expressing strain successfully penetrated the rice leaves, but was unable to produce proper secondary invasive hyphae, thus underscoring the importance of CpkA in growth and differentiation in planta. The Cpk2-GFP localized to the nuclei and cytoplasmic vesicles in conidia and germ tubes. The Cpk2-GFP colocalized with CpkA-mCherry on vesicles in the cytosol, but such overlap was not evident in the nuclei. Our studies indicate that CpkA and Cpk2 share overlapping functions, but also play distinct roles during pathogenesis-associated signaling and morphogenesis in the rice blast fungus.

Reactive astrogliosis and microgliosis after spinal cord injury (SCI) contribute to glial scar formation that impedes axonal regeneration. The mechanisms underlying reactive astrocyte and microglia proliferation upon injury remain partially understood. Protein phosphatase 1, catalytic subunit, gamma isozyme (PPP1CC) participates in cell proliferation, differentiation and apoptosis. However, the expression and functions of PPP1CC following SCI are still unknown. In this study, an acute spinal cord contusion injury model in adult rats was established to investigate the potential role of PPP1CC during the pathological process of SCI. The palpable expression increase of PPP1CC after SCI was found by western blot and immunohistochemistry staining. Double immunofluorescence staining showed that PPP1CC expression mainly increased in astrocytes and microglia, as well as PPP1CC and proliferating cell nuclear antigen (PCNA) co-localized in astrocytes and microglia. Furthermore, PCNA expression also elevated after SCI in a similar manner as PPP1CC. In vitro, PPP1CC and PCNA expression in primary rat spinal cord astrocytes and microglia changed in a similar concentration- and time-dependent manner according to LPS treatment. In addition, PPP1CC knockdown in astrocytes and microglia resulted in the decrease of PCNA expression and the number of Brdu positive cells after LPS stimulation, showing that PPP1CC promoted astrocyte and microglia proliferation after inflammation. In a word, PPP1CC might be associated with astrocyte and microglia proliferation after SCI, implying that PPP1CC is a potential molecular target for the therapy of SCI. [ABSTRACT FROM AUTHOR]

The proteasome is a nuclear-cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions, but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit-selective inhibitors and dual-color fluorescent activity-based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant-microbe interactions. Our data reveal that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1-1 [Pto DC3000(ΔhQ)] whilst the activity profile of the β1 subunit changes. Infection with wild-type Pto DC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community, and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species. [ABSTRACT FROM AUTHOR]

The stress proteasome in the animal kingdom facilitates faster conversion of oxidized proteins during stress conditions by incorporating different catalytic β subunits. Plants deal with similar kind of stresses and also carry multiple paralogous genes encoding for each of the three catalytic β subunits. Here, we investigated the existence of stress proteasomes upon abiotic stress (salt stress) in tomato roots. In contrast to Arabidopsis thaliana, tomato has a simplified proteasome gene set with single genes encoding each β subunit except for two genes encoding β2. Using proteasome activity profiling on tomato roots during salt stress, we discovered a transient modification of the catalytic subunits of the proteasome coinciding with a loss of cell viability. This stress-induced active proteasome disappears at later time points and coincides with the need to degrade oxidized proteins during salt stress. Subunit-selective proteasome probes and MS analysis of fluorescent 2D gels demonstrated that the detected stress-induced proteasome is not caused by an altered composition of subunits in active proteasomes, but involves an increased molecular weight of both labeled β2 and β5 subunits, and an additional acidic pI shift for labeled β5, whilst labeled b1 remains mostly unchanged. Treatment with phosphatase or glycosidases did not affect the migration pattern. This stress-induced proteasome may play an important role in PCD during abiotic stress. [ABSTRACT FROM AUTHOR]