Protein Kinase A Phosphorylation Research Articles

Clarifying a Novel PKA Phosphorylation Site on the L-type Calcium Channel

Persistent sympathetic activation leads to the development of hypertrophy and heart failure. Activation of the L-type calcium channel is essential for the increase in contractility and heart rate however the mechanism for the increase in channel function has remained controversial for over 40 years. The downstream effector of β-adrenergic signalling pathway is the cyclic AMP-dependent protein kinase A (PKA). We recently identified a novel candidate PKA phosphorylation site on Cav1.2 protein (the main pore forming and ion conducting alpha subunit). We reconstituted the purified channel isoforms in liposomes. Using single channel patch-clamp analysis, mutation of serine 1458 to alanine attenuated the effect of PKA on channel function (Po = 0.052 ± 0.01 vs 0.043 ± 0.007, n = 5; p = NS). In vitro thermal stability assay performed on the S1458A mutant demonstrated similar melting peak profiles. Following 1 hr in vitro PKA treatment the melting temperature of the WT isoform was altered, but not the mutant isoform, demonstrating that protein-protein interactions between PKA and the Cav1.2 had occurred. Mass spectrometry analysis was performed to demonstrate that serine 1458 could be phosphorylated by PKA. We performed in vitro PKA phosphorylation of the non-phosphorylated synthetic peptide YLTRDWSILGPHHL, corresponding to aa 1452-1465 of the short NT isoform then performed MS/MS analysis. Peptide ions for YLTRDWSILGPHHL were observed with a mass of 1706.86 in control and 1786.84 in PKA treated samples, and we could track the mass difference to the serine residue in the MS/MS spectra. In conclusion, we identified a novel, PKA specific phosphorylation site on the proximal C terminus of Cav1.2 protein.

The impact of tau hyperphosphorylation at Ser262 on memory and learning after global brain ischaemia in a rat model of reversible cardiac arrest

An increase in phosphorylated tau (p-tau) is associated with Alzheimer's disease (AD), and brain hypoxia. Investigation of the association of residue-specific tau hyperphosphorylation and changes in cognition, leads to greater understanding of its potential role in the pathology of memory impairment. The aims of this study are to investigate the involvement of the main metabolic kinases, Liver Kinase B1 (LKB1) and Adenosine Monophosphate Kinase Protein Kinase (AMPK), in tau phosphorylation-derived memory impairment, and to study the potential contribution of the other tau kinases and phosphatases including Glycogen Synthase Kinase (GSK-3β), Protein kinase A (PKA) and Protein Phosphatase 2A (PP2A). Spatial memory and learning were tested in a rat global brain ischemic model of reversible cardiac arrest (CA). The phosphorylation levels of LKB1, AMPK, GSK-3β, PP2A, PKA and tau-specific phosphorylation were assessed in rats, subjected to ischaemia/reperfusion and in clinically diagnosed AD and normal human brains. LKB1 and AMPK phosphorylation increased 4 weeks after CA as did AMPK related p-tau (Ser262). The animals showed unchanged levels of GSK-3β specific p-tau (Ser202/Thr205), phospho-PP2A (Tyr307), total GSK-3β, PP2A, phospho-cAMP response element-binding protein (CREB) which is an indicator of PKA activity, and no memory deficits. AD brains had hyperphosphorylated tau in all the residues of Ser262, Ser202 and Thr205, with increased phosphorylation of both AMPK (Thr172) and GSK-3β (Ser9), and reduced PP2A levels. Our data suggests a crucial role for a combined activation of tau kinases and phosphatases in adversely affecting memory and that hyperphosphorylation of tau in more than one specific site may be required to create memory deficits.

A Role for cAMP and Protein Kinase A in Experimental Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating intestinal disease that has been associated with Cronobacter sakazakii and typically affects premature infants. Although NEC has been actively investigated, little is known about the mechanisms underlying the pathophysiology of epithelial injury and intestinal barrier damage. Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) are important mediators and regulators of apoptosis. To test the hypothesis that C. sakazakii increases cAMP and PKA activation in experimental NEC resulting in increased epithelial apoptosis, we investigated the effects of C. sakazakii on cAMP and PKA invitro and invivo. Specifically, rat intestinal epithelial cells and a human intestinal epithelial cell line were infected with C. sakazakii, and cAMP levels and phosphorylation of PKA were measured. An increase in cAMP was demonstrated after infection, as well as an increase in phosphorylated PKA. Similarly, increased intestinal cAMP and PKA phosphorylation were demonstrated in a rat pup model of NEC. These increases were correlated with increased intestinal epithelial apoptosis. The additional of a PKA inhibitor (KT5720) significantly ameliorated these effects and decreased the severity of experimental NEC. Findings were compared with results from human tissue samples. Collectively, these observations indicate that cAMP and PKA phosphorylation are associated with increased apoptosis in NEC and that inhibition of PKA activation protects against apoptosis and experimental NEC.

PKA-Mediated Phosphorylation of APOBEC3B Suppresses Its DNA Mutagenic Potential in Myeloma Cells

Multiple myeloma (MM) is an incurable plasma cell malignancy and the reason for this is not yet completely understood. It might be partially attributable to heterogeneity and clonal evolution through DNA mutation accumulation. Recent next generation sequencing (NGS) studies have revealed that apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) 3B (A3B) is one of the cause of clonal evolution in MM. APOBEC protein family consists of 11 proteins and most of them, including A3B, have cytidine deaminase (CDA) activity on single-strand DNA, which catalyzes deamination of cytosine to uridine, resulting in C-to-T or C-to-G mutations. We have previously shown in human cell line models that A3B induces genomic mutations (Shinohara et al., Sci Rep, 2012). Therefore, controlling A3B CDA activity is an attractive therapeutic strategy to overcome the unfavorable prognosis of patients with these malignancies.Enzymatic activity of other APBEC family proteins, activation-induced cytidine deaminase (AID) and APOBEC3G (A3G) is regulated by protein kinase A (PKA)-mediated phosphorylation. A3B also has two consensus PKA phosphorylation motifs conserved upstream of each CDA domain. Thus, we hypothesized that PKA phosphorylates A3B which may alter A3B CDA activity.Using NetPhosK1.0 and ScanSite programs, two A3B residues (serine at the 46th and threonine at the 214th position) are predicted to be PKA phosphorylation sites. Firstly, we examined the binding between A3B and PKA. We transfected HEK293T cells with expression vectors for C-terminal HA-tagged A3B and N-terminal FLAG-tagged PKA catalytic subunit a (PKACA) and used CoIP assays with anti-HA or anti-FLAG antibodies to demonstrate physical binding between A3B and PKACA. Secondly, to determine whether the putative residues are phosphorylated, we overexpressed PKACA and A3B alanine mutants in HEK293T cells and performed immunoblotting analysis with anti-phospho PKA motif antibodies and found that threonine-214 is phosphorylated by PKA. To confirm that PKA phosphorylates A3B directly, we employed in vitro phosphorylation assays using purified C-terminal A3B and PKACA and found that PKACA phosphorylated A3B, but not the A3B T214A mutant, thus we concluded that PKACA directly phosphorylates threonine-214 in A3B.Next, to investigate the effect of this phosphorylation on the CDA activity of A3B, we constructed phosphomimetic mutants of these residues, T214D and T214E. We carried out in vitro CDA assays using cell lysates from HEK293T cells overexpressing A3B or mutants (T214A, T214D, T214E), and found that T214D and T214E almost completely lost their CDA activity. We also performed in vitro CDA assays using purified C-terminal A3B and mutant proteins. The purified A3B T214D mutant also lost CDA activity, suggesting that PKA-mediated phosphorylation of A3B might also lead to loss of its CDA activity.To confirm this result in cell lines, we used differential DNA denaturation PCR (3D-PCR) of DNA from HEK293T cells transfected with vectors for uracil DNA glycosylase inhibitor, EGFP and A3B wild type or mutants. 3D-PCR products of the EGFP sequence were amplified at a lower denaturation temperature in cells expressing the wild type A3B compared to those in cells expressing phosphomimetic A3B or catalytically inactivated A3B. These results indicate that phosphomimetic A3B no longer has exogenous DNA editing activity. We further plan to evaluate the PKA phosphorylation of A3B and the consequences on its mutagenic potential in myeloma cell lines.In conclusion, PKA induces phosphorylation at threonine-214 in A3B, and this phosphorylation suppresses its CDA activity. PKA-mediated phosphorylation of A3B may be an attractive therapeutic target for inhibiting A3B-induced clonal evolution in lymphoid malignancies, especially in MM. DisclosuresTakaori-Kondo:Kyowa Kirin: Research Funding; Alexion Pharmaceuticals: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Mochida Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Toyama Chemical: Research Funding; Cognano: Research Funding.

A cannabinoid link between mitochondria and memory.

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB1) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB1 receptors. Genetic exclusion of CB1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB1 receptors signal through intra-mitochondrial Gαi protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants.

Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation.

NLRP3 inflammasome inhibition is disrupted in a group of auto-inflammatory disease CAPS mutations.

Inflammasomes are positioned to rapidly escalate the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. However, negative regulation of inflammasomes remains poorly understood, as is the signaling cascade that dampens inflammasome activity. We found that rapid NLRP3 inflammasome activation was directly inhibited by protein kinase A (PKA), which was induced by prostaglandin E2 (PGE2) signaling via the PGE2 receptor E-prostanoid 4 (EP4). PKA directly phosphorylated the cytoplasmic receptor NLRP3 and attenuated its ATPase function. We found that Ser295 in human NLRP3 was critical for rapid inhibition and PKA phosphorylation. Mutations in NLRP3-encoding residues adjacent to Ser295 have been linked to the inflammatory disease CAPS (cryopyrin-associated periodic syndromes). NLRP3-S295A phenocopied the human CAPS mutants. These data suggest that negative regulation at Ser295 is critical for restraining the NLRP3 inflammasome and identify a molecular basis for CAPS-associated NLRP3 mutations.

Cardiac β2-Adrenergic Receptor Phosphorylation at Ser355/356 Regulates Receptor Internalization and Functional Resensitization.

Previous studies have demonstrated that β2-adrenergic receptors (β2ARs) can be phosphorylated by G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA), affecting β2AR internalization and desensitization. However, the exact physiological function of β2ARs in cardiomyocytes is unknown. In this study, we showed that neonatal mouse cardiomyocytes had different contraction and internalization responses to sustained or repeated, transient agonist stimulation. Specifically, short-time stimulation (10 min) with epinephrine or norepinephrine increased the cardiomyocyte contraction rate, reaching a maximum at 5 min, followed by a slow decline. When the agonist was re-added after a 60-min wash-out period, the increase in the cardiomyocyte contraction rate was similar to the initial response. In contrast, when cardiomyocytes were exposed continuously to epinephrine or norepinephrine for 60 min, the second agonist stimulation did not increase the contraction response. These results indicated that continuous β2AR stimulation caused functional desensitization. Phosphorylation of β2ARs at serine (Ser)355/356 GRK phosphorylation sites, but not at Ser345/346 PKA phosphorylation sites increased with continuous epinephrine stimulation for 60 min. Accordingly, β2AR internalization increased. Interestingly, β2AR internalization was blocked by mutations at the GRK phosphorylation sites, but not by mutations at the PKA phosphorylation sites. Furthermore, inhibition of β2AR dephosphorylation by okadaic acid, a phosphatase 2A inhibitor, impaired the recovery of internalized β2ARs and reduced the cardiomyocyte contraction rate in response to epinephrine. Finally, epinephrine treatment induced the physical interaction of β-arrestin with internalized β2ARs in cardiomyocytes. Together, these data revealed the essential role of the Ser355/356 phosphorylation status of β2ARs in regulating receptor internalization and physiological resensitization in neonatal cardiomyocytes to contraction functions.

Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR mRNA molecules via dual hybridization at the 3′- and 5′-ends and reveals an unexpectedly high frequency of 1:1 pairing with mitochondria marked by the matrix StAR protein. This pairing may be central to translation-coupled cholesterol transfer. Altogether, our results show that adrenal cells exhibit high-efficiency StAR activity that needs to integrate rapid cholesterol transfer with homeostasis and pulsatile hormonal stimulation. StAR NBD, the extended 3.5-kb mRNA, SIK1, and Tis11b play important roles.

Protein kinase A modulation of CaV1.4 calcium channels.

The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family.

Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

The Steroid Hormone 20-Hydroxyecdysone Enhances Gene Transcription through the cAMP Response Element-binding Protein (CREB) Signaling Pathway

Animal steroid hormones regulate gene transcription through genomic pathways by binding to nuclear receptors. These steroid hormones also rapidly increase intracellular calcium and cyclic adenosine monophosphate (cAMP) levels and activate the protein kinase C (PKC) and protein kinase A (PKA) nongenomic pathways. However, the function and mechanism of the nongenomic pathways of the steroid hormones are unclear, and the relationship between the PKC and PKA pathways is also unclear. We propose that the steroid hormone 20-hydroxyecdysone (20E) activates the PKA pathway to enhance 20E-induced gene transcription in the lepidopteran insect Helicoverpa armigera The expression of the catalytic subunit 1 of PKA (PKAC1) increased during metamorphosis, and PKAC1 knockdown blocked pupation and repressed 20E-responsive gene expression. 20E regulated PKAC1 phosphorylation at threonine 200 and nuclear translocation through an ecdysone-responsive G-protein-coupled receptor 2. PKAC1 induced cAMP response element-binding protein (CREB) phosphorylation at serine 143, which bound to the cAMP response element on DNA to enhance 20E-responsive gene transcription. Through ecdysone-responsive G-protein-coupled receptor 2, 20E increased cAMP levels, which induced CREB PKA phosphorylation and 20E-responsive gene expression. This study demonstrates that the PKA/CREB pathway tightly and critically regulates 20E-induced gene transcription as well as its relationship with the 20E-induced PKC pathway.

GW627368X inhibits proliferation and induces apoptosis in cervical cancer by interfering with EP4/EGFR interactive signaling.

PGE2, the major product of cyclooxygenases implicated in carcinogenesis, is significantly upregulated in cervical cancer. PGE2 via prostanoid receptor EP4 stimulates proliferation and motility while inhibiting apoptosis and immune surveillance. It promotes angiogenesis by stimulating the production of pro-angiogenic factors. The present study demonstrates GW627368X, a highly selective competitive EP4 antagonist, which hinders cervical cancer progression by inhibiting EP4/epithelial growth factor receptor (EGFR) interactive signaling. GW627368X reduced protein kinase A (PKA) phosphorylation which in turn leads to decreased cAMP response element-binding protein (CREB) activation. Decreased PKA phosphorylation also directly enhanced Bax activity and in part reduced glycogen synthase kinase 3 (GSK3)β phosphorylation. Owing to the interactive signaling between EP4 and EGFR, GW627368X lowered EGFR phosphorylation in turn reducing Akt, mitogen-activated protein kinase (MAPK) and GSK3β activity significantly. Sublethal dose of GW627368X was found to reduce the nuclear translocation of β-catenin in a time dependent manner along with time-dependent decrease in cytoplasmic as well as whole-cell β-catenin. Decreased CREB and β-catenin transcriptional activity restricts the aberrant transcription of key genes like EP4, cyclooxygenase (COX)-2, vascular endothelial growth factor and c-myc, which ultimately control cell survival, proliferation and angiogenesis. Reduced activity of EGFR resulted in enhanced expression of 15-hydroxyprostaglandin dehydrogenase increasing PGE2 degradation thereby blocking a positive feedback loop. In xenograft model, dose-dependent decrease in cancer proliferation was observed characterized by reduction in tumor mass and volume and a marked decrease in Ki67 expression. A diminished CD31 specific staining signified decreased tumor angiogenesis. Reduced expression of pAkt, pMAPK, pEGFR and COX-2 validated in vitro results. GW627368X therefore effectively inhibits tumor survival, motility, proliferation and angiogenesis by blocking EP4/EGFR interactive signaling. EP4 is a potent therapeutic target in cervical cancer and can be explored in combination with conventional therapies to attain superior outcomes and to overcome complications associated with organ toxicities, therapeutic resistance and disease relapse.

Protein kinase A inhibitor, H89, enhances survival and clonogenicity of dissociated human embryonic stem cells through Rho-associated coiled-coil containing protein kinase (ROCK) inhibition.

Can cell survival of dissociated human embryonic stem cells (hESCs) be increased during culture? A protein kinase A (PKA) inhibitor, H89, can significantly enhance survival and clonogenicity of dissociated hESCs without affecting their pluripotency. hESCs are vulnerable to massive cell death upon cellular detachment and dissociation. hESCs were dissociated into single cells and then cultured in feeder-dependent and -independent manners. H89 was added to the culture medium at different concentrations for 1 day. The statistical results were obtained from at least three independent experiments (n ≥ 4). The group without treatment was used as the negative control. 4 µM H89 was added in the culture medium to promote cell survival and colony formation of dissociated hESCs. MTT method and propidium iodide (PI) staining were used to determine cell proliferation, cell death and cell cycle, respectively. To count colony formation, alkaline phosphatase (AP) staining was carried out. Western blot was performed to determine protein expression. Except AP staining, immunofluorescence, RT-PCR and karyotype analysis were used to confirm the pluripotent state of H89 treated hESCs. H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK), myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1), significantly increases cell survival and colony formation, and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. Appropriate H89 concentration should be used and 1 day of H89 treatment is sufficient for promoting survival and colony formation of dissociated hESCs. These results provide an alternative for human pluripotent stem cell (hPSC) culture, broaden the scope of participants in the cell death of single hES cells after dissociation and further enlighten clues to understand the mechanism of dissociation-induced cell death. This research was supported by the National Natural Science Foundation of China (21176238, 21576266), and Chinese Academy of Sciences. There is no conflict of interest to declare. Nil.

Structural Measurements within the M-Domain Reveal Unique Details of Cardiac Myosin Binding Protein-C Phosphorylation

We have used molecular dynamics (MD) simulations in atomic-level detail to resolve the structural changes associated with cardiac myosin binding protein-C (cMyBP-C) phosphorylation by protein kinase A (PKA), focusing on the N-terminal phosphorylation motif (pm/M-domain). The results are supported by spectroscopic measurements, and have implications for cMyBP-C's role in myocardial contraction, with particular relevance to -adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. PKA phosphorylation in the pm mediates the affinity of cMyBP-C interactions with actin and the myosin-neck that modulate cardiac muscle contractility. Our spectroscopic studies using time-resolved FRET (TR-FRET) had previously detected distance distributions between probe pairs engineered at sites of cMyBP-C domains C0 through C2. Phosphorylation reduces the level of molecular disorder and the distribution of N-terminal inter-domain distances become more compact (e.g., C1-to-C2). Here, we gain further insight from 20 independent microsecond-long MD simulations of the intrinsically disordered pm. We modeled the motif structure (residues His255-Lys356) and simulated the unphosphorylated and phosphorylated (Ser- 273, 282, and 302) characteristics of molecular disorder, secondary and tertiary structure, intra-motif distance measurements, surface accessibility, and predictions of binding probability. A large structural change in the disordered region of pm was revealed, whereby phosphorylation unmasks a surface of positively-charged residues on an α-helix within the ordered region of pm. Distance measurements between residues in the disordered N-terminal portion of the motif to the helix residues of the motif C-terminus changed proximity to one another by as much as 2 nm upon phosphorylation. These findings help to elucidate cMyBP-C structural dynamics, providing new molecular insight into the modulatory role of cMyBP-C in force development. This work was supported by NIH grants to DDT (R01 AR032961) and to BC (R00 HL122397) and to LME-F from the American Heart Association (12SDG12060656).

The N-Terminal Domains of MyBPC3 Restrict Actin Dynamics and Increase Resilience in a Phosphorylation-Dependent Manner

We have determined the effects of myosin binding protein-C (MyBP-C) N-terminal domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C modulates muscle contractility and is capable of interacting with thin filaments. Protein kinase A (PKA) phosphorylates cardiac and slow skeletal MyBP-C, altering myofilament structure and function. We previously used TPA to show that full-length MyBP-C restricts actin torsional dynamics, and effects by cardiac and slow skeletal MyBP-C (cMyBP-C; ssMyBP-C) are relieved by PKA phosphorylation. To determine the effects of MyBP-C N-terminal domains on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin flexibility at saturation of the TPA effect. PKA phosphorylation of ssMyBP-C domains C1-C2 and cMyBP-C domains C0-C2 relieved the restriction of torsional amplitude but also increased the rate of torsional motion, thus increasing actin resilience (rate/amplitude; less brittle). Moreover, effects of cardiac C0-C2 on actin resilience were also PKA-dependent, whereas slow skeletal C1-C2 effects which promote actin resilience persisted independently of phosphorylation. In the case of fast skeletal C1-C2, actin resilience was unaffected and its effect to restrict actin dynamics was unchanged by phosphorylation. The N-terminal domains of cMyBP-C (C0-C2) and skeletal MyBP-C's (C1-C2) had effects on actin dynamics similar to those determined for the full-length MyBP-C isoforms. Effects of phosphomimetic mutations were also investigated. These unique isoform-dependent MyBP-C-induced changes in actin dynamics may play a role in the functional effects of MyBP-C on contraction. This work was supported by NIH grants to DDT (R01 AR032961), to SS (R01 AR067279), and to BC (R00 HL122397).

A Single Protein Kinase A or Calmodulin Kinase II Site Does Not Control the Cardiac Pacemaker Ca2+ Clock.

Fight or flight heart rate (HR) increases depend on protein kinase A (PKA)- and calmodulin kinase II (CaMKII)-mediated enhancement of Ca(2+) uptake and release from sarcoplasmic reticulum (SR) in sinoatrial nodal cells (SANC). However, the impact of specific PKA and CaMKII phosphorylation sites on HR is unknown. We systematically evaluated validated PKA and CaMKII target sites on phospholamban and the ryanodine receptor using genetically modified mice. We found that knockin alanine replacement of ryanodine receptor PKA (S2808) or CaMKII (S2814) target sites failed to affect HR responses to isoproterenol or spontaneous activity in vivo or in SANC. Similarly, selective mutation of phospholamban amino acids critical for enhancing SR Ca(2+) uptake by PKA (S16) or CaMKII (T17) to alanines did not affect HR in vivo or in SANC. In contrast, CaMKII inhibition by expression of AC3-I has been shown to slow SANC rate responses to isoproterenol and decrease SR Ca(2+) content. Phospholamban deficiency rescued SR Ca(2+) content and SANC rate responses to isoproterenol in mice with AC3-I expression, suggesting that CaMKII affects HR by modulation of SR Ca(2+) content. Consistent with this, mice expressing a superinhibitory phospholamban mutant had low SR Ca(2+) content and slow HR in vivo and in SANC. SR Ca(2+) depletion reduces HR and SR Ca(2+) repletion restores physiological SANC rate responses, despite CaMKII inhibition. PKA and CaMKII do not affect HR by a unique target site governing SR Ca(2+) uptake or release. HR acceleration may require an SR Ca(2+) content threshold.

Langevin Dynamics Simulation of AKAP-PKA Complex: Re-Envisioning the Local Concentration Mechanism for Directing PKA Phosphorylation

The second messenger cAMP and its effector cAMP-dependent protein kinase A (PKA) constitute a ubiquitous cell signaling system. Many receptor types existing within the same cell may use this signaling system and compartmentation of signaling is thought to occur due to A-Kinase Anchoring Proteins (AKAPs), which act to co-localize PKA with specific substrates. The molecular mechanism allowing AKAPs to direct PKA phosphorylation to a particular substrate remains unknown. A large body of evidence suggests that the catalytic subunit, which is highly diffusible, is released in response to cAMP. Recent studies have shown however that inside living cells, the catalytic subunit may not be released from the AKAP complex, and alterations in the structure of the PKA regulatory subunit tether affect substrate phosphorylation. We used a novel computational software based on Langevin dynamics to simulate the AKAP-PKA complex to investigate the possible molecular mechanisms for tether length and flexibility influence on phosphorylation and whether or not AKAPs can direct PKA phosphorylation to a particular substrate if the catalytic subunit is released from the complex. We find that short and flexible tethers contribute to a decrease in the average characteristic time of binding, which yields faster characteristic times of phosphorylation. We further demonstrate that release of the catalytic subunit from the AKAP complex abrogates the effect of tethering, with characteristic times of phosphorylation similar to non-AKAP bound PKA. The data suggests that AKAPs likely do not release the catalytic subunit and target PKA activity by increasing the efficiency of phosphorylation of AKAP-bound substrates.

Selective phosphorylation of PKA targets after β-adrenergic receptor stimulation impairs myofilament function in Mybpc3-targeted HCM mouse model.

Hypertrophic cardiomyopathy (HCM) has been associated with reduced β-adrenergic receptor (β-AR) signalling, leading downstream to a low protein kinase A (PKA)-mediated phosphorylation. It remained undefined whether all PKA targets will be affected similarly by diminished β-AR signalling in HCM. We aimed to investigate the role of β-AR signalling on regulating myofilament and calcium handling in an HCM mouse model harbouring a gene mutation (G > A transition on the last nucleotide of exon 6) in Mybpc3 encoding cardiac myosin-binding protein C. Cardiomyocyte contractile properties and phosphorylation state were measured in left ventricular permeabilized and intact cardiomyocytes isolated from heterozygous (HET) or homozygous (KI) Mybpc3-targeted knock-in mice. Significantly higher myofilament Ca²⁺sensitivity and passive tension were detected in KI mice, which were normalized after PKA treatment. Loaded intact cardiomyocyte force-sarcomere length relation was impaired in both HET and KI mice, suggesting a reduced length-dependent activation. Unloaded cardiomyocyte function revealed an impaired myofilament contractile response to isoprenaline (ISO) in KI, whereas the calcium-handling response to ISO was maintained. This disparity was explained by an attenuated increase in cardiac troponin I (cTnI) phosphorylation in KI, whereas the increase in phospholamban (PLN) phosphorylation was maintained to wild-type values. These data provide evidence that in the KI HCM mouse model, β-AR stimulation leads to preferential PKA phosphorylation of PLN over cTnI, resulting in an impaired inotropic and lusitropic response.