Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

The cellular activation of conventional protein kinase C (PKC) isozymes is initiated by the binding of their C2 domains to membranes in response to elevations in intracellular Ca²⁺. Following this C2 domain-mediated membrane recruitment, the C1 domain binds its membrane-embedded ligand diacylglycerol, resulting in activation of PKC. Here we explore the molecular mechanisms by which the C2 domain controls the initial step in the activation of PKC. Using stopped-flow fluorescence spectroscopy to measure association and dissociation rate constants, we show that hydrophobic interactions are the major driving force in the binding of the C2 domain to anionic membranes, whereas electrostatic interactions dominate in membrane retention. Specifically, mutation of select hydrophobic or select basic residues in the Ca²⁺-binding loops reduces membrane affinity by distinct mechanisms; mutation of hydrophobic residues primarily alters association rate constants, whereas mutation of charged residues affects dissociation rate constants. Live cell imaging reveals that introduction of these mutations into full-length PKCα not only reduces the Ca²⁺-dependent translocation to plasma membrane but, by impairing the plasma membrane-sensing role of the C2 domain, causes phorbol ester-triggered redistribution of PKCα to other membranes, such as the Golgi. These data underscore the key role of the C2 domain in driving conventional PKC isozymes to the plasma membrane and reveal that not only the amplitude but also the subcellular location of conventional PKC signaling can be tuned by altering the affinity of this module for membranes.     

Protein kinase C {delta}-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases

Conventional and novel protein kinase C (PKC) isozymes transduce the abundance of signals mediated by phospholipid hydrolysis; however redundancy in regulatory mechanisms confounds dissecting the unique signaling properties of each of the eight isozymes constituting these two subgroups. Previously, we created a genetically encoded reporter (C kinase activity reporter (CKAR)) to visualize the rate, amplitude, and duration of agonist-evoked PKC signaling at specific locations within the cell. Here we designed a reporter, δCKAR, that specifically measures the activation signature of one PKC isozyme, PKC δ, in cells, revealing unique spatial and regulatory properties of this isozyme. Specifically, we show two mechanisms of activation: 1) agonist-stimulated activation at the plasma membrane (the site of most robust PKC δ signaling), Golgi, and mitochondria that is independent of Src and can be triggered by phorbol esters and 2) agonist-stimulated activation in the nucleus that requires Src kinase activation and cannot be triggered by phorbol esters. Translocation studies reveal that the G-protein-coupled receptor agonist UTP induces the translocation of PKC δ into the nucleus by a mechanism that depends on the C2 domain and requires Src kinase activity. However, translocation from the cytosol into the nucleus is not required for the Src-dependent regulation of nuclear activity; a construct of PKC δ prelocalized to the nucleus continues to be activated by UTP by a mechanism dependent on Src kinase activity. These data identify the nucleus as a signaling hub for PKC δ that is driven by receptor-mediated signaling pathways (but not phorbol esters) and differs from signaling at plasma membrane and Golgi in that it is controlled by Src family kinases.     

Protein Kinase C�� Negatively Regulates Store-independent Ca2+ Entry and Phosphatidylserine Exposure Downstream of Glycoprotein VI in Platelets

Platelet activation must be tightly controlled to provide an effective, but not excessive, response to vascular injury. Cytosolic calcium is a critical regulator of platelet function, including granule secretion, integrin activation, and phosphatidylserine (PS) exposure. Here we report that the novel protein kinase C isoform, PKCθ, plays an important role in negatively regulating Ca²⁺ signaling downstream of the major collagen receptor, glycoprotein VI (GPVI). This limits PS exposure and so may prevent excessive platelet procoagulant activity. Stimulation of GPVI resulted in significantly higher and more sustained Ca²⁺ signals in PKCθ^−/− platelets. PKCθ acts at multiple distinct sites. PKCθ limits secretion, reducing autocrine ADP signaling that enhances Ca²⁺ release from intracellular Ca²⁺ stores. PKCθ thereby indirectly regulates activation of store-operated Ca²⁺ entry. However, PKCθ also directly and negatively regulates store-independent Ca²⁺ entry. This pathway, activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, was enhanced in PKCθ^−/− platelets, independently of ADP secretion. Moreover, LOE-908, which blocks 1-oleoyl-2-acetyl-sn-glycerol-induced Ca²⁺ entry but not store-operated Ca²⁺ entry, blocked the enhanced GPVI-dependent Ca²⁺ signaling and PS exposure seen in PKCθ^−/− platelets. We propose that PKCθ normally acts to restrict store-independent Ca²⁺ entry during GPVI signaling, which results in reduced PS exposure, limiting platelet procoagulant activity during thrombus formation.     

Classical protein kinases C are regulated by concerted interaction with lipids: the importance of phosphatidylinositol-4,5-bisphosphate

Classical protein kinase C (PKC) enzymes are known to be important factors in cell physiology both in terms of health and disease. They are activated by triggering signals that induce their translocation to membranes. The consensus view is that several secondary messengers are involved in this activation, such as cytosolic Ca²⁺ and diacylglycerol. Cytosolic Ca²⁺ bridges the C2 domain to anionic phospholipids as phosphatidylserine in the membrane, and diacylglycerol binds to the C1 domain. Both diacylglycerol and the increase in Ca²⁺ concentration are assumed to arise from the extracellular signal that triggers the hydrolysis of phosphatidylinositol-4,5-bisphosphate. However, results obtained during the last decade indicate that this phosphoinositide itself is also responsible for modulating classical PKC activity and its localization in the plasma membrane.     

Raf-1 Kinase Inhibitory Protein (RKIP) Mediates Ethanol-induced Sensitization of Secretagogue Signaling in Pancreatic Acinar Cells

Excessive alcohol consumption is associated with most cases of chronic pancreatitis, a progressive necrotizing inflammatory disease that can result in pancreatic insufficiency due to acinar atrophy and fibrosis and an increased risk of pancreatic cancer. At a cellular level acute alcohol exposure can sensitize pancreatic acinar cells to secretagogue stimulation, resulting in dysregulation of intracellular Ca²⁺ homeostasis and premature digestive enzyme activation; however, the molecular mechanisms by which ethanol exerts these toxic effects have remained undefined. In this study we identify Raf-1 kinase inhibitory protein as an essential mediator of ethanol-induced sensitization of cholecystokinin- and carbachol-regulated Ca²⁺ signaling in pancreatic acinar cells. We show that exposure of rodent acinar cells to ethanol induces protein kinase C-dependent Raf-1 kinase inhibitory protein phosphorylation, sensitization of cholecystokinin-stimulated Ca²⁺ signaling, and potentiation of both basal and cholecystokinin-stimulated extracellular signal-regulated kinase activation. Furthermore, we show that either suppression of Raf-1 kinase inhibitory protein expression using short hairpin RNA or gene ablation prevented the sensitizing effects of ethanol on cholecystokinin- and carbachol-stimulated Ca²⁺ signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of Raf-1 inhibitory protein expression may have future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.     

Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary ⁴⁵Ca fluxes and free-cytosolic Ca²⁺ ([Ca²⁺] _i ) measurements were used to study the effect of Ca²⁺-mobilizing hormones on plasma membrane Ca²⁺ permeability and the plasma membrane Ca²⁺ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca²⁺ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca²⁺. In the present study, we show that activation of this pathway increases the plasma membrane Ca²⁺ permeability by approximately sevenfold. Despite that, the cells reduce [Ca²⁺]i back to near resting levels. To compensate for the increased plasma membrane Ca²⁺ permeability, a plasma membrane Ca²⁺ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca²⁺ pump. Activation of the plasma membrane Ca²⁺ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca²⁺ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca⁺ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca²⁺ pump.     

Compartmentalized crosstalk of CFTR and TMEM16A (ANO1) through EPAC1 and ADCY1

Airway epithelial cells express both Ca²⁺ activated TMEM16A/ANO1 and cAMP activated CFTR anion channels. Previous work suggested a significant crosstalk of intracellular Ca²⁺ and cAMP signaling pathways, leading to activation of both chloride channels. We demonstrate that in airway epithelial cells, stimulation of purinergic or muscarinic G-protein coupled receptors (GPCRs) activates TMEM16A and CFTR. Additional expression of G_q/11 and phospholipase C coupled GPCRs strongly enhanced the crosstalk between Ca²⁺- and cAMP-dependent signaling. Knockdown of endogenous GRCRs attenuated crosstalk and functional coupling between TMEM16A and CFTR. The number of receptors did not affect expression or membrane localization of TMEM16A or CFTR, but controlled assembly of the local signalosome. GPCRs translocate Ca²⁺-sensitive adenylate cyclase type 1 (ADCY1) and exchange protein directly activated by cAMP (EPAC1) to particular plasma membrane domains containing GPCRs, CFTR and TMEM16A, thereby producing compartmentalized Ca²⁺ and cAMP signals and significant crosstalk. While biosynthesis and membrane trafficking of CFTR requires a functional Golgi apparatus, maturation and membrane trafficking of TMEM16A may occur independent of the Golgi. Because Ca²⁺ activated TMEM16A currents are only transient, continuous Cl⁻ secretion by airway epithelial cells requires CFTR. The present data also explain why receptor-dependent activation of TMEM16A is more efficient than direct stimulation by Ca²⁺.     

Trafficking of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor (AMPA) Receptor Subunit GluA2 from the Endoplasmic Reticulum Is Stimulated by a Complex Containing Ca2+/Calmodulin-activated Kinase II (CaMKII) and PICK1 Protein and by Release of Ca2+ from Internal Stores

The GluA2 subunit of the AMPA receptor (AMPAR) dominantly blocks AMPAR Ca²⁺ permeability, and its trafficking to the synapse regulates AMPAR-dependent synapse Ca²⁺ permeability. Here we show that GluA2 trafficking from the endoplasmic reticulum (ER) to the plasma membrane of cultured hippocampal neurons requires Ca²⁺ release from internal stores, the activity of Ca²⁺/calmodulin activated kinase II (CaMKII), and GluA2 interaction with the PDZ protein, PICK1. We show that upon Ca²⁺ release from the ER via the IP3 and ryanodine receptors, CaMKII that is activated enters a complex that contains PICK1, dependent upon the PICK1 BAR (Bin-amphiphysin-Rvs) domain, and that interacts with the GluA2 C-terminal domain and stimulates GluA2 ER exit and surface trafficking. This study reveals a novel mechanism of regulation of trafficking of GluA2-containing receptors to the surface under the control of intracellular Ca²⁺ dynamics and CaMKII activity.     

Divergence in Endothelin-1- and Bradykinin-Activated Store-Operated Calcium Entry in Afferent Sensory Neurons

Endothelin-1 (ET-1) and bradykinin (BK) are endogenous peptides that signal through Gαq/11-protein coupled receptors (GPCRs) to produce nociceptor sensitization and pain. Both peptides activate phospholipase C to stimulate Ca²⁺ accumulation, diacylglycerol production, and protein kinase C activation and are rapidly desensitized via a G-protein receptor kinase 2-dependent mechanism. However, ET-1 produces a greater response and longer lasting nocifensive behavior than BK in multiple models, indicating a potentially divergent signaling mechanism in primary afferent sensory neurons. Using cultured sensory neurons, we demonstrate significant differences in both Ca²⁺ influx and Ca²⁺ release from intracellular stores following ET-1 and BK treatments. As intracellular store depletion may contribute to the regulation of other signaling cascades downstream of GPCRs, we concentrated our investigation on store-operated Ca²⁺ channels. Using pharmacological approaches, we identified transient receptor potential canonical channel 3 (TRPC3) as a dominant contributor to Ca²⁺ influx subsequent to ET-1 treatment. On the other hand, BK treatment stimulated Orai1 activation, with only minor input from TRPC3. Taken together, data presented here suggest that ET-1 signaling targets TRPC3, generating a prolonged Ca²⁺ signal that perpetuates nocifensive responses. In contrast, Orai1 dominates as the downstream target of BK receptor activation and results in transient intracellular Ca²⁺ increases and abridged nocifensive responses.     

Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca²⁺-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg²⁺-dependent, Ca²⁺-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.     

Diacylglycerol kinase in plasma membranes from wheat

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg²⁺ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl₂. The Mg²⁺ requirement could be substituted only partially by Mn²⁺ and not at all by Ca²⁺. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

Functional organization of TRPC-Ca channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1–TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca²⁺ influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) hydrolysis, generation of IP₃ and DAG, and IP₃-induced Ca²⁺ release from the intracellular Ca²⁺ store via inositol trisphosphate receptor (IP₃R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca²⁺ entry mechanisms. The former is regulated by the emptying/refilling of internal Ca²⁺ store(s) while the latter depends on PIP₂ hydrolysis (due to changes in PIP₂ per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca²⁺ entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca²⁺]_i signals which are critical for precise control of downstream cellular functions.     

The dynamics of PKC‐induced phosphorylation triggered by Ca2+ oscillations in mouse eggs

Fertilization of mammalian eggs is characterized by a series of Ca²⁺ oscillations triggered by a phospholipase C activity. These Ca²⁺ increases and the parallel generation of diacylglycerol (DAG) stimulate protein kinase C (PKC). However, the dynamics of PKC activity have not been directly measured in living eggs. Here, we have monitored the dynamics of PKC‐induced phosphorylation in mouse eggs, alongside Ca²⁺ oscillations, using fluorescent C‐kinase activity reporter (CKAR) probes. Ca²⁺ oscillations triggered either by sperm, phospholipase C zeta (PLCζ) or Sr²⁺ all caused repetitive increases in PKC‐induced phosphorylation, as detected by CKAR in the cytoplasm or plasma membrane. The CKAR responses lasted for several minutes in both the cytoplasm and plasma membrane then returned to baseline values before subsequent Ca²⁺ transients. High frequency oscillations caused by PLCζ led to an integration of PKC‐induced phosphorylation. The conventional PKC inhibitor, Gö6976, could inhibit CKAR increases in response to thapsigargin or ionomycin, but not the repetitive responses seen at fertilization. Repetitive increases in PKCδ activity were also detected during Ca²⁺ oscillations using an isoform‐specific δCKAR. However, PKCδ may already be mostly active in unfertilized eggs, since phorbol esters were effective at stimulating δCKAR only after fertilization, and the PKCδ‐specific inhibitor, rottlerin, decreased the CKAR signals in unfertilized eggs. These data show that PKC‐induced phosphorylation outlasts each Ca²⁺ increase in mouse eggs but that signal integration only occurs at a non‐physiological, high Ca²⁺ oscillation frequency. The results also suggest that Ca²⁺‐induced DAG formation on intracellular membranes may stimulate PKC activity oscillations at fertilization. J. Cell. Physiol. 228: 110–119, 2013. © 2012 Wiley Periodicals, Inc.     

Targets of oxidative stress in cardiovascular system

Although oxidants such as superoxide (O₂.^-) and hydrogen peroxide (H₂O₂) play a role in host-mediated destruction of foreign pathogens yet excessive generation of oxidants may lead to a variety of pathological complications in the cardiovascular system. An important mechanism by which oxidants cause dysfunction of the cardiovascular system appears to be due to the increase in intracellular free Ca²⁺ concentration. Oxidants cause cellular Ca²⁺ mobilization by modulating activities of a variety of regulators such as Na⁺/H⁺ and Na⁺/Ca²⁺ exchangers, Na⁺/K⁺ ATPase and Ca²⁺ ATPase and Ca²⁺ channels that are associated with Ca²⁺ transport in the plasma membrane and the sarco(endo)plasmic reticular membrane of myocardial cells. Recent research have suggested that the increase in Ca²⁺ level by oxidants plays a pivotal role in indicing several protein kinases such as protein kinase C, tyrosine kinase and mitogen activated protein kinases. Oxindant-mediated alteration of different signal transduction systems and their interations eventually regulate a variety of pathological conditoins such as atherosclerosis, apoptosis and necrosis in the myocardium     

Activation of the Ca2+-sensing receptor stimulates the activity of the epithelial Ca2+ channel TRPV5

The extracellular Ca²⁺-sensing receptor (CaR) is a key-player in plasma Ca²⁺ homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca²⁺ reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca²⁺-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca²⁺ reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca²⁺ concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR^R185Q. Interestingly, the activity of TRPV6, TRPV5′ closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca²⁺ influx via a PMA-insensitive PKC isoform pathway.     

A Novel Cross-talk in Diacylglycerol Signaling: THE RAC-GAP β2-CHIMAERIN IS NEGATIVELY REGULATED BY PROTEIN KINASE Cδ-MEDIATED PHOSPHORYLATION*

Although the family of chimaerin Rac-GAPs has recently gained significant attention for their involvement in development, cancer, and neuritogenesis, little is known about their molecular regulation. Chimaerins are activated by the lipid second messenger diacylglycerol via their C1 domain upon activation of tyrosine kinase receptors, thereby restricting the magnitude of Rac signaling in a receptor-regulated manner. Here we identified a novel regulatory mechanism for β2-chimaerin via phosphorylation. Epidermal growth factor or the phorbol ester phorbol 12-myristate 13-acetate caused rapid phosphorylation of β2-chimaerin on Ser¹⁶⁹ located in the SH2-C1 domain linker region via protein kinase Cδ, which retained β2-chimaerin in the cytosol and prevented its C1 domain-mediated translocation to membranes. Furthermore, despite the fact that Ser¹⁶⁹ phosphorylation did not alter intrinsic Rac-GAP activity in vitro, a non-phosphorylatable β2-chimaerin mutant was highly sensitive to translocation, and displayed enhanced association with activated Rac, enhanced Rac-GAP activity, and anti-migratory properties when expressed in cells. Our results not only revealed a novel regulatory mechanism that facilitates Rac activation, but also identified a novel mechanism of cross-talk between diacylglycerol receptors that restricts β2-chimaerin relocalization and activation.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters pancreatic membrane tyrosine phosphorylation following acute treatment

To understand the basic mechanisms of TCDD's action to cause hypoinsulinemia in several experimental animals, we have studied TCDD-induced changes in various protein kinase activities in membrane preparations of guinea pig pancreas. For this purpose, young male guinea pigs were treated through a single intraperitoneal in jection with 1 or 3 μg/kg of TCDD in vivo, and, after given time periods, pancreas samples were obtained and membranes were isolated through homogenization and centrifugation procedures. Several sets of incubation conditions were selected for protein kinase activity assay, each favoring a specific type of protein kinase. It was found that overall protein phosphorylation activities were higher in the preparation from TCDD-treated an imals as compared to those found in pair-fed controls and that this trend was more pronounced when the assay medium contained Mn²⁺ in place of Mg²⁺ and EGTA. These are the conditions that are known to favor protein tyrosine kinases. Other types of protein kinases from the treated animals did not show any significant differences from the pair-fed control animals, though that of protein kinase C in the treated preparation showed a modest increase. To establish that the type of protein kin ases stimulated by TCDD are protein tyrosine kin ases, we have carried out phosphoamino acid analyses, KOH digestion, and western blot analyses using an antibody to phosphotyrosine. All the results were consistent in supporting the idea that TCDD causes a rise in protein-tyrosine kinases in pancreas at early stages of poisoning.     

Targeted Activation of Conventional and Novel Protein Kinases C through Differential Translocation Patterns

Activation of the two ubiquitous families of protein kinases, protein kinase A (PKA) and protein kinase C (PKC), is thought to be independently coupled to stimulation of Gα_s and Gα_q, respectively. Live-cell confocal imaging of protein kinase C fluorescent protein fusion constructs revealed that simultaneous activation of Gα_s and Gα_q resulted in a differential translocation of the conventional PKCα to the plasma membrane while the novel PKCδ was recruited to the membrane of the endoplasmic reticulum (ER). We demonstrate that the PKCδ translocation was driven by a novel Gα_s-cyclic AMP-EPAC-RAP-PLCε pathway resulting in specific diacylglycerol production at the membrane of the ER. Membrane-specific phosphorylation sensors revealed that directed translocation resulted in phosphorylation activity confined to the target membrane. Specific stimulation of PKCδ caused phosphorylation of the inositol-1,4,5-trisphosphate receptor and dampening of global Ca²⁺ signaling revealed by graded flash photolysis of caged inositol-1,4,5-trisphosphate. Our data demonstrate a novel signaling pathway enabling differential decoding of incoming stimuli into PKC isoform-specific membrane targeting, significantly enhancing the versatility of cyclic AMP signaling, thus demonstrating the possible interconnection between the PKA and PKC pathways traditionally treated independently. We thus provide novel and elementary understanding and insights into intracellular signaling events.