Localization Of Protein Kinase Research Articles

Loss of habenular Prkar2a reduces hedonic eating and increases exercise motivation.

The habenula (Hb) is a bilateral, evolutionarily conserved epithalamic structure connecting forebrain and midbrain structures that has gained attention for its roles in depression, addiction, rewards processing, and motivation. Of its 2 major subdivisions, the medial Hb (MHb) and lateral Hb (LHb), MHb circuitry and function are poorly understood relative to those of the LHb. Prkar2a codes for cAMP-dependent protein kinase (PKA) regulatory subunit IIα (RIIα), a component of the PKA holoenzyme at the center of one of the major cell-signaling pathways conserved across systems and species. Type 2 regulatory subunits (RIIα, RIIβ) determine the subcellular localization of PKA, and unlike other PKA subunits, Prkar2a has minimal brain expression except in the MHb. We previously showed that RIIα-knockout (RIIα-KO) mice resist diet-induced obesity. In the present study, we report that RIIα-KO mice have decreased consumption of palatable, “rewarding” foods and increased motivation for voluntary exercise. Prkar2a deficiency led to decreased habenular PKA enzymatic activity and impaired dendritic localization of PKA catalytic subunits in MHb neurons. Reexpression of Prkar2a in the Hb rescued this phenotype, confirming differential roles for Prkar2a in regulating the drives for palatable foods and voluntary exercise. Our findings show that in the MHb decreased PKA signaling and dendritic PKA activity decrease motivation for palatable foods, while enhancing the motivation for exercise, a desirable combination of behaviors.

A Combined Approach Reveals a Regulatory Mechanism Coupling Src’s Kinase Activity, Localization, and Phosphotransferase-Independent Functions

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Morphine Induces Differential Activation and Cellular Localization of Protein Kinases in the Mouse Periaqueductal Gray

Morphine is a commonly used potent opioid analgesic with very high propensity for tolerance development. The analgesic and tolerance properties of opioids are partially regulated by the midbrain ventrolateral periaqueductal gray (vlPAG). Here, we investigate the intracellular kinase activation and localization patterns in the mouse vlPAG following acute and chronic morphine treatment. Wild‐type mice were treated with morphine (10 mg/kg, i.p.) or saline in a chronic or acute administration paradigm. We performed fluorescence immunohistochemistry to map the localization and activation of extracellular signal‐regulated kinase 1/2 (ERK 1/2), protein kinase‐C (PKC), and protein kinase‐A (PKA). We observed significantly greater activated ERK 1/2 in the vlPAG chronic morphine‐treated animals. We also visualized the co‐localization of ERK 1/2 with the endosomal marker Eea1. We note that phosphorylated PKC tends to localize to the plasma membrane in the vlPAG following chronic morphine treatment and interacts with the μ‐opioid receptor. Further, we observed that although PKA activation is increased following chronic morphine treatment, there is a significant reduction in nuclear translocation of this kinase. Additionally, we examined the cell‐type specificity of kinase activation by co‐localizing these kinases to GAD67 and VGluT neurons that project from the PAG downstream to the Rostral Ventromedial Medulla. Taken together, this study demonstrates a differential activation and localization of ERK 1/2, PKC and PKA, and opens avenues to explore the role of chronic morphine treatment on G‐protein signaling and kinase nuclear transport.Support or Funding InformationThis research is supported by the Department of Biology at Utah State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RHOA activity in expanding blastocysts is essential to regulate HIPPO-YAP signaling and to maintain the trophectoderm-specific gene expression program in a ROCK/actin filament-independent manner.

What molecular signals are required to maintain the functional trophectoderm (TE) during blastocyst expansion of the late stage of preimplantation development? The activity of ras homology family member A (RHOA) GTPases is necessary to retain the expanded blastocyst cavity and also to sustain the gene expression program specific to TE. At the early stages of preimplantation development, the precursor of the TE lineage is generated through the molecular signals that integrate RHOA, RHO-associated coiled-coil containing protein kinase (ROCK), the apicobasal cell polarity, and the HIPPO-Yes-associated protein (YAP) signaling pathway. By contrast, molecular mechanisms regulating the maintenance of the TE characteristics at the later stage, which is crucial for blastocyst hatching and implantation, are scarcely understood. Expanding mouse blastocysts, obtained from crosses of the F1 (C57BL6 × DBA/2) strain, were exposed to chemical agents that interfere with RHOA, ROCK, or the actin cytoskeleton for up to 8 h, and effects on the blastocyst cavity, HIPPO-YAP signaling, and cell lineage-specific gene expression profiles were examined. Mouse embryos at the embryonic stage E3.5 (expanding blastocysts) and E4.5 (fully expanded blastocysts) were treated with RHOA inhibitor (C3 exoenzyme), ROCK inhibitor (Y27632), or actin filament disruptors (cytochalasin B and latrunculin A). The integrity of the blastocyst cavity was evaluated based on the gross morphology. Effects on HIPPO-YAP signaling were assessed based on the presence of nuclearized YAP protein by immunofluorescence staining and the expression of YAP/TEA domain family member (TEAD) target genes by quantitative RT-PCR (qRT-PCR). The impact of these disruptors on cell lineages was evaluated based on expression of the TE-specific and inner cell mass-specific marker genes by qRT-PCR. The integrity of the apicobasal cell polarity was assessed by localization of protein kinase C zeta (PRKCZ; apical) and scribbled planar cell polarity (SCRIB; basal) proteins by immunofluorescence staining. For comparisons, cultured cell lines, NIH/3T3 (mouse fibroblast) and P19C5 (mouse embryonal carcinoma), were also treated with RHOA inhibitor, ROCK inhibitor, and actin filament disruptors for up to 8 h, and effects on HIPPO-YAP signaling were assessed based on expression of YAP/TEAD target genes by qRT-PCR. Each experiment was repeated using three independent batches of embryos (n = 40-80 per batch) or cell collections. Statistical analyses of data were performed, using one-way ANOVA and two-sample t-test. Inhibition of RHOA deflated the cavity, diminished nuclear YAP (P < 0.01), and down-regulated the YAP/TEAD target and TE-specific marker genes in both E3.5 and E4.5 blastocysts (P < 0.05), indicating that the maintenance of the key TE characteristics is dependent on RHOA activity. However, inhibition of ROCK or disruption of actin filament only deflated the blastocyst cavity, but did not alter HIPPO-YAP signaling or lineage-specific gene expressions, suggesting that the action of RHOA to sustain the TE-specific gene expression program is not mediated by ROCK or the actomyosin cytoskeleton. By contrast, ROCK inhibitor and actin filament disruptors diminished YAP/TEAD target gene expressions in cultured cells to a greater extent than RHOA inhibitor, implicating that the regulation of HIPPO-YAP signaling in expanding blastocysts is distinctly different from that in the cell lines. Furthermore, the apicobasal cell polarity proteins in the expanding blastocyst were mislocalized by ROCK inhibition but not by RHOA inhibition, indicating that cell polarity is not linked to regulation of HIPPO-YAP signaling. Taken together, our study suggests that RHOA activity is essential to maintain the TE lineage in the expanding blastocyst and it regulates HIPPO-YAP signaling and the lineage-specific gene expression program through mechanisms that are independent of ROCK or actomyosin cytoskeleton. Not applicable. This study was conducted using one species, the mouse. Direct translation of the experiments and findings to human fertility preservation and ART requires further investigations. The elucidation of the mechanisms of TE formation is highly pertinent to fertility preservation in women. Our findings may raise awareness among providers of ART that the TE is sensitive to disturbance even in the late stage of blastocyst expansion and that rational approaches should be devised to avoid conditions that may impair the TE and its function. This study was funded by grants from the Ingeborg v.F. McKee Fund of the Hawaii Community Foundation (16ADVC-78882 to V.B.A.), and the National Institutes of Health (P20 GM103457 and R03 HD088839 to V.B.A.). The authors have no conflict of interest to declare.

Type II PKAs are anchored to mature insulin secretory granules in INS-1 β-cells and required for cAMP-dependent potentiation of exocytosis.

Specificity of the cAMP-dependent protein kinase (PKA) pathway relies on an extremely sophisticated compartmentalization mechanism of the kinase within a given cell, based on high-affinity binding of PKA tetramer pools to different A-Kinase Anchoring Proteins (AKAPs). We and others have previously shown that AKAPs-dependent PKA subcellular targeting is a requisite for optimal cAMP-dependent potentiation of insulin exocytosis. We thus hypothesized that a PKA pool may directly anchor to the secretory compartment to potentiate insulin exocytosis. Here, using immunofluorescence analyses combined to subcellular fractionations and purification of insulin secretory granules (ISGs), we identified discrete subpools of type II PKAs, RIIα and RIIβ PKAs, along with the catalytic subunit, physically associated with ISGs within pancreatic insulin-secreting β-cells. Ultrastructural analysis of native rodent β-cells confirmed invivo the occurrence of PKA on dense-core ISGs. Isoform-selective disruption of binding of PKAs to AKAPs reinforced the requirement of type II PKA isoforms for cAMP potentiation of insulin exocytosis. This granular localization of PKA was of critical importance since siRNA-mediated depletion of either RIIα or RIIβ PKAs resulted in a significant reduction of cAMP-dependent potentiation of insulin release. The present work provides evidence for a previously unrecognized pool of type II PKAs physically anchored to the β-cell ISGs compartment and supports a non-redundant function for type II PKAs during cAMP potentiation of exocytosis.

Mycobacterium indicus pranii (Mw) inhibits invasion by reducing matrix metalloproteinase (MMP-9) via AKT/ERK-1/2 and PKCα signaling: A potential candidate in melanoma cancer therapy

ABSTRACTInvasion and metastasis via induction of matrix metalloproteinases are the main causes of death in melanoma cancer. In this study, we investigated the inhibitory effects of heat killed saprophytic bacterium Mycobacterium indicus pranii (Mw) on B16F10 melanoma cell invasion. Mw reported to be an immunomodulator has antitumor activity however, its effect on cancer cell invasion has not been studied. Highly invasive B16F10 melanoma was found sensitive to Mw which downregulated MMP-9 expression. Mw treatment inhibited nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) transcriptional activity and respective DNA binding to MMP-9 promoter. Moreover, Mw also overcame the promoting effects of PMA on B16F10 cell invasion. Mw decreased PMA-induced transcriptional activation of NF-κB and AP-1 by inhibiting phosphorylation of AKT and ERK-1/2. Furthermore, Mw strongly suppressed PMA-induced membrane localization of protein kinase C α (PKCα) since PKCα inhibition caused a marked decrease in PMA-induced MMP-9 secretion as well as AKT/ERK-1/2 activation. These results suggest that Mw may be a promising anti-invasive agent as it blocks tumor growth and inhibits B16F10 cell invasion by reducing MMP-9 activation through inhibition of PKCα/ AKT/ ERK-1/2 phosphorylation and NF-κB/AP-1 activation.

Abstract 20504: Dysfunctional cAMP-Dependent Protein Kinase (PKA) Signaling Causes Impaired Autonomic Control in Diabetic Heart Failure

Autonomic control of cardiac function primarily occurs through the regulation of cAMP-dependent protein kinase (PKA) via the adrenergic and muscarinic pathways. The diabetic heart has a suppressed β-adrenergic response that is classically attributed to β-adrenergic receptor internalization, but little is known about how the PKA signaling pathway is affected. We assessed the hypothesis that β-adrenergic impairment in diabetes occurs through PKA signaling dysfunction. Four-month streptozotocin (STZ)-induced diabetic mice were used in this study as a model of diabetic cardiomyopathy. Although the diabetic mice have substantial cardiac impairment, as measured by pressure catheter, cAMP levels are not reduced, suggesting the pathway upstream of PKA is intact. We next developed a novel system using acute 8-min treatments with a cAMP analog, 8Br-cAMP, to activate PKA in a receptor-independent manner and increase ventricular inotropy. Diabetic mice have an impaired response to 8Br-cAMP treatment as shown by minimal change in PKA substrate phosphorylation (Fig. 1) and PKA activity by enzyme assay (% increase, C: 65±3.5, STZ: 30±5, n=5). Interestingly, cytosolic phosphorylation of PFK-2 is absent (% increase, C: 142±32, STZ: -15.7±13, n=5), yet nuclear phosphorylation of CREB is maintained suggesting a difference in localization of PKA. Although total PKA content is not different in the diabetic hearts, there is reduced cytosolic content of PKA (-35±1%, n=5) and an increase in nuclear translocation of PKA with treatment. The improper localization of PKA in the diabetic mice explains the differing phosphorylation. A major contributor is the reduction of nuclear PKA inhibitors/exporters, PKIα (-33±6.2%, n=5) and PKIγ (-20±7.2%, n=5), which normally regulate the nuclear content of PKA. In conclusion, PKA signaling is greatly impaired in diabetes and is attributable to altered enzyme localization, which is at least in part a result of PKI loss.

Localization of protein kinase C isoforms in the optic pathway of mouse embryos and their role in axon routing at the optic chiasm

Protein kinase C (PKC) plays a key role in many receptor-mediated signaling pathways that regulate cell growth and development. However, its roles in guiding axon growth and guidance in developing neural pathways are largely unknown. To investigate possible functions of PKC in the growth and guidance of axons in the optic chiasm, we first determined the localization of major PKC isoforms in the retinofugal pathway of mouse embryos, at the stage when axons navigate through the midline. Results showed that PKC was expressed in isoform specific patterns in the pathway. PKC-α immunoreactivity was detected in the chiasm and the optic tract. PKC-βΙΙ was strong in the optic stalk but was attenuated on axons in the diencephalon. Immunostaining for PKC-ε showed a colocalization in the chiasmatic neurons that express a surface antigen stage specific embryonic antigen-1 (SSEA-1). These chiasmatic neurons straddled the midline of the optic chiasm, and have been shown in earlier studies a role in regulation of axon growth and guidance. Expression levels of PKC-βΙ, -δ and -γ were barely detectable in the pathway. Blocking of PKC signaling with Ro-32-0432, an inhibitor specific for PKC-α and -β at nanomolar concentration, produced a dramatic reduction of ipsilateral axons from both nasal retina and temporal crescent. We conclude from these studies that PKC-α and -βΙΙ are the predominant forms in the developing optic pathway, whereas PKC-ε is the major form in the chiasmatic neurons. Furthermore, PKC-α and -βΙΙ are likely involved in signaling pathways triggered by inhibitory molecules at the midline that guide optic axons to the uncrossed pathway.

Cytoplasmic Anchoring of cAMP-Dependent Protein Kinase (PKA) by A-Kinase Anchor Proteins (AKAPs) Is Required for Meiotic Arrest of Porcine Full-Grown and Growing Oocytes1

Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. We previously hypothesized that the meiotic incompetence of porcine GOs was attributed to complex spatial-temporal regulation of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs), but found that AKAP1 is not involved in the meiotic incompetence of porcine GOs. In the present study, we cloned porcine cDNAs of AKAP5 and AKAP7alpha, and found that inhibiting the expression of these AKAPs induced PKA translocation into the nucleus and promoted meiotic resumption of porcine GOs without affecting the total PKA activity of GOs, whereas overexpressing these AKAPs had no effect. Because AKAPs regulate PKA localization through binding with regulatory subunits of PKA (PKA-Rs), PKA-R binding with AKAPs was inhibited by AKAP-binding inhibition peptides or PKA-R expression inhibition by antisense RNAs. We found that the expression inhibition and binding inhibition of PRKAR1A, an isoform of mammalian PKA-R, promoted meiotic resumption of porcine GOs, whereas these inhibitions of PRKAR2A, another PKA-R isoform, had no effect. In contrast, the expression inhibition and binding inhibition of PRKAR2A had higher effects than those of PRKAR1A on meiotic resumption of porcine full-grown oocytes. These results suggest that cytoplasmic anchoring of PKA by AKAPs is required for meiotic arrest of oocytes and that the PKA-R isoform working for the maintenance of meiotic arrest changed from PRKAR1A to PRKAR2A during the acquisition of meiotic competence.

Functional recovery and alterations in the expression and localization of protein kinase C following voluntary exercise in rat with cerebral infarction

Recently, it has become widely known that rehabilitative training after stroke brings about some improvement of paralysis and disability; however, not much is known about the relationship between paralysis recovery and the participation of plasticity-related molecules. Hence, the localization and level of expression of several proteins in the cerebral cortex of rat groups with/without voluntary exercise using a running wheel after photo thrombotic infarction were examined in this study. In behavioral evaluation, the mean latency until falling from a rotating rod in the group with voluntary exercise at 6 days after infarction was significantly longer than that in the group without exercise. Immunohistochemical localization of c-Fos protein after behavioral test occurred in the area surrounding the infarction core in the exercise group. In protein expression analysis, protein kinase C (PKC), growth-associated protein 43 (GAP43) and phosphorylated at serine 41 GAP43 (p-GAP43) were significantly increased after voluntary exercise compared with those in rats without exercise. Expression of PKC immunoreactivity was observed in layer III of the perilesional cortex in rats with exercise, and the intracellular localization in the pyramidal neurons was mainly translocated to the plasma membrane. The expression and localization of these proteins may be related to the underlying mechanisms of exercise-induced paralysis recovery, that is, neuronal plasticity and remodeling of cortical connections through the phosphorylation of GAP43 by interaction with PKC. In the present study, the participation of at least some of the modulators associated with the improvement of motor deficit adjacent to the brain lesion might have been detected.

A-Kinase Anchor Protein 1 (AKAP1) Regulates cAMP-Dependent Protein Kinase (PKA) Localization and Is Involved in Meiotic Maturation of Porcine Oocytes1

In mammalian oocytes, cAMP-dependent protein kinase (PKA) has critical functions in meiotic arrest and meiotic maturation. Although subcellular localization of PKA is regulated by A-kinase anchor proteins (AKAPs) and PKA compartmentalization is essential for PKA functions, the role of AKAPs in meiotic regulation has not been fully elucidated. In the present study, we performed far-Western blot analysis using porcine PRKAR2A for detection of AKAPs and found, to our knowledge, several novel signals in porcine oocytes. Among these signals, a 150-kDa AKAP showed the major expression and was the product of porcine AKAP1. Overexpression of AKAP1 changed the PKA localization and promoted meiotic resumption of porcine oocytes even in the presence of a high concentration of cAMP, which inhibits meiotic resumption by inducing high PKA activity. On the contrary, knockdown of AKAP1 showed inhibitory effects on meiotic resumption and oocyte maturation. In addition, the expression level of AKAP1 in porcine growing oocytes, which show meiotic incompetence and PKA mislocalization, was significantly lower than that in fully grown oocytes. However, AKAP1 insufficiency was not the primary cause of the meiotic incompetence of the growing oocytes. These results suggest that the regulation of PKA localization by AKAP1 may be involved in meiotic resumption and oocyte maturation but not in meiotic incompetence of porcine growing oocytes.

Dynamic expression and localization of Protein Kinase A regulatory subunit RIα in cardiac mitochondria controls response to oxidative stress

Recent studies have implicated many possible roles for protein kinase A (PKA) activity localized to mitochondria, especially in organs such as the heart with high metabolic activity and/or increased susceptibility to oxidative stress. Because mitochondria serve as a critical control point for cellular adaption to environmental stress, we hypothesize that regulation of mitochondrial‐localized PKA (mt‐PKA) modifies mitochondrial integrity and cellular homeostasis. In particular, we propose that PKA associated proteins are actively regulated in response to oxidative stress in order to modulate mt‐PKA activity. Using biochemical and proteomic analysis of differing mitochondrial subpopulations within cardiac tissue, we found a striking difference between the relative localization and expression of PKA and its associated proteins. By combining acute oxidative stress models, biochemical, microscopic and pharmacological assessment, as well as physiological and metabolic functional analysis methods, our data suggests that a stress‐induced loss of mitochondrial localized PKA RIα (mt‐RIα) is directly correlated to deterioration of mitochondria integrity, decrease in respiratory activity, increase in ROS production, and an overall reduction in cell viability and heart function. Therefore, we conclude that mt‐RIα is a necessary component for regulating mitochondrial response to oxidative stress. Support for this work was provided by HHMI and NIH funding.

Complex Formation, Phosphorylation, and Localization of Protein Kinase A ofSchizosaccharomyces pombeupon Glucose Starvation

Nine sam mutants that undergo sexual differentiation without requiring starvation in Schizosaccharomyces pombe were previously isolated. In this study, we identified a nonsense mutation on the pka1 locus in the sam6 mutant. pka1 encodes a catalytic subunit of protein kinase A (PKA). Replacement and overexpression of pka1 suppressed the KCl sensitivity and hyper-mating phenotype of sam6, confirming that sam6 is an allele of pka1. To characterize further the regulation of Pka1, we tested the physical interaction between Pka1 and Cgs1 (a regulatory subunit of PKA). Pka1 and Cgs1 physically interacted under glucose-limited conditions but not under glucose-rich conditions. In addition, the formation of a Pka1-Cgs1 complex was detected under glucose-limited conditions by Blue Native PAGE. Furthermore, the Pka1 protein was found to be phosphorylated under glucose-starved conditions, and at the same time its localization shifted from the nucleus towards the cytoplasm (mainly the vacuoles), suggesting a strong relationship among phosphorylation, complex formation, and the cytoplasmic distribution of Pka1.

Regulation of cyclooxygenase-2 and cytosolic phospholipase A2 gene expression by lipopolysaccharide through the RNA-binding protein HuR: involvement of NADPH oxidase, reactive oxygen species and mitogen-activated protein kinases.

BACKGROUND AND PURPOSE Lipopolysaccharide (LPS)-induced expression of cyclooxygenase-2 (COX-2) and cytosolic phospholipase A(2) (cPLA(2) ) has been implicated in several respiratory diseases. HuR is known to enhance the expression of genes by binding to 3'-untranslated region (3'-UTR) of mRNA and stabilizing mRNA. However, the exact mechanisms by which HuR affects the stability of mRNA and modulates LPS-induced COX-2 and cPLA(2) expression in human tracheal smooth muscle cells (HTSMCs) are not known. EXPERIMENTAL APPROACH The expression of prostaglandin E(2) (PGE(2) ) was measured by ELISA, and pro-inflammatory proteins were determined by use of a promoter assay, PCR or Western blot analysis. Overexpression of siRNAs to knock down the target components was used to manipulate the expression of HuR. Release of reactive oxygen species (ROS) was detected by fluorescence dye. The activation of signalling components was assessed by comparing phosphorylation levels, localization of protein kinases or coimmunoprecipitation assay. KEY RESULTS LPS induced COX-2 and cPLA(2) expression via post-translational regulation of mRNA stabilization, which were attenuated by transfection with HuR siRNA in HTSMCs. In addition, LPS-stimulated NADPH oxidase activation and ROS generation were attenuated by the NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and apocynin (APO). Generation of ROS induced phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK and JNK1/2, which was attenuated by DPI and APO and the ROS scavenger N-acetylcysteine. CONCLUSIONS AND IMPLICATIONS These results suggested that in HTSMCs, LPS-induced COX-2 and cPLA(2) expression is mediated through NADPH oxidase/ROS-dependent MAPKs associated with HuR accumulation in the cytoplasm. Activated MAPKs may regulate the nucleocytoplasmic shuttling of HuR, and thus induce the cytoplasmic accumulation of HuR.

Immunohistochemical localization of protein kinase C (PKC) beta I in the pig retina during postnatal development

In order to investigate the expression of protein kinase C (PKC) beta I in the retinas of pigs during postnatal development, we analyzed retinas sampled from 3-day-old and 6-month-old pigs by Western blotting and immunohistochemistry. Western blot analysis detected the expression of PKC beta I in the retinas of 3-day-old piglets and it was increased significantly in the retinas of 6-month-old adult pigs. Immunohistochemical staining showed PKC beta I in the retinas of both groups. Immunohistochemistry of 3-day-old retinas revealed weak PKC beta I reactivity in the ganglion cell layer, inner plexiform layer, inner nuclear cell layer, outer plexiform layer and rod and cone cell layer. In the 6-month-old pig retina, the cellular localization of PKC beta I immunostaining was similar to that of the 3-day-old retina, where PKC beta I was localized in some glial fibrillary acidic protein-positive cells, glutamine synthetase-positive cells, parvalbumin-positive cells, and PKC alpha-positive cells in the retina. This is the first study to show the expression and cellular localization of PKC beta I in the retina of pigs with development, and these results suggest that PKC beta I, in accordance with PKC alpha, plays important roles in signal transduction pathways in the pig retina with development.

Coming to a Location within You: Localization of Protein Kinase A and DPY‐30

Signal transduction is an essential process in cells. One critical signaling molecule, protein kinase A (PKA), phosphorylates target proteins, thereby changing their conformations and modifying their functions. PKA is a component of multiple signaling pathways that regulate a variety of proteins. Since the broad substrate specificity of PKA can lead to phosphorylation of unintended proteins, PKA activity must be limited to specific times and places. A‐kinase anchoring proteins (AKAPs) bind and help localize PKA to specific areas. The RIIa domain in PKA provides a shallow groove for an amphipathic helix of AKAP to bind via interactions of hydrophobic side chains. A similar binding motif is found in the DPY‐30 domain, which suggests this domain may also play a localization role. The ability of AKAP to interact with PKA and regulate its activity is essential for the specificity of many cellular responses. The ability of a cell to localize proteins containing a DPY‐30 domain may also be important for proper function. If localization is disrupted, serious problems like heart disease and cancer may result. To further understand the impact of structural interactions on localization, physical models of RIIa, DPY‐30, and AKAP amphipathic helix have been designed and built by the Cedarburg High School SMART (Students Modeling a Research Topic) Team using 3D printing technology. Supported by a grant from NIH‐NCRR‐SEPA.

Activation and localization of protein kinase C in Neurospora crassa

The Neurospora crassa protein kinase C (NPKC) is reported to be a regulator of light responsive genes. It phosphorylates the light receptor WC-1 and regulates the levels of the circadian clock protein FRQ and transcription of the light-induced albino-2 gene. In mammals, the conventional and novel isoforms of PKC are activated by diacylglycerol (DAG), which induces PKC translocation from the cytoplasm to membranes. To investigate the interaction of NPKC and DAG in Neurospora, we constructed a strain that expresses a PKC-GFP fusion protein. We found that NPKC localizes to growing tips and sub-apical plasma membrane in actively growing hyphae, and actively participates in septum development. NPKC is activated by exogenous DAG and phorbol esters, and translocates to the plasma membrane from the cytoplasm. We have previously reported that choline depletion of the chol-1 mutant of Neurospora increases DAG levels and lengthens the period of the circadian rhythm of conidiation. We have found that the activity of NPKC is rhythmic, and that NPKC levels are increased on choline depletion. However, over-expression of NPKC did not lengthen the conidiation period, indicating that PKC in Neurospora may not be responsible for the lengthened period in low choline cultures.

Disruption of Protein Kinase A Localization Using a Trans-activator of Transcription (TAT)-conjugated A-kinase-anchoring Peptide Reduces Cardiac Function

Localization of protein kinase A (PKA) via A-kinase-anchoring proteins (AKAPs) is important for cAMP responsiveness in many cellular systems, and evidence suggests that AKAPs play an important role in cardiac signaling. To test the importance of AKAP-mediated targeting of PKA on cardiac function, we designed a cell-permeable peptide, which we termed trans-activator of transcription (TAT)-AKAD for TAT-conjugated A-kinase-anchoring disruptor, using the PKA binding region of AKAP10 and tested the effects of this peptide in isolated cardiac myocytes and in Langendorff-perfused mouse hearts. We initially validated TAT-AKAD as a PKA localization inhibitor in cardiac myocytes by the use of confocal microscopy and cellular fractionation to show that treatment with the peptide disrupts type I and type II PKA regulatory subunits. Knockdown of PKA activity was demonstrated by decrease in phosphorylation of phospholamban and troponin I after beta-adrenergic stimulation in isolated myocytes. Treatment with TAT-AKAD reduced myocyte shortening and rates of contraction and relaxation. Injection of TAT-AKAD (1 microM), but not scrambled control peptide, into the coronary circulation of isolated perfused hearts rapidly (<1 min) and reversibly decreased heart rate and peak left ventricular developed pressure. TAT-AKAD also had a pronounced effect on developed pressure (-dP/dt), consistent with a delayed relaxation of the heart. The effects of TAT-AKAD on heart rate and contractility persisted in hearts pretreated with isoproterenol. Disruption of PKA localization with TAT-AKAD thus had negative effects on chronotropy, inotropy, and lusitropy, thereby indicating a key role for AKAP-targeted PKA in control of heart rate and contractile function.

Abstract 4144: The treatment with a derivative of Vitamin A (ATRA) modulates the expression and subcellular localization of Protein Kinase C (PKC) delta and induces growth inhibition in normal and tumor derived mammary cells

Abstract Retinoids may exert some of their effects on cell differentiation and malignant phenotype reversion through the interaction with different PKC isoforms. In the present work we addressed the following issues: A)- the role of ATRA on the in vitro growth potential of LM3, a murine mammary tumor-derived cell line; NMuMG, a normal murine mammary cell line and MDA-MB231, a human breast cancer-derived cell line. B)- the effect of retinoid treatment on the expression and subcellular distribution of PKCδ and C)- the importance of PKCδ in the activity of retinoid receptors. ATRA treatment (1μM, 72h) induced a significant growth inhibition in the analyzed murine cell lines. This phenomenon was associated with the reduction of pERK1 levels (80±9% and 40±5% in LM3 and NMuMG cells respectively) and the increase of the cell cycle inhibitor p27 in the nuclear fraction without altering the expression of Cyclin D1 in both murine cell lines. None of these modulations were observed in the ATRA unresponsive MDA-MB241 cell line. By Western blot we could determine that 72h treatment with ATRA induced an increase of PKCδ protein levels both in the nuclear fraction of LM3 cells (4-fold) and in the cytoplasm (2- and 3-fold for LM3 and NMuMG cells respectively). On the contrary, in MDA-MB241 cells PKCδ expression was reduced after ATRA exposure. Interestingly, pharmacological silencing of PKCδ in LM3 cells prevented the activation of retinoid receptors by ATRA, as evidenced by a reporter gene assay (RARE-Luciferase). Our results suggest that, in ATRA-responsive cells, the treatment with this vitamin A derivate inhibits in vitro growth, by reducing mitogenic signaling, by increasing the expression of a cell-cycle inhibitory protein as well as by increasing PKCδ an isoform usually associated with growth inhibition and apoptosis induction. The subcellular localization was also altered by this treatment in LM3 cells. Moreover, the effect of ATRA exerted through the activation of its specific nuclear receptors would depend on the nuclear translocation of this PKC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4144.

Sonic-hedgehog-mediated proliferation requires the localization of PKA to the cilium base

Cerebellar granular neuronal precursors (CGNPs) proliferate in response to the mitogenic activity of Sonic hedgehog (Shh), and this proliferation is negatively regulated by activation of cAMP-dependent protein kinase (PKA). In the basal state, the PKA catalytic subunits (C-PKA) are inactive because of their association with the regulatory subunits (R-PKA). As the level of cAMP increases, it binds to R-PKA, displacing and thereby activating the C-PKA. Here we report that, in the presence of Shh, inactive C-PKA accumulates at the cilium base of proliferative CGNPs whereas removal of Shh triggers the activation of PKA at this particular location. Furthermore, we demonstrate that the anchoring of the PKA holoenzyme to the cilium base is mediated by the specific binding of the type II PKA regulatory subunit (RII-PKA) to the A-kinase anchoring proteins (AKAPs). Disruption of the interaction between RII-PKA and AKAPs inhibits Shh activity and, therefore, blocks proliferation of CGNP cultures. Collectively, these results demonstrate that the pool of PKA localized to the cilium base of CGNP plays an essential role in the integration of Shh signal transduction.