PKC-mediated Signal Transduction Research Articles

The ε-Isozyme of Protein Kinase C (PKCε) Is Impaired in ALS Motor Cortex and Its Pulse Activation by Bryostatin-1 Produces Long Term Survival in Degenerating SOD1-G93A Motor Neuron-like Cells.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease, characterized by a progressive depletion of upper and lower motor neurons (MNs) in the brain and spinal cord. The aberrant regulation of several PKC-mediated signal transduction pathways in ALS has been characterized so far, describing either impaired expression or altered activity of single PKC isozymes (α, β, ζ and δ). Here, we detailed the distribution and cellular localization of the ε-isozyme of protein kinase C (PKCε) in human postmortem motor cortex specimens and reported a significant decrease in both PKCε mRNA (PRKCE) and protein immunoreactivity in a subset of sporadic ALS patients. We furthermore investigated the steady-state levels of both pan and phosphorylated PKCε in doxycycline-activated NSC-34 cell lines carrying the human wild-type (WT) or mutant G93A SOD1 and the biological long-term effect of its transient agonism by Bryostatin-1. The G93A-SOD1 cells showed a significant reduction of the phosphoPKCε/panPKCε ratio compared to the WT. Moreover, a brief pulse activation of PKCε by Bryostatin-1 produced long-term survival in activated G93A-SOD1 degenerating cells in two different cell death paradigms (serum starvation and chemokines-induced toxicity). Altogether, the data support the implication of PKCε in ALS pathophysiology and suggests its pharmacological modulation as a potential neuroprotective strategy, at least in a subgroup of sporadic ALS patients.

天然物の構造単純化による有用分子の創製：天然プロテインキナーゼC活性化剤のアナログ設計

Indolactam-V (IL-V), bryostatin 1 (Bryo-1), and aplysiatoxin (ATX) are structurally diverse natural products that potently activate protein kinase C (PKC). Although they exhibit scientifically and therapeutically attractive bioactivities through PKC activation, their application for research and therapeutic uses has been limited by poor synthetic accessibility and complex biological profile. Recognizing this issue, we have designed structurally simpler and thus synthetically more accessible analogs of these PKC activators that can emulate or even improve biological performance of the parent natural products. IL-V analog was proved to be a useful tool for tracing PKC-mediated cellular signal transduction. Bryo-1 and ATX analogs have become promising leads for anti-cancer drugs. This study demonstrates that structural simplification of natural products provides biologically and therapeutically valuable molecules that are better suitable for human use.

Phosphorylation of Noxo1 at threonine 341 regulates its interaction with Noxa1 and the superoxide‐producing activity of Nox1

Superoxide production by Nox1, a member of the Nox family NAPDH oxidases, requires expression of its regulatory soluble proteins Noxo1 (Nox organizer 1) and Noxa1 (Nox activator 1) and is markedly enhanced upon cell stimulation with phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC). The mechanism underlying PMA-induced enhancement of Nox1 activity, however, remains to be elucidated. Here we show that, in response to PMA, Noxo1 undergoes phosphorylation at multiple sites, which is inhibited by the PKC inhibitor GF109203X. Among them, Thr341 in Noxo1 is directly phosphorylated by PKC invitro, and alanine substitution for this residue reduces not only PMA-induced Noxo1 phosphorylation but also PMA-dependent enhancement of Nox1-catalyzed superoxide production. Phosphorylation of Thr341 allows Noxo1 to sufficiently interact with Noxa1, an interaction that participates in Nox1 activation. Thus phosphorylation of Noxo1 at Thr341 appears to play a crucial role in PMA-elicited activation of Nox1, providing a molecular link between PKC-mediated signal transduction and Nox1-catalyzed superoxide production. Furthermore, Ser154 in Noxo1 is phosphorylated in both resting and PMA-stimulated cells, and the phosphorylation probably participates in a PMA-independent constitutive activity of Nox1. Ser154 may also be involved in protein kinase A (PKA) mediated regulation of Nox1; this serine is the major residue that is phosphorylated by PKA invitro. Thus phosphorylation of Noxo1 at Thr341 and at Ser154 appears to regulate Nox1 activity in different manners. Noxo1 binds to p22phox by pull down (1, 2, 3) Noxo1 binds to Noxo1 by pull down (View interaction) Noxa1 binds to Noxo1 by pull down (1, 2, 3, 4, 5).

Inhibitory effect of sanguinarine on PKC-CPI-17 pathway mediating by muscarinic receptors in dispersed intestinal smooth muscle cells

This study investigated the inhibitory effects of sanguinarine (SA) on PKC-CPI-17 pathway in rat intestinal smooth muscle cells (ISMC). Previous studies indicate that the inhibitory effects of SA on ISMC contraction are possibly mediated by the Ca2+ influx. ISMC was treated with 1μM SA for 24h remarkably inhibited the mRNA expression of m2 and m3 receptors. ISMC treated with 1 or 3μM SA for 30min significantly decreased the mRNA expression of PKC-δ, PKC-ε, PKC-η, and CPI-17. 1μM SA could markedly inhibit carbachol (CCh)-mediated increase PKC-δ, PKC-η, and CPI-17 mRNA but had no effect in PKC-ε.Treatment of ISMC with SA (1μM, 30min) caused a decrease in protein expression of PKC-δ. However, the expression of CPI-17 was significantly inhibited in a time-dependent manner. These results demonstrate that the inhibitory effect of SA is coupled with alteration of PKC-mediated signal transduction and intracellular Ca2+ concentration.

Targeting PKC-mediated signal transduction pathways using enzastaurin to promote apoptosis in acute myeloid leukemia-derived cell lines and blast cells.

Recent studies in acute myeloid leukemia (AML) suggest activation of pro-proliferative signaling cascades including those mediated by protein kinase C (PKC) represent a poor prognostic factor for patients. The classical PKC isoforms α and β generally support survival signaling and have emerged as important targets for anti-cancer therapy. Enzastaurin is a PKC β inhibitor and is in clinical trials for lymphomas, gliomas, and lung cancer. Presently, it is not known if enzastaurin could be effective against AML. In the current study, we found that high dose enzastaurin was found to promote apoptosis in the AML-derived cell lines and in blast cells from AML patients. The mechanism of cell death, however, likely does not involve PKC β as another PKC β inhibitor was not toxic to AML cell lines and did not promote enzastaurin-induced cell killing. While enzastaurin is fairly specific for PKC β, the agent can inhibit other PKC isoforms at higher concentrations. Enzastaurin was effective at inhibiting PKC α phosphorylation and membrane localization in the AML cell lines and suppressed phosphorylation of BCL2. Furthermore, enzastaurin suppressed activation of ERK (which can be activated by PKC α). Analysis of the serine/threonine phosphorylation profile in HL60 cells after enzastaurin treatment revealed that the drug inhibits the phosphorylation of a distinct set of proteins while promoting phosphorylation of another set of proteins. This suggests the drug may regulate multiple signaling pathways. Taken together, these findings suggest that enzastaurin could be effective in the therapy of AML.

RACK1 is involved in β-amyloid impairment of muscarinic regulation of GABAergic transmission

RACK1 (receptor for activated C-kinase 1), an anchoring protein that shuttles activated PKC to cellular membranes, plays an important role in PKC-mediated signal transduction pathways. A significant loss of RACK1 has been found in the brain of aging animals and Alzheimer's disease (AD) patients, which implicates the potential involvement of RACK1 in altered PKC activation associated with dementia. Our previous studies have demonstrated that GABAergic inhibition in prefrontal cortex, which is important for cognitive processes like "working memory", is regulated by muscarinic receptors via a PKC-dependent mechanism, and this effect is impaired by β-amyloid peptide (Aβ). In this study, we found that Aβ oligomers decreased RACK1 distribution in the membrane fraction of cortical neurons. Moreover, overexpression of RACK1 rescued the effect of muscarinic receptors on GABAergic transmission in Aβ-treated cortical cultures in vitro and Aβ-injected cortical neurons in vivo. These results suggest that the Aβ-induced loss of RACK1 distribution in the cell membrane may underlie the Aβ impairment of muscarinic regulation of PKC and GABAergic transmission. Thus, RACK1 provides a potential therapeutic target that can restore some of the impaired cellular processes by Aβ.

Aquaporin 4 knockout resists negative regulation of neural cell proliferation by cocaine in mouse hippocampus

Our previous study revealed that aquaporin 4 (AQP4) knockout attenuated locomotor activity in cocaine exposure mice and reduced the extracellular dopamine levels in the nucleus accumbens, suggesting that AQP4 might participate in cocaine addiction. The aim of the present study was to investigate the impact of AQP4 on cell proliferation of dentate gyrus in the mouse hippocampus after repeated cocaine treatment and withdrawal. The immunohistochemistry results showed that repeated cocaine administration significantly decreased cellular proliferation in the subgranular zone, which was followed by a rebound increase after 2-wk withdrawal and a return to normal level after 3-wk withdrawal. AQP4 knockout resisted cocaine-induced reductions of neural cell proliferation. Further studies through immunohistochemistry and immunoblot analysis showed that AQP4 knockout sustained the levels of glial fibrillary acidic protein in the hippocampus, and suppressed the enhancement of extracellular signal-regulated kinase phosphorylation induced by repeated cocaine administration. Notably, AQP4 knockout increased protein kinase C activity examined by substrate protein phosphorylation method, which was not affected by cocaine administration or withdrawal. We also found that repeated cocaine administration could elevate the expression of AQP4 in wild-type mice. In conclusion, it is reported for the first time that AQP4 knockout resisted cocaine-mediated inhibition of neural cell proliferation via up-regulating PKC-mediated signal transduction, suggesting that AQP4 might regulate neurogenesis during drug addiction. Our findings may have helpful implications in the cell biology of neurogenesis.

Deletion of SM-B, the high ATPase isoform of myosin, upregulates the PKC-mediated signal transduction pathway in murine urinary bladder smooth muscle

Detrusor smooth muscle (DSM) hypertrophy induced by partial bladder outlet obstruction (PBOO) is associated with changes in the NH2-terminal myosin heavy chain isoform from predominantly SM-B to SM-A, alteration in the Ca2+ sensitization pathway, and the contractile characteristics from phasic to tonic in rabbits. We utilized the SM-B knockout (KO) mouse to determine whether a shift from SM-B to SM-A without PBOO is associated with changes in the signal transduction pathway mediated via PKC and CPI-17, which keeps the myosin phosphorylation (MLC20) level high by inhibiting the myosin phosphatase. DSM strips from SM-B KO mice generated more force in response to electrical field stimulation, KCl, carbachol, and phorbol 12,13-dibutyrate than that of age-matched wild-type mice. There was no difference in the ED50 for carbachol but the maximum response was greater for the SM-B KO mice. DSM from SM-B KO mice revealed increased mass and hypertrophy. The KO mice also showed an overexpression of PKC-alpha, increased levels of phospho-CPI-17, and an elevated level of IP3 and DAG upon stimulation with carbachol. Two-dimensional gel electrophoresis revealed an increased level of MLC20 phosphorylation in response to carbachol. Together, these changes may be responsible for the higher level of force generation and maintenance by the DSM from the SM-B KO bladders. In conclusion, our data show that ablation of SM-B is associated with alteration of PKC-mediated signal transduction and CPI-17-mediated Ca2+ sensitization pathway that regulate smooth muscle contraction. Interestingly, similar changes are also present in PBOO-induced DSM compensatory response in the rabbit model in which SM-B is downregulated.

Leishmania donovani: Role of CD2 on CD4 + T-cell function in Visceral Leishmaniasis

In this study, we investigated whether alteration in the CD2 mediated coordination of an immune response was associated with down regulation of CD4 associated Th1 cell response during Visceral Leishmaniasis (VL). Leishmania donovani ( Ld) infection in VL patients markedly reduced expression of CD2 cell surface antigen on CD4 + cells. T-cells of VL patients were mostly in G0/G1 stage of the cell cycle (98.20%) with little or no activity of protein kinase C-α (PKC-α) isoform. However, pre-incubation with activating anti-CD2 monoclonal antibody (MAb) resulted in a corresponding increase up to 2.52-fold in T-cells of G2/M population supported by both activity and expression of PKC-α isoform. Furthermore, we observed that co-incubation of T-cell with anti-CD2 increased the lymphocyte-blast population in patients in whom the CD4 cells became more antigen responsive (CD4 + CD69 + cells). Consistent with these observations, it was shown that 59.3% of CD4 cells from patients responded to Ld by producing IFN-γ. Even in the culture condition, when the T-cells from patients were depleted of APC, IFN-γ production was noticed after CD2 activation. On the other hand, IL-4 production became low in the anti-CD2 antibody supplemented peripheral blood mononuclear cells (PBMNCs) culture. These findings imply that infection with L. donovani induces less CD2 on the surface of CD4 + T-cells, which once activated orchestrate the protective IFN-γ dominant host defense mechanism via PKC-mediated signal transduction and cell cycle.

Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCα, PKCβ1, PKCγ, and PKCε are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.

159: Deletion of SM-B, the High Atpase Isoform of Myosin, Upregulates the PKC-Mediated Signal Transduction Pathway in the Urinary Bladder Smooth Muscle

You have accessJournal of UrologyDiscussed Poster, Sunday, May 21, 2006, 9:00 am - 12:00 pm1 Apr 2006159: Deletion of SM-B, the High Atpase Isoform of Myosin, Upregulates the PKC-Mediated Signal Transduction Pathway in the Urinary Bladder Smooth Muscle Joseph A. Hypolite, Shaohua Chang, Edward Labelle, Gopal J. Babu, Muthu Periasamy, Alan J. Wein, and Samuel Chacko Joseph A. HypoliteJoseph A. Hypolite More articles by this author , Shaohua ChangShaohua Chang More articles by this author , Edward LabelleEdward Labelle More articles by this author , Gopal J. BabuGopal J. Babu More articles by this author , Muthu PeriasamyMuthu Periasamy More articles by this author , Alan J. WeinAlan J. Wein More articles by this author , and Samuel ChackoSamuel Chacko More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)32426-1AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "159: Deletion of SM-B, the High Atpase Isoform of Myosin, Upregulates the PKC-Mediated Signal Transduction Pathway in the Urinary Bladder Smooth Muscle." The Journal of Urology, 175(4S), p. 51 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 175Issue 4SApril 2006Page: 51 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Joseph A. Hypolite More articles by this author Shaohua Chang More articles by this author Edward Labelle More articles by this author Gopal J. Babu More articles by this author Muthu Periasamy More articles by this author Alan J. Wein More articles by this author Samuel Chacko More articles by this author Expand All Advertisement PDF downloadLoading ...

Characterization of protein kinase C isoforms in primary cultured cerebellar granule cells

Protein kinase C (PKC) is a family of serine/threonine kinases comprised of 10 isoforms. Although commercial antibodies are available for all 10 isoforms, the specificity of these antibodies has been questioned. We have identified immunoblot conditions in which commercially purchased PKC antibodies are specific for their respective isoform. We then used these conditions to determine that PKC isoforms α, βI, βII, δ, ε, γ, λ, θ, and ζ are present in rat primary cultured cerebellar granule cells (CGCs) 6–14 days in vitro (DIV). This PKC profile is identical to that observed in cerebellar homogenates taken from 6-, 14- and 21-day-old rats. Western blot analysis indicated that the classical and the atypical PKC isoforms were more prevalent in the cytosolic subcellular fraction compared to the particulate fraction under basal conditions. Immunoreactivity for the novel isoforms tended to be higher in the particulate fraction under basal conditions. Phorbol 12-myristate 13-acetate (PMA) treatment resulted in translocated immunoreactivity from the cytosolic to the particulate fraction for all of the classical and novel PKC isoforms, but not for the atypical isoforms. However, the degree of translocation as well as the speed of translocation varied among the isoforms. The stability of the individual isoforms after PMA-induced activation also varied among the isoforms. Differences in these parameters were dependent upon culture batches and PKC isoform groups. We have identified experimental conditions in which reproducible results can be obtained with primary cultured CGCs in the study of PKC. We discuss possible solutions for problems encountered when utilizing primary cultured neurons to study PKC-mediated signal transduction.

Analysis of PKC targeting mechanism using PKC fused with fluorescent proteins

Protein kinase C (PKC) is a family, which consists of at least ten subtypes. To elucidate subtype-specific functions of PKC, we have developed the methods to observe PKC translocation in real time and in the living state using PKC fused with fluorescent proteins, including GFP and DsRed. Based on the live imaging of PKC translocation, we have demonstrated that PKC showed stimulus- and subtype-specific translocation, which can recognize its specific substrate and induce its specific cellular response (PKC targeting). These findings suggest that PKC targeting is the molecular basis underlying the diversity of PKC functions. Live imaging of PKC translocation has been proved to be a beneficial tool for understanding not only PKC functions, but also PKC-mediated signal transduction pathways. We have further analyzed PKC functions in the central nervous system using transgenic mice, which can express PKC-GFP in a brain-region-specific manner.

Mitogen-stimulated TIS21 protein interacts with a protein-kinase-Calpha-binding protein rPICK1.

TIS21 is induced transiently by PMA and a number of extracellular stimuli. Yeast two-hybrid screening has identified three TIS21 interacting clones from a rat cDNA library [Lin, Gary, Yang, Clarke and Herschman (1996) J. Biol. Chem 271, 15034-15044]. The amino acid sequence deduced from clone 5A shows 96.9% identity with the murine PICK1, a protein kinase Calpha (PKCalpha)-binding protein postulated to act as an intracellular receptor for PKC. A fusion protein of glutathione S-transferase and rPICK1 associates with the TIS21 translated in vitro, suggesting a direct physical interaction between these two proteins. TIS21 and rPICK1 are co-immunoprecipitated from NIH 3T3 cells overexpressing these two proteins. This indicates that the interaction also occurs in mammalian cells. Deletion of the PDZ domain at the N-terminus of rPICK1 abolishes its interaction with TIS21. A putative carboxylate-binding loop required for PICK1 to bind PKCalpha [Staudinger, Lu and Olson (1997) J. Biol. Chem 272, 32019-32024] is within this deleted region. Our results suggest a potential competition between TIS21 and PKC for binding to PICK1. We show that recombinant TIS21 is phosphorylated by PKC in vitro. The catalytic activity of PKC towards TIS21 is significantly decreased in the presence of rPICK1, whereas phosphorylation of histone by PKC is not affected. rPICK1 seems to modulate the phosphorylation of TIS21 through specific interactions between these two proteins. TIS21 might have a role in PKC-mediated extracellular signal transduction through its interaction with rPICK1.

Mitogen-stimulated TIS21 protein interacts with a protein-kinase-Cα-binding protein rPICK1

TIS21 is induced transiently by PMA and a number of extracellular stimuli. Yeast two-hybrid screening has identified three TIS21 interacting clones from a rat cDNA library [Lin, Gary, Yang, Clarke and Herschman (1996) J. Biol. Chem 271, 15034–15044]. The amino acid sequence deduced from clone 5A shows 96.9% identity with the murine PICK1, a protein kinase Cα (PKCα)-binding protein postulated to act as an intracellular receptor for PKC. A fusion protein of glutathione S-transferase and rPICK1 associates with the TIS21 translated in vitro, suggesting a direct physical interaction between these two proteins. TIS21 and rPICK1 are co-immunoprecipitated from NIH 3T3 cells overexpressing these two proteins. This indicates that the interaction also occurs in mammalian cells. Deletion of the PDZ domain at the N-terminus of rPICK1 abolishes its interaction with TIS21. A putative carboxylate-binding loop required for PICK1 to bind PKCα [Staudinger, Lu and Olson (1997) J. Biol. Chem 272, 32019–32024] is within this deleted region. Our results suggest a potential competition between TIS21 and PKC for binding to PICK1. We show that recombinant TIS21 is phosphorylated by PKC in vitro. The catalytic activity of PKC towards TIS21 is significantly decreased in the presence of rPICK1, whereas phosphorylation of histone by PKC is not affected. rPICK1 seems to modulate the phosphorylation of TIS21 through specific interactions between these two proteins. TIS21 might have a role in PKC-mediated extracellular signal transduction through its interaction with rPICK1.

Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events.

Leishmania donovani, an intracellular protozoan parasite, challenges host defense mechanisms by impairing the signal transduction of macrophages. In this study we investigated whether interleukin-10 (IL-10)-mediated alteration of signaling events in a murine model of visceral leishmaniasis is associated with macrophage deactivation. Primary in vitro cultures of macrophages infected with leishmanial parasites markedly elevated the endogenous release of IL-10. Treatment with either L. donovani or recombinant IL-10 (rIL-10) inhibited both the activity and expression of the Ca2+-dependent protein kinase C (PKC) isoform. However, preincubation with neutralizing anti-IL-10 monoclonal antibody (MAb) restored the PKC activity in the parasitized macrophage. Furthermore, we observed that coincubation of macrophages with rIL-10 and L. donovani increased the intracellular parasite burden, which was abrogated by anti-IL-10 MAb. Consistent with these observations, generation of superoxide (O2-) and nitric oxide and the release of murine tumor necrosis factor-alpha were attenuated in response to L. donovani or rIL-10 treatment. On the other hand, preincubation of the infected macrophages with neutralizing anti-IL-10 MAb significantly blocked the inhibition of nitric oxide and murine tumor necrosis factor-alpha release by the infected macrophages. These findings imply that infection with L. donovani induces endogenous secretion of murine IL-10, which in turn facilitates the intracellular survival of the protozoan and orchestrates several immunomodulatory roles via selective impairment of PKC-mediated signal transduction.

Effect of protein kinase C inhibitor (PKCI) on radiation sensitivity and c- fos transcription

Purpose: The human genetic disorder ataxia-telangiectasia (AT) is a multisystem disease characterized by extreme radiosensitivity. Although ionizing radiation was known to induce c- fos transcription and cellular protein kinase C (PKC) induces the expression of this immediate response gene, little is known about how mutated AT (ATM) or PKC-mediated signal transduction pathway modulates the c-fos gene transcription and gene expression. Here we have studied the effect of PKC inhibitor (PKCI) on radiation sensitivity and c-fos transcription in normal and AT cells, and also studied whether PKCI effect on c- fos occurs in Ras-dependent pathway. Methods and Materials: Normal (LM217) and AT (AT5BIVA) cells were transfected with PKCI expression plasmid and integration and overexpression of PKCI was evaluated by polymerase chain reaction and northern blotting, respectively. Cells were irradiated at a dose of 5 Gy/min with 137Cs irradiator and harvested 48 h after irradiation and investigated apoptosis with TUNEL method. The c- fos transcription activity was studied by performing compute assisted tomography (CAT) assay of reporter gene after transfection of c- fos CAT plasmid into LM and AT cells. Overexpression of Ras protein in transfected cells was shown by western blotting. Results: Our results demonstrated for the first time a role of PKCI on the radiation sensitivity and c-fos transcription in LM and AT cells. PKCI increased radiation induced apoptosis in LM cells (5% to 20%) but reduced apoptosis slightly in AT cells. The basal c-fos transcription activity is 70 times lower in AT cells than in LM cells. This c-fos transcription activity was repressed by overexpression of PKCI in LM cells but not in AT cells. After induction of c-fos by Ras protein, overexpression of PKCI repressed c- fos transcription in LM cells but not in AT cells. Conclusions: Overexpression of PKCI increased radiation sensitivity and repressed c- fos transcription in LM cells but not in AT cells, and this is related with Ras. These results suggest that the effect of PKCI on c-fos transcription activity is related with Ras dependent signal transduction pathways and these mechanisms are different between normal fibroblasts, LM and ATM mutated, AT cells. The data obtained by this study provided evidence for novel transcriptional difference between LM and AT cells and this may be a reason for increased radiation sensitivity of AT cells.

Site-Specific Localization of Protein Kinase C Isoforms in Rat Pancreas

Background: Protein kinase C (PKC), a major signal-transducing enzyme, is recognized to play an important role in the regulation of pancreatic exocrine and endocrine secretion, and yet the distribution of PKC isoforms in rat pancreas has remained unclarified. Aim of the Study: We examined the precise localization of PKC isoforms to elucidate the role of PKC in the normal rat pancreas. Methods: Male Sprague-Dawley rats were used throughout the experiment. For Western blot analysis, the islet of Langerhans and the acinar tissue were separated by the collagenase digestion method. Also, the whole pancreas was taken out and immunohistochemistry performed. Results: According to Western blot analysis, PKC-α, -γ, -δ, -Ε, -ς, and -λ were detected in both acinar and islet cells while PKC-βII were observed exclusively in the islet. PKC-βI was not observed. On immunohistochemistry, the immunoreactivities of PKC isoforms were observed as follows: PKC-α, weakly in some endocrine cells and ductal epithelium; PKC-βII, mainly in the islet center; PKC-γ, in the islet, intrapancreatic ganglia and ductal epithelium; PKC-δ, in the islet periphery, weakly in some acinar cells and ductal epithelium; PKC-Ε, strongly in the islet, acinar cell and ductal epithelium; PKC-ς, in the islet, acinar cell and ductal epithelium; PKC-λ in some endocrine cells and ductal epithelium. Conclusion: These results suggest that the intrapancreatic site-specific existence of PKC isoforms may regulate pancreatic exocrine and endocrine functions via a PKC-mediated signal transduction.

Protein Kinase C Recognizes the Protein Kinase A-binding Motif of Nonstructural Protein 3 of Hepatitis C Virus

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) inhibits the nuclear transport and the enzymatic activity of the catalytic subunit of protein kinase A. This inhibition is mediated by an arginine-rich domain localized between amino acids 1487-1500 of the HCV polyprotein. The data presented here indicate that the arginine-rich domain, when embedded in recombinant fragments of NS3, interacts with the catalytic site of protein kinase C (PKC) and inhibits the phosphorylation mediated by this enzyme in vitro and in vivo. Furthermore, a direct binding of PKC to the NS3 fragments leads to an inhibition of the free shuttling of the kinase between the cytoplasm and the particulate fraction. In contrast, a peptide corresponding to the arginine-rich domain (HCV (1487-1500)), despite also being a PKC inhibitor, did not influence the PKC shuttling process and was transported to the particulate fraction by the translocating kinase upon activation with tetradecanoylphorbol-13-acetate. Using the tetradecanoylphorbol-13-acetate -stimulated respiratory burst of NS3-introduced neutrophils as a model system, we could demonstrate that NS3 is able to block PKC-mediated functions within intact cells. Our data support the possibility that NS3 disrupts the PKC-mediated signal transduction.

Demonstration of selective protein kinase C-dependent activation of Src and Lck tyrosine kinases during ischemic preconditioning in conscious rabbits.

Src tyrosine kinases have been shown to mediate cellular responses to stress in noncardiac cells. However, the effect of myocardial ischemia on Src tyrosine kinases is unknown. Furthermore, the identity of the tyrosine kinase(s) involved in the genesis of ischemic preconditioning (PC) remains obscure. Here, we present the first evidence that ischemic PC (6 cycles of 4-minute coronary occlusion and 4-minute reperfusion) induces selective activation of 2 members of the Src family of tyrosine kinases, Src and Lck, in the heart of conscious rabbits. The activation of Src in the particulate fraction was not evident at 5 minutes after ischemic PC but became apparent at 30 minutes (+119% versus control), whereas the activation of Lck in the particulate fraction was apparent both at 5 minutes (+103% versus control) and at 30 minutes (+89%) after ischemic PC. The activity of the other 5 members of the Src tyrosine kinases expressed in the rabbit heart (Fyn, Fgr, Yes, Lyn, and Blk) was not affected by ischemic PC. Ischemic PC had no effect on the activity of epidermal growth factor receptor kinases, either at 5 or at 30 minutes. The activation of Src and Lck was completely abrogated by the tyrosine kinase inhibitor lavendustin A, given at doses that have previously been shown to block the protective effect of ischemic PC in this same conscious rabbit model, suggesting that Src and Lck kinases are essential for the development of ischemic PC. The activity of the epsilon isoform of protein kinase C (PKC) in the particulate fraction increased at 5 minutes (+72%) and at 30 minutes (+67%) after ischemic PC. Pretreatment with lavendustin A had no effect on the activation of PKCepsilon, whereas pretreatment with the PKC inhibitor chelerythrine (given at doses that have previously been shown to block ischemic PC) blocked not only the activation of PKCepsilon but also that of Src and Lck, indicating that Src and Lck are downstream of PKCepsilon in the signaling cascade of ischemic PC. This study identifies a new component of the signaling mechanism of ischemic PC. The results support the concept that, in conscious rabbits, 2 specific members of the Src family of tyrosine kinases, Src and Lck, play an important role in the genesis of late PC by serving as downstream elements of PKC-mediated signal transduction.