Protein Kinase C Kinase Activity Research Articles

Abstract P2-24-03: Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-03.

6,6'-Dihydroxythiobinupharidine (DTBN) Purified from Nuphar lutea Leaves Is an Inhibitor of Protein Kinase C Catalytic Activity.

Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6′-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer.

Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis

AbstractWe previously reported the discovery of a novel lipid deacetylase in platelets, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), and that its inhibition impairs agonist-induced platelet aggregation, Rap1 GTP loading, protein kinase C (PKC) activation, and ex vivo thrombus growth. However, precise mechanisms by which AADACL1 impacts platelet signaling and function in vivo are currently unknown. Here, we demonstrate that AADACL1 regulates the accumulation of ether lipids that impact PKC signaling networks crucial for platelet activation in vitro and in vivo. Human platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5′-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12−/− and CalDAG GEFI−/− mice revealed that the P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed weak agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 distinct PKC isoforms and blocked PKC kinase activity in vitro. Finally, AADACL1 inhibition in rats reduced platelet aggregation, protected against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP accumulation that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage.

Metformin regulates atrial SK2 and SK3 expression through inhibiting the PKC/ERK signaling pathway in type 2 diabetic rats

BackgroundOur previous study showed that metformin regulates the mRNA and protein levels of type 2 small conductance calcium-activated potassium channel (SK2) and type 3 small conductance calcium-activated potassium channels (SK3) in atrial tissue as well as the ion current of atrial myocytes in rats with type 2 diabetes mellitus (T2DM), but the underlying signaling mechanism is unknown. This study aimed to investigate whether metformin regulates atrial SK2 and SK3 protein expression in T2DM rats though the protein kinase C (PKC)/extracellular signal-regulated kinase (ERK) signaling pathway.MethodsA T2DM rat model was established using a high-fat and high-sugar diet combined with a low-dose intraperitoneal injection of streptozotocin (STZ). The rats were randomly divided into the following five groups: the control group, the untreated T2DM group, the metformin-treated only group, the phorbol 12-myristate 13-acetate (PMA; a PKC agonist administered by intraperitoneal injection) treatment group, and the recombinant human epidermal growth factor (rh-EGF; an ERK agonist administered by tail vein injection) treatment group. The activity of PKC in atrial tissues was assayed by a PKC kinase activity assay kit. The protein expression of SK2, SK3, and phosphorylated ERK (pERK) were determined by western blotting and immunohistochemistry.ResultsCompared with the Control group, atrial PKC activity and pERK and SK3 protein expression were increased, while SK2 protein expression was decreased in atrial tissues of T2DM rats. Eight weeks of metformin treatment inhibited the PKC activity and pERK and SK3 expression, and elevated SK2 expression compared with the T2DM group. Compared with the metformin-treated only group, the injection of rh-EGF increased pERK and SK3 expression, and decreased SK2 expression; the injection of PMA increased PKC activity and SK3 expression, and decreased SK2 expression. In addition, the injection with PMA significantly elevated the expression of pERK.ConclusionsThe PKC/ERK signaling pathway is involved in the downregulation of SK2 expression and the upregulation of SK3 expression in the atrium of T2DM rats. Long-term metformin treatment prevents the SK2 downregulation and the SK3 upregulation through inhibiting the PKC/ERK signaling pathway.

Increased PKCα activity by Rack1 overexpression is responsible for chemotherapy resistance in T-cell acute lymphoblastic leukemia-derived cell line.

Chemoresistant mechanisms in T-cell acute lymphoblastic leukemia (T-ALL) patients are not clarified. The apoptotic signaling mediated by receptor of activated C kinase 1 (Rack1), protein kinase C (PKC) and FEM1 homolog b (FEM1b) was investigated in two T-ALL-derived cell lines (Jurkat and CCRF-CEM) following treatment with chemotherapy drugs vincristine and prednisone. Serum starvation or chemotherapeutic drugs significantly reduced Rack1 level and PKC activation, while promoted cellular apoptosis in both cell lines. Rack1 overexpression protected T-ALL cell against starvation or chemotherapeutic drug-induced apoptosis. Moreover, Rack1 overexpression reduced the level of cytochrome c and active caspase 3 as well as FEM1b and apoptotic protease activating factor-1 (Apaf-1), and inhibited induction of cellular apoptosis in chemotherapeutic drug-treated Jurkat cell. Interaction of Rack1 and PKCα, not PKCβ, was detected in both cell lines. Of note, Rack1 overexpression abrogated reduction of PKC kinase activity in chemotherapeutic drug-treated T-ALL cell. PKC kinase inhibitor Go6976 or siPKCα inhibited downregulation of FEM1b and/or Apaf-1, and thus increased cellular apoptosis in Rack1-overexpressed T-ALL cell receiving chemotherapeutic drugs. Accordingly, our data provided evidence that increased Rack1-mediated upregulation of PKC kinase activity may be responsible for the development of chemoresistance in T-ALL-derived cell line potentially by reducing FEM1b and Apaf-1 level.

Inhibition of superoxide anion generation by CHS-111 via blockade of the p21-activated kinase, protein kinase B/Akt and protein kinase C signaling pathways in rat neutrophils

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O 2 − ) generation, which was not mediated by scavenging the generated O 2 − or by a cytotoxic effect, and attenuated migration. CHS-111 had no effect on the arachidonic acid-induced NADPH oxidase activation or the GTPγS-stimulated Rac2 membrane translocation in cell-free systems, whereas it effectively attenuated the membrane recruitment of p40 phox, p47 phox and p67 phox, phosphorylation of Ser residues in p47 phox, association between p47 phox and p22 phox, and Rac activation in fMLP-stimulated neutrophils. Moreover, the phosphorylation and membrane recruitment of p21-activated kinase (PAK), PAK kinase activity and the interaction of PAK with p47 phox were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47 phox, moderately inhibited the membrane recruitment of Akt and phospho-PDK1, and slightly attenuated Akt (Thr308) phosphorylation, whereas it had no effect on Akt (Ser473) phosphorylation or p110γ membrane translocation. The membrane recruitment of protein kinase C (PKC)-α, -βI, -βII, -δ and -ζ, PKC phosphorylation and PKC kinase activity was attenuated by CHS-111, whereas CHS-111 did not affect the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or downstream MAPK-activated protein kinase-2. Higher concentrations of CHS-111 were required to decrease fMLP-stimulated intracellular free Ca 2+ concentration ([Ca 2+] i) elevation in the presence but not in the absence of extracellular Ca 2+, and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O 2 − generation in rat neutrophils through the blockade of PAK, Akt and PKC signaling pathways.

Novel Chimeric Peptide Inhibits Protein Kinase C and Induces Apoptosis in Human Immune Cells

Protein kinase C (PKC) participates in a myriad of cellular processes. Protein kinase C isoforms play different roles based on their cellular expression balance and activation. The activity of classical PKC isoforms has been shown to be crucial for immune cell population homeostasis, playing a positive role in survival and proliferation. Protein kinase C inhibitors have been used for conditions where up-regulated PKC results in a pathological state. The most commonly investigated PKC inhibitors are highly effective in inhibiting PKC function but they are relatively unspecific, some of them even inhibiting other kinase families. Protein kinase C pseudosubstrates are auto-inhibitory domains which have been used to inhibit more specifically PKC in vitro but they do not freely penetrate cells. This could be resolved by using cell-permeable PKC pseudosubstrates which would more accurately modulate cellular PKC activity and PKC-related functions in intact cells. Here we show the development of a chimeric peptide inhibitor of classical PKC isoforms, consisting of a cell permeable sequence and a pseudosubstrate sequence which was able to translocate into cells, inhibiting PKC kinase activity and PKC T-cell-specific substrate phosphorylation. We also demonstrate a dramatic reduction in T-cell proliferation at high chimeric peptide concentration; this was attributed to apoptosis induction, as demonstrated by cell shrinking, phosphatidylserine exposure and DNA fragmentation. As expected, the control peptide (pseudosubstrate) did not penetrate cells, affect cell proliferation or survival. We also show that a neoplastic T-cell line which expresses higher levels of PKC is more resistant to chimeric peptide-mediated cell death than normal cells, corroborating a PKC role in apoptosis resistance. This chimeric peptide could be useful for the specific modulation of the PKC signalling pathway in pathological conditions.

Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors

ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCalpha, PKCbetaII, PKCdelta and PKCepsilon, but not atypical PKCzeta, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCalpha-GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.

Target lethal effect of recombinant soluble Fas coupled with protein kinase C inhibitor on colorectal carcinoma cells

To study the target killing effect of soluble Fas(sFas) coupled with protein kinase C(PKC) inhibitor on colorectal carcinoma cells. The extracellular region of Fas protein was cloned and amplified by RT-PCR, and the expressing vector pGEX-4T-1-sFas was constructed. The sFas protein was purified by GST fusion protein purification system and coupled with Calphostin C(one kind of PKC inhibitor). The killing effect of soluble Fas coupled with PKC inhibitor on FasL-positive colorectal carcinoma cells was detected. After amplifying and cloning, the extracellular region of Fas protein, a 571 bp fragment, was proved by limited enzyme cutting and DNA sequencing. The expressed and purified protein was identified by Western Blot after transformed into E. coli BL21. The coupled sFas-Calphostin C showed suppressant activity on PKC kinase by the PKC kinase activity assay kit. The growth suppression rate of FasL-positive colorectal carcinoma HR-8348 cells treated with sFas-Calphostin C was significantly higher than that of FasL-negative cells, but the killing effect of sFas-Calphostin C on normal human monocyte was not obvious. Compared with 5-Fu alone, the growth suppression rate of FasL-positive colorectal carcinoma HR-8348 cells was significantly raised by sFas-Calphostin C combined with 5-Fu. The recombinant of soluble Fas and PKC inhibitor shows target killing effect on colorectal carcinoma cells.

Metformin reduces angiotensin-mediated intracellular production of reactive oxygen species in endothelial cells through the inhibition of protein kinase C

Oxidative stress plays a major role in the pathogenesis and in the onset of macrovascular complications of diabetes. We previously reported that the antihyperglycaemic drug metformin was able to decrease significantly intracellular reactive oxygen species (ROS) production of bovine aortic endothelial cells (BAEC) activated by high levels of glucose and angiotensin II (ANG). The aim of the present study was to investigate whether the antioxidant effect of metformin on BAEC could be mediated through a modulation of protein kinase C (PKC) activity, which plays a key role in the pathophysiology of diabetes. The effects of metformin on intracellular ROS production, PKC translocation and activity were studied on endothelial cells stimulated by PMA (a direct PKC activator), ANG or high levels of glucose as pathophysiological stimuli of endothelial dysfunction in diabetes. We showed that metformin decreased ROS production on PMA-, ANG- and glucose-stimulated BAEC in a similar manner to that obtained by PKC specific inhibitors (calphostin C, chelerythrine) alone. On the other hand, metformin reduced both PKC membrane translocation and kinase activity in ANG-stimulated cells. In PMA-activated cells, metformin reduced membrane PKC activity but we did not observe any alteration of PKC membrane translocation. Finally, in vitro incubation with purified PKC indicated that metformin had no direct effect on PKC activity. Taken together, our results suggest that metformin exerted intracellular antioxidant properties by decreasing ROS production through the inhibition of PKC activity.

Heterogeneity of Cellular Proliferation within Transitional Cell Carcinoma: Correlation of Protein Kinase C Alpha/betal Expression and Activity

A total of 18 histological samples containing both transitional cell carcinoma (TCC) and normal urothelial epithelium were analyzed for protein kinase C (PKC)-alpha and -betaI expression, and for their phosphorylated substrates. The results showed an increased expression of PKC-alpha in 13 out of 18 samples and -betaI in 11 out of 18 TCC samples when compared with normal urothelium. In addition, 11 out of 18 of the TCC tumors displayed heterogeneous expression of the PKC isoenzymes, with different levels of immunosignal in different areas of the tumor. Within the same sample, areas of highest PKC isoenzyme expression also showed highest classical PKC activity, as estimated by immunodetection of phosphorylated forms of PKC substrates. The areas of highest expression of PKC-alpha and/or -betaI isoenzymes showed also the highest number of cells positive for Ki67, an indicator of proliferation. Immunofluorescence and Western blotting demonstrated that in cultured TCC cells, PKC-alpha was located in the cytoplasm, whereas PKC-betaI was located primarily in the nucleus as a 65-kDa fragment and in the cytoplasm as a full-size 79-kDa protein. Our results indicate that increased expression of PKC-alpha and -betaI leads to increased total classical PKC kinase activity and suggest that increased activity of the isoenzymes plays a role in accelerated growth of TCC. Furthermore, these results suggest that even in carcinoma tissue, PKC expression and activity are under strict control.

Dexamethasone-Induced Ras Protein 1 Negatively Regulates Protein Kinase C δ: Implications for Adenylyl Cyclase 2 Signaling

We identified dexamethasone-induced Ras protein 1 (Dexras1) as a negative regulator of protein kinase C (PKC) delta, and the consequences of this regulation have been examined for adenylyl cyclase (EC 4.6.1.1) type 2 (AC2) signaling. Dexras1 expression in human embryonic kidney 293 cells completely abolished dopamine D2 receptor-mediated potentiation of AC2 activity, which is consistent with previous reports of its ability to block receptor-mediated Gbetagamma signaling pathways. In addition, Dexras1 significantly reduced phorbol 12-myristate 13-acetate (PMA)-stimulated AC2 activity but did not alter Galpha(s)-mediated cAMP accumulation. Dexras1 seemed to inhibit PMA stimulation of AC2 by interfering with PKCdelta autophosphorylation. This effect was selective for the delta isoform because Dexras1 did not alter autophosphorylation of PKCalpha or PKCepsilon. Dexras1 disruption of PKCdelta autophosphorylation resulted in a significant blockade of PKC kinase activity as measured by [gamma-32P]ATP incorporation using a PKC-specific substrate. Moreover, Dexras1 and PKCdelta coimmunoprecipitated from whole-cell lysates. Dexras1 did not alter the membrane translocation of PKCdelta; however, the ability of Dexras1 to interfere with PKCdelta autophosphorylation was isoprenylation-dependent as determined using the farnesyltransferase inhibitor methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate (FTI-277) and a CAAX box-deficient Dexras1 (C277S) mutant. PMA-stimulated AC2 activity was also not affected by Dexras1 C277S. Taken as a whole, these data suggest that Dexras1 functionally interacts with PKCdelta at the cellular membrane through an isoprenylation-dependent mechanism to negatively regulate PKCdelta activity. Moreover our study suggests that Dexras1 acts to modulate the activation of AC2 in an indirect fashion by inhibiting both Gbetagamma- and PKC-stimulated AC2 activity. The current study provides a novel role for Dexras1 in signal transduction.

A Novel Diacylglycerol-lactone Shows Marked Selectivity in Vitro among C1 Domains of Protein Kinase C (PKC) Isoforms α and δ as Well as Selectivity for RasGRP Compared with PKCα

Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms alpha, beta, gamma, delta, and epsilon, with apparent Ki values ranging from 340 nm for PKCalpha to 29 nm for PKCepsilon. When binding to isolated C1 domains of PKCalpha and -delta, 130C037 showed good affinity (Ki= 1.78 nm) only for deltaC1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of deltaC1b. 130C037 bound intact RasGRP1 and RasGRP3 with Ki values of 3.5 and 3.8 nm, respectively, reflecting 8- and 90-fold selectivity relative to PKCepsilon and PKCalpha. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED50 = 286 nm), partial reduction of PKCepsilon (ED50 > 10 microm), and no effect on PKCalpha. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKCepsilon, and no translocation of PKCalpha. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets.

Protein kinase C δ is activated in mouse ovarian surface epithelial cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Interactions between the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and protein kinase C (PKC) signaling pathways are governed in cell and tissue-specific manners, albeit the physiological significance of which is unclear. This research sought to define the effects of TCDD on the PKC pathway using a mouse ovarian surface epithelial cancer cell line (ID8). Phorbol-12-myristate-13-acetate (PMA) potentiated (1 nM) TCDD-induced 7-ethoxyresorufin-O-deethylase (EROD) activity after 24h of treatment, and pre-treatment with (1 microM) of either a general PKC inhibitor (BisI) or PKCdelta-specific inhibitor (Rotterlin) abolished the potentiation indicating that activation of PKC enhances TCDD signal transduction. Western blot analysis revealed that unstimulated ID8 cells express PKCalpha, beta, epsilon, tau, lambda and RACK1. PKCgamma, eta, theta and DGKtheta were not detected. TCDD (1 nM) increased PKCdelta protein approximately eight-fold after 24h of treatment and this effect was dose-dependent (0.1-100 nM); other PKC isoforms and related signaling proteins tested were unaffected by TCDD treatment. Immunofluorescent microscopy revealed that TCDD (1 nM) promoted the subcellular redistribution of PKCdelta, from the cytoplasm and the nucleus to the perinuclear area after 2h of treatment, however, after 24h of treatment PKCdelta was observed in nuclear structures that resembled nucleoli. TCDD (1 nM) also increased total PKC and PKCdelta-specific kinase activities in biphasic time-responsive manners. Total PKC and PKCdelta-specific activities increased after 1-2h of treatment. Then TCDD increased the total PKC activity again after 12h of treatment, whereas, PKCdelta-specific activity resurged at 24h and remained elevated at 48 h after treatment. The results indicate that TCDD preferentially induces PKCdelta protein expression and phosphotransferase activity, and its membrane translocation, indicating a potential intracellular role for PKCdelta as an effector molecule for TCDD-mediated biological events in this ovarian cancer cell line.

Physical and Functional Interaction between Protein Kinase C δ and Fyn Tyrosine Kinase in Human Platelets

An increasing number of tyrosine kinases have been shown to associate with isoforms of the protein kinase C (PKC) family. Here, we show evidence for physical and functional interaction between PKCdelta and the Src family kinase Fyn in human platelets activated by alboaggregin-A, a snake venom capable of activating both GPIb-V-IX and GPVI adhesion receptors. This interaction involves phosphorylation of PKCdelta on tyrosine and is specific in that other isoforms of PKC, PKCepsilon and lambda, which also become tyrosine-phosphorylated, do not interact with Fyn. In addition, PKCdelta does not interact with other platelet-expressed tyrosine kinases Syk, Src, or Btk. Stimulation also leads to activation of both Fyn and PKCdelta and to serine phosphorylation of Fyn within a PKC consensus sequence. Alboaggregin-A-dependent activation of Fyn is blocked by bisindolylmaleimide I, suggesting a role for PKC isoforms in regulating Fyn activity. Platelet activation with alboaggregin-A induces translocation of the two kinases from cytoplasm to the plasma membrane of platelets, as observed by confocal immunofluorescence microscopy. Translocation of Fyn and PKCdelta are blocked by PP1 and bisindolylmaleimide I, showing a dependence upon Src and PKC kinase activities. Although PKC activity is required for translocation, it is not required for association between the two kinases, because this was not blocked by bisindolylmaleimide I. Rottlerin, which inhibited PKCdelta activity, did not block translocation of either PKCdelta or Fyn but potentiated platelet aggregation, 5-hydroxytryptamine secretion, and the calcium response induced by alboaggregin-A, indicating that this kinase plays a negative role in the control of these processes.

Fibroblast Growth Factor-2-induced Signaling through Lipid Raft-associated Fibroblast Growth Factor Receptor Substrate 2 (FRS2)

The plasma membrane is not homogeneous but contains specific subcompartments characterized by their unique lipid and protein composition. Based on their enrichment in various signaling molecules, these membrane microdomains are recognized to be sites of localized signal transduction for a number of extracellular stimuli. We have previously shown that fibroblast growth factor-2 (FGF2) induced a specific signaling response within a lipid raft membrane microdomain in human neuroblastoma cells characterized by the tyrosine phosphorylation of a p80 phosphoprotein. Herein, we show that this protein is the signaling adaptor FRS2 and that it is localized exclusively to lipid rafts in vitro and in vivo. We have examined how the tyrosine phosphorylation and serine-threonine phosphorylation of FRS2 within lipid rafts affect the response of cells to FGF2 signaling. Our data suggest that activation of protein kinase C, Src family kinases, and MEK1/2 are involved in regulating serine-threonine phosphorylation of FRS2, which can indirectly affect FRS2 phosphotyrosine levels. We also show that Grb2 is recruited to lipid rafts during signaling events and that activation of MEK1/2 by different mechanisms within lipid rafts may lead to different cellular responses. This work suggests that compartmentalized signaling within lipid rafts may provide a level of specificity for growth factor signaling.

Upregulation of PKC genes and isozymes in cardiovascular tissues during early stages of experimental diabetes.

The protein kinase C (PKC) pathway has recently been recognized as an important mechanism in the development of diabetic complications including cardiomyopathy and angiopathy. Although an increase in PKC kinase activity has been detected in the cardiovascular system of diabetic patients and animals, it is unclear whether the same pathological condition alters PKC at the transcriptional and translational levels. In this study we assessed quantitatively the mRNA and protein expression profiles of PKC isozymes in the heart and vascular tissues from streptozotocin-induced diabetic pigs. Partial regions of the porcine PKCalpha, beta1, and beta2 mRNAs were sequenced, and real-time RT-PCR assays were developed for PKC mRNA quantification. The results showed a significant increase in the mRNA levels of PKCalpha, beta1, and beta2 in the heart at 4-8 wk of diabetes. In concomitance, the PKCbeta1 and beta2 genes, but not the PKCalpha gene, were upregulated in the diabetic aorta. Correspondingly, there was a significant increase in the protein expression of PKCalpha and beta2 in the heart and PKCbeta2 in the aorta with a time course correlated to that of mRNA expression. In summary, PKCbeta2 was significantly upregulated in the heart and aorta at both the transcriptional and translational levels during early stages of experimental diabetes, suggesting that PKCbeta2 may be a prominent target of diabetic injury in the cardiovascular system.

Phenylarsine oxide and H2O2 plus vanadate induce reverse translocation of phorbol-ester-activated PKCbetaII.

The intracellular localization of protein kinase C (PKC) is important for the regulation of its biological activity. Recently, it was reported that, whereas phorbol esters such as PMA induce prolonged translocation of PKC to the plasma membrane, with physiological stimuli, the translocation of PKC is transient and followed by rapid return to the cytoplasm. In addition, this membrane dissociation of PKC was shown to require both the kinase activity of PKC and the phosphorylation of its carboxyl terminus autophosphorylation sites. However, the detailed molecular mechanism of PKC reverse translocation remains obscure. We demonstrated that in porcine polymorphonuclear leucocytes (PMNs), phenylarsine oxide (PAO), a putative protein tyrosine phosphatase (PTPase) inhibitor, induced reverse translocation of PMA-stimulated PKCbetaII. Hydrogen peroxide (H(2)O(2)) in combination with vanadate, both of which are PTPase inhibitors, also induced reverse translocation of PKCbetaII. H(2)O(2) or vanadate alone had little effect on PMA-induced PKCbetaII translocation. Furthermore, genistein and ethanol, which are inhibitors of tyrosine kinase and phospholipase D, respectively, prevented the PKCbetaII reverse translocation induced by the PTPase inhibitors. These results indicate, for the first time, that the tyrosine phosphorylation/phospholipase D pathway may be involved in the process of membrane dissociation of PKC.

Regulation of α 1-adrenoceptor-linked phosphoinositide metabolism in cultured glia: involvement of protein phosphatases and kinases

Noradrenaline-stimulated phosphoinositide breakdown in cultured glia was found to be mediated by α 1A-adrenoceptors. The α 1A-selective agonist A61603 was as effective as noradrenaline in eliciting 3H-inositol phosphate (IP) accumulation but was approximately 50-fold more potent. In addition, the use of selective antagonists revealed a clear rank order of potency in the ability of these drugs to reverse the effect of noradrenaline on phosphoinositide breakdown: RS17053 (α 1A-selective)≫AH11110A (α 1B-selective)>BMY7378 (α 1D-selective). Pre-treatment of cultured glia with the protein phosphatase inhibitor okadaic acid resulted in a concentration- and time-dependent reduction in noradrenaline-evoked 3H-IP accumulation. This effect was mimicked by, but was not additive with, a phorbol ester, was reversed by protein kinase C (PKC) inhibitors and was not evident in cells which had been PKC depleted. The ability of cell extracts to dephosphorylate radiolabelled glycogen phosphorylase revealed the presence of the phosphatases PP1 and PP2A in almost equal abundance. Okadaic acid pre-treatment of intact cultures elicited a marked reduction in total phosphatase activity, particularly that mediated by PP2A. We also determined the effect of okadaic acid pre-treatment on PKC and cyclic AMP-dependent protein kinase (PKA) activities in these cells. PKC and PKA activities in cell extracts were assessed by determining the incorporation of 32P into histone and kemptide, respectively. Okadaic acid elicited increases in both Ca 2+-dependent and Ca 2+-independent PKC activity; in addition, increases in both initial and total PKA activities were also recorded. The effect of okadaic acid on noradrenaline-stimulated 3H-IP accumulation were not, however, mimicked by either forskolin or 8-bromo-cyclic AMP, suggesting that this event is not regulated by PKA. Our data point to roles for both PKC and PP2A in the regulation of α 1A-adrenoceptor-linked phosphoinositide metabolism in cultured cortical glia.

Protein Kinase Cμ Regulation of the JNK Pathway Is Triggered via Phosphoinositide-dependent Kinase 1 and Protein Kinase Cε

The protein kinase C (PKC)-related enzyme PKC(mu)/PKD (protein kinase D) is activated by activation loop phosphorylation through PKC(eta). Here we demonstrate that PKC(mu) is activated by the direct phosphorylation of PKC(epsilon). PKC(mu) colocalizes with PKC(epsilon) in HEK293 and MCF7 cells as shown by confocal immunofluorescence analyses. PDK1, known as the upstream kinase for several PKC isozymes, associates intracellularly with PKC(epsilon) and PKC(eta). PKC(eta) is phosphorylated by PDK1 in vitro, leading to kinase activation as similarly reported for PKC(epsilon) activation by PDK1. Coexpression of PDK1, PKC(epsilon) and PKC(mu) in HEK293 cells results in PKC(mu) activation. In contrast, the coexpression of PDK1 and PKC(eta) with PKC(mu) does not activate PKC(eta) or consequently PKC(mu). PDK1/PKC(epsilon)-triggered activation of PKC(mu) inhibits JNK, a downstream effector of PKC(mu), whereas upon transient expression of PDK1, PKC(eta), and PKC(mu), JNK is not affected. These data implicate PKC(epsilon) as the biologically important upstream kinase for PKC(mu) in HEK293 cells, regulating downstream effectors. Our results further indicate a PDK1/PKC(eta)/PKC(mu) controlled negative regulation of PKC(eta) kinase activity. In this study, we show that differentially activated kinase cascades involving PDK1 and novel PKC isotypes are responsible for the regulation of PKC(mu) activity and consequently inhibit the JNK pathway.