PKCζ Activation Research Articles

Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation.

Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ.

IRS-1 Functions as a Molecular Scaffold to Coordinate IGF-I/IGFBP-2 Signaling During Osteoblast Differentiation.

Insulin like growth factor I (IGF-I) and insulin like growth factor binding protein-2 (IGFBP-2) function coordinately to stimulate AKT and osteoblast differentiation. IGFBP-2 binding to receptor protein tyrosine phosphatase β (RPTPβ) stimulates polymerization and inactivation of phosphatase activity. Because phosphatase and tensin homolog (PTEN) is the primary target of RPTPβ, this leads to enhanced PTEN tyrosine phosphorylation and inactivation. However RPTPβ inactivation also requires IGF-I receptor activation. The current studies were undertaken to determine the mechanism by which IGF-I mediates changes in RPTPβ function in osteoblasts. IGFBP-2/IGF-I stimulated vimentin binding to RPTPβ and this was required for RPTPβ polymerization. Vimentin serine phosphorylation mediated its binding to RPTPβ and PKCζ was identified as the kinase that phosphorylated vimentin. To determine the mechanism underlying IGF-I stimulation of PKCζ-mediated vimentin phosphorylation, we focused on insulin receptor substrate-1 (IRS-1). IGF-I stimulated IRS-1 phosphorylation and recruitment of PKCζ and vimentin to phospho-IRS-1. IRS-1 immunoprecipitates containing PKCζ and vimentin were used to confirm that activated PKCζ directly phosphorylated vimentin. PKCζ does not contain a SH-2 domain that is required to bind to phospho-IRS-1. To determine the mechanism of PKCζ recruitment we analyzed the role of p62 (a PKCζ binding protein) that contains a SH2 domain. Exposure to differentiation medium plus IGF-I stimulated PKCζ/p62 association. Subsequent analysis showed the p62/PKCζ complex was co-recruited to IRS-1. Peptides that disrupted p62/PKCζ or p62/IRS-1 inhibited IGF-I/IGFBP-2 stimulated PKCζ activation, vimentin phosphorylation, PTEN tyrosine phosphorylation, AKT activation, and osteoblast differentiation. The importance of these signaling events for differentiation was confirmed in primary mouse calvarial osteoblasts. These results demonstrate the cooperative interaction between RPTPβ and the IGF-I receptor leading to a coordinated series of signaling events that are required for osteoblast differentiation. Our findings emphasize the important role IRS-1 plays in modulating these signaling events and confirm its essential role in facilitating osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.

The Na(+)-Taurocholate Cotransporting Polypeptide Traffics with the Epidermal Growth Factor Receptor.

Na(+)-taurocholate cotransporting polypeptide (ntcp) mediates bile acid transport, also serving as the hepatitis B virus receptor. It traffics in vesicles along microtubules, requiring activity of protein kinase C (PKC)ζ for motility. We have now found that the epidermal growth factor receptor (EGFR) is the target of PKCζ activity and that EGFR and ntcp colocalize in vesicles. ntcp-containing vesicles that are not associated with EGFR have reduced microtubule-based motility, consistent with intracellular accumulation and reduced surface expression of ntcp in cells following EGFR knockdown.

IL-6 Activates PI3K and PKCζ Signaling and Determines Cardiac Differentiation in Rat Embryonic H9c2 Cells.

IL-6 influences several biological processes, including cardiac stem cell and cardiomyocyte physiology. Although JAK-STAT3 activation is the defining feature of IL-6 signaling, signaling molecules such as PI3K, PKCs, and ERK1/2 are also activated and elicit different responses. Moreover, most studies on the specific role of these signaling molecules focus on the adult heart, and few studies are available on the biological effects evoked by IL-6 in embryonic cardiomyocytes. The aim of this study was to clarify the biological response of embryonic heart derived cells to IL-6 by analyzing the morphological modifications and the signaling cascades evoked by the cytokine in H9c2 cells. IL-6 stimulation determined the terminal differentiation of H9c2 cells, as evidenced by the increased expression of cardiac transcription factors (NKX2.5 and GATA4), structural proteins (α-myosin heavy chain and cardiac Troponin T) and the gap junction protein Connexin 43. This process was mediated by the rapid modulation of PI3K, Akt, PTEN, and PKCζ phosphorylation levels. PI3K recruitment was an upstream event in the signaling cascade and when PI3K was inhibited, IL-6 failed to modify PKCζ, PTEN, and Akt phosphorylation. Blocking PKCζ activity affected only PTEN and Akt. Finally, the overexpression of a constitutively active form of PKCζ in H9c2 cells largely mimicked the morphological and molecular effects evoked by IL-6. This study demonstrated that IL-6 induces the cardiac differentiation of H9c2 embryonic cells though a signaling cascade that involves PI3K, PTEN, and PKCζ activities.

Hyperglycemia stimulates p62/PKCζ interaction, which mediates NF-κB activation, increased Nox4 expression, and inflammatory cytokine activation in vascular smooth muscle.

Hyperglycemia leads to vascular smooth muscle cell (VSMC) dedifferentiation and enhances responses to IGF-I. Prior studies showed that hyperglycemia stimulated NADPH oxidase 4 (Nox4) synthesis, and IGF-I facilitated its recruitment to a signaling complex where it oxidized src, leading to AKT and MAPK activation. To determine the mechanism that led to these changes, we analyzed the roles of p62 (sequestrosome1) and PKCζ. Hyperglycemia induced a 4.9 ± 1.0-fold increase in p62/PKCζ association, and disruption of PKCζ/p62 using a peptide inhibitor or p62 knockdown reduced PKCζ activation (78 ± 6%). 3-Phosphoinoside-dependent protein kinase 1 was also recruited to the p62 complex and directly phosphorylated PKCζ, leading to its activation (3.1 ± 0.4-fold). Subsequently, activated PKCζ phosphorylated p65 rel, which led to increased Nox4 synthesis. Studies in diabetic mice confirmed these findings (6.0 ± 0.4-fold increase in p62/PKCζ) and their disruption of attenuated Nox4 synthesis (76 ± 9% reduction). PKCζ/p62 activation stimulated inflammatory cytokine production and enhanced IGF-I-stimulated VSMC proliferation. These results define the molecular mechanism by which PKCζ is activated in response to hyperglycemia and suggest that this could be a mechanism by which other stimuli such as cytokines or metabolic stress function to stimulate NF-κB activation, thereby altering VSMC sensitivity to IGF-I.

Bcl10 crucially nucleates the pro-apoptotic complexes comprising PDK1, PKCζ and caspase-3 at the nuclear envelope of etoposide-treated human cervical carcinoma C4-I cells.

Protein kinase (PK)Cζ signaling at various subcellular levels affects cell survival, differentiation, growth and/or apoptosis. However, the mechanisms modulating PKCζ activity at the nuclear membrane (NM) are not yet fully understood. Previously, we demonstrated that PKCζ interacts with the B‑cell lymphoma 10 (Bcl10) protein at the NM of human cervical carcinoma (HCC) C4‑I cells. In the present study, we aimed to further clarify the interactions between PKCζ, Bcl10 and other proteins co-immunoprecipitated from NMs isolated from untreated and etoposide (also known as VP‑16; 2.0 µg/ml)‑treated C4‑I cells using biochemical and proteomics analyses. Aside from the Bcl10 protein, 3‑phosphoinositide‑dependent protein kinase‑1 (PDK1) also co-immunoprecipitated with PKCζ from NMs of C4‑I cells, indicating the assembly of a heterotrimeric complex, which increased with time in VP‑16‑exposed cells, as did the activity of PDK1‑phosphorylated‑PKCζ. In turn, PKCζ‑phosphorylated‑Bcl10 straddled an enlarged complex which comprised caspase‑3. Subsequently, activity‑enhanced caspase‑3 cleaved and inactivated PKCζ. Finally, the suppression of Bcl10 using specific siRNA or lentiviral transduction prevented the increase in the PDK1•PKCζ association, the increase in the activity of PKCζ and caspase‑3, as well as the caspase‑3‑mediated PKCζ proteolysis and inactivation from occurring at the NMs of the VP‑16‑exposed C4‑I cells. Our observations provide evidence that Bcl10 acts as a pivotal pro-apoptotic protein which crucially nucleates complexes comprising PDK1, PKCζ and active caspase‑3 at the NMs of VP‑16‑exposed C4‑I cells. Hence, our data suggest that Bcl10 and PKCζ are potential therapeutic targets in the treatment of HCC.

PKCα-Mediated Signals Regulate the Motile Responses of Cochlear Outer Hair Cells

There is strong evidence that changes in the actin/spectrin-based cortical cytoskeleton of outer hair cells (OHCs) regulate their motile responses as well as cochlear amplification, the process that optimizes the sensitivity and frequency selectivity of the mammalian inner ear. Since a RhoA/protein kinase C (PKC)-mediated pathway is known to inhibit the actin-spectrin interaction in other cell models, we decided to investigate whether this signaling cascade could also participate in the regulation of OHC motility. We used high-speed video microscopy and confocal microscopy to explore the effects of pharmacological activation of PKCα, PKCβI, PKCβII, PKCδ, PKCε, and PKCζ with lysophosphatidic acid (LPA) and their inhibition with bisindolylmaleimide I, as well as inhibition of RhoA and Rho-associated protein kinase (ROCK) with C3 and Y-27632, respectively. Motile responses were induced in isolated guinea pig OHCs by stimulation with an 8 V/cm external alternating electrical field as 50 Hz bursts of square wave pulses (100 ms on/off). We found that LPA increased expression of PKCα and PKCζ only, with PKCα, but not PKCζ, phosphorylating the cytoskeletal protein adducin of both Ser-726 and Thr-445. Interestingly, however, inhibition of PKCα reduced adducin phosphorylation only at Ser-726. We also determined that LPA activation of a PKCα-mediated signaling pathway simultaneously enhanced OHC electromotile amplitude and cell shortening, and facilitated RhoA/ROCK/LIMK1-mediated cofilin phosphorylation. Altogether, our results suggest that PKCα-mediated signals, probably via adducin-mediated inhibition of actin-spectrin binding and cofilin-mediated depolymerization of actin filaments, play an essential role in the homeostatic regulation of OHC motility and cochlear amplification.

The Coordinate Cellular Response to Insulin-like Growth Factor-I (IGF-I) and Insulin-like Growth Factor-binding Protein-2 (IGFBP-2) Is Regulated through Vimentin Binding to Receptor Tyrosine Phosphatase β (RPTPβ)

Insulin-like growth factor-binding protein-2 (IGFBP-2) functions coordinately with IGF-I to stimulate cellular proliferation and differentiation. IGFBP-2 binds to receptor tyrosine phosphatase β (RPTPβ), and this binding in conjunction with IGF-I receptor stimulation induces RPTPβ polymerization leading to phosphatase and tensin homolog inactivation, AKT stimulation, and enhanced cell proliferation. To determine the mechanism by which RPTPβ polymerization is regulated, we analyzed the protein(s) that associated with RPTPβ in response to IGF-I and IGFBP-2 in vascular smooth muscle cells. Proteomic experiments revealed that IGF-I stimulated the intermediate filament protein vimentin to bind to RPTPβ, and knockdown of vimentin resulted in failure of IGFBP-2 and IGF-I to stimulate RPTPβ polymerization. Knockdown of IGFBP-2 or inhibition of IGF-IR tyrosine kinase disrupted vimentin/RPTPβ association. Vimentin binding to RPTPβ was mediated through vimentin serine phosphorylation. The serine threonine kinase PKCζ was recruited to vimentin in response to IGF-I and inhibition of PKCζ activation blocked these signaling events. A cell-permeable peptide that contained the vimentin phosphorylation site disrupted vimentin/RPTPβ association, and IGF-I stimulated RPTPβ polymerization and AKT activation. Integrin-linked kinase recruited PKCζ to SHPS-1-associated vimentin in response to IGF-I and inhibition of integrin-linked kinase/PKCζ association reduced vimentin serine phosphorylation. PKCζ stimulation of vimentin phosphorylation required high glucose and vimentin/RPTPβ-association occurred only during hyperglycemia. Disruption of vimetin/RPTPβ in diabetic mice inhibited RPTPβ polymerization, vimentin serine phosphorylation, and AKT activation in response to IGF-I, whereas nondiabetic mice showed no difference. The induction of vimentin phosphorylation is important for IGFBP-2-mediated enhancement of IGF-I-stimulated proliferation during hyperglycemia, and it coordinates signaling between these two receptor-linked signaling systems.

Low-dose endothelial monocyte-activating polypeptide-II increases permeability of blood–tumor barrier via a PKC-ζ/PP2A-dependent signaling mechanism

Our previous study demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood–tumor barrier (BTB) opening via the RhoA/Rho kinase/protein kinase C (PKC)-α/β signaling pathway and that PKC-ζ is involved in this process via other mechanisms. In the present study, using an in vitro BTB model, we detected the exact signaling mechanisms by which PKC-ζ activation affects EMAP-II-induced BTB hyperpermeability. Our results showed that three types of serine/threonine (Ser/Thr) protein phosphatases (PPs), namely PP1, PP2A, and PP2B, were expressed by rat brain microvascular endothelial cells (RBMECs). There was an interaction between PKC-ζ and PP2A in RBMECs. In addition, EMAP-II induced a significant increase in both the expression and the activity of PP2A in RBMECs. Inhibition of PKC-ζ with PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI) completely blocked EMAP-II-induced PP2A activation. Conversely, inhibition of PP2A with okadaic acid (OA) had no effect on EMAP-II-induced PKC-ζ activation. Like PKC-ζ-PI, OA partially prevented EMAP-II-induced BTB hyperpermeability and occludin redistribution in RBMECs. Neither PKC-ζ-PI nor OA affected EMAP-II-induced phosphorylation of myosin light chain and redistribution of actin cytoskeleton in RBMECs. Taken together, our present study demonstrated that low-dose EMAP-II increases BTB permeability by activating the PKC-ζ/PP2A signaling pathway, which consequently leads to the disruption of TJs and impairment of endothelial barrier function.

Su1885 Prolonged High-Fat Feeding Decreases SGLT1 and GLUT2 Activity and Blunts Their Upregulation by Glucose-Dependent Insulinotropic Polypeptide (GIP) in Obese Mice

generate PCTV to transport the chylomicron from ER to Golgi. The chylomicron output into lymph is correlated to intestinal luminal phosphatidylcholine (PC). Luminal PC is absorbed as lyso-phosphatidylcholine (lyso-PC). We previously showed that the dietary lipids are absorbed from the apical membrane by Caveolin-1 containing Cytosolic Endocytic Vesicles (CEV). We tested the hypothesis that lyso-PC activates the PKCζ; detach it from CEV, enabling PKCζ to phosphorylate Sar1b. Methods: Cytosol was isolated from rats whose intestinal PC was altered by (A) bile diversion, no PC (B) saline infusion, low PC (C) chow fed, normal PC (D) fat fed, high PC and (E) PC infusion, very high PC. PKCζ activity was measured by phosphorylation of PKCζ pseudo substrate. Lyso-PC was measured by HPLC. Results: PKCζ was activated by lyso-PC, non-linear regression curve for PKCζ vs. lyso-PC, calculated as Km=1.49 ± 0.244 nM and Vmax = 1.12 ± 1.058 nM. The amount of cytosolic lyso-PC in A to E ranges from 0 to 0.45 nM, suggesting that the amount of cytosolic lysoPC is always within a range to control PKCζ activation. PKCζ activity ranges from 0 to 0.7 Arbitrary Unit in A to E. Post absorptive CEV contain PKCζ by western blot but PKCζ detachment from the CEV is proportional to cytosolic lyso-PC as estimated by western blot of CEV. Biotinylation of r-PKCζ showed a conformational change on activation by lyso-PC. We conclude that PKCζ on CEV was activated by lyso-PC, changes its conformation and eluted from vesicles, enter into the cytosol and phosphorylate Sar1b, split the heteroquatramer protein complex to release FABP1. Now free FABP1 can bind to ER membrane for PCTV formation, which transports chylomicrons from ER to Golgi. Fig. 1 illustrated the scheme for control of dietary lipid transport.

Su1887 Mechanism of Inhibition of Na-Glutamine Co-Transport by Prostaglandin E2 in Intestinal Epithelial Cells

generate PCTV to transport the chylomicron from ER to Golgi. The chylomicron output into lymph is correlated to intestinal luminal phosphatidylcholine (PC). Luminal PC is absorbed as lyso-phosphatidylcholine (lyso-PC). We previously showed that the dietary lipids are absorbed from the apical membrane by Caveolin-1 containing Cytosolic Endocytic Vesicles (CEV). We tested the hypothesis that lyso-PC activates the PKCζ; detach it from CEV, enabling PKCζ to phosphorylate Sar1b. Methods: Cytosol was isolated from rats whose intestinal PC was altered by (A) bile diversion, no PC (B) saline infusion, low PC (C) chow fed, normal PC (D) fat fed, high PC and (E) PC infusion, very high PC. PKCζ activity was measured by phosphorylation of PKCζ pseudo substrate. Lyso-PC was measured by HPLC. Results: PKCζ was activated by lyso-PC, non-linear regression curve for PKCζ vs. lyso-PC, calculated as Km=1.49 ± 0.244 nM and Vmax = 1.12 ± 1.058 nM. The amount of cytosolic lyso-PC in A to E ranges from 0 to 0.45 nM, suggesting that the amount of cytosolic lysoPC is always within a range to control PKCζ activation. PKCζ activity ranges from 0 to 0.7 Arbitrary Unit in A to E. Post absorptive CEV contain PKCζ by western blot but PKCζ detachment from the CEV is proportional to cytosolic lyso-PC as estimated by western blot of CEV. Biotinylation of r-PKCζ showed a conformational change on activation by lyso-PC. We conclude that PKCζ on CEV was activated by lyso-PC, changes its conformation and eluted from vesicles, enter into the cytosol and phosphorylate Sar1b, split the heteroquatramer protein complex to release FABP1. Now free FABP1 can bind to ER membrane for PCTV formation, which transports chylomicrons from ER to Golgi. Fig. 1 illustrated the scheme for control of dietary lipid transport.

(+)-Nootkatone inhibits tumor necrosis factor α/interferon γ-induced production of chemokines in HaCaT cells

Chemokines are important mediators of cell migration, and thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well-known typical inflammatory chemokines involved in atopic dermatitis (AD). (+)-Nootkatone is the major component of Cyperus rotundus. (+)-Nootkatone has antiallergic, anti-inflammatory, and antiplatelet activities. The purpose of this study was to investigate the effect of (+)-nootkatone on tumor necrosis factor α (TNF-α)/interferon γ (IFN-γ)-induced expression of Th2 chemokines in HaCaT cells. We found that (+)-nootkatone inhibited the TNF-α/IFN-γ-induced expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells. It also significantly inhibited TNF-α/IFN-γ-induced activation of nuclear factor kappa B (NF-κB), p38 mitogen-activated protein kinase (MAPK), and protein kinase Cζ (PKCζ). Furthermore, we showed that PKCζ and p38 MAPK contributed to the inhibition of TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression by blocking IκBα degradation in HaCaT cells. Taken together, these results suggest that (+)-nootkatone may suppress TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression in HaCaT cells by inhibiting of PKCζ and p38 MAPK signaling pathways that lead to activation of NF-κB. We propose that (+)-nootkatone may be a useful therapeutic candidate for inflammatory skin diseases such as AD.

The mechanism of serine protease‐induced increases in epithelial barrier function in intestinal epithelial cells (60.7)

Apical serine proteases increase barrier function in intestinal epithelial cells (IECs). We investigated the requirement for proteolytic, protein kinase C (PKC)ζ, and epidermal growth factor receptor (EGFR) activities in the prolonged transepithelial resistance (TER) response to trypsin in IECs.Methods. 1. SCBN epithelial cell monolayers grown on Transwells were treated with trypsin (45BAU/ml) and collected for measurement of phospho‐PKCζ at 2, 5, 10, 15, 30, and 60 min using Western blot. 2. TER across SCBN monolayers was measured in Ussing chambers. Trypsin activity was inhibited by soybean trypsin inhibitor (SBTI) treatment at 10 min (start of plateau phase of TER response) or 30 min (after plateau phase had been established). 3. To investigate the role of EGFR, cells were pretreated with an EGFR inhibitor (PD15305) before addition of trypsin.Results. 1. Upregulation of phospho‐PKCζ was observed in trypsin‐treated SCBNs for up to 60 min. 2. Apical trypsin caused an increase in TER that reached a sustained plateau by 10 min. SBTI at 30 or 60 μg/ml decreased the trypsin‐induced increase in TER by 65% and 80% (respectively) at 10 min and by 70% and 80% at 30 min. 3. Inhibition of EGFR with PD153035 reduced the trypsin‐induced TER by 55%.Our data show the requirement for ongoing proteolytic activity, sustained PKCζ activation and EGFR activity in the trypsin‐induced increase in intestinal epithelial barrier function.Grant Funding Source: Supported by the Canadian Institutes of Health Research

Ceramide inhibits insulin-stimulated Akt phosphorylation through activation of Rheb/mTORC1/S6K signaling in skeletal muscle

Ceramide is a negative regulator of insulin activity. At the molecular level, it causes a decrease in insulin-stimulated Akt Ser473 phosphorylation in C2C12 myotubes. Interestingly, we found that the phosphorylation of S6K at Thr389 was increased under the same conditions. Utilizing both rapamycin to inhibit mTORC1 activity and shRNA to knock down Rheb, we demonstrated that the decrease in Akt Ser473 phosphorylation stimulated by insulin after C2-ceramide incubation can be prevented. The mechanism by which C2-ceramide impairs signaling would seem to involve a negative feedback of activated S6K via phosphorylation of insulin receptor substrate-1 at Ser636/639, since S6K inhibitor can block this phenomenon. Finally, rapamycin treatment was found not to affect C2-ceramide-induced PKCζ activation, suggesting that the pathway revealed in this study is parallel to the one involving PKCζ activation. We proposed a novel pathway/mechanism involving Rheb/mTORC1/S6K signaling to explain how C2-ceramide impairs insulin signaling via Akt phosphorylation. The existence of multiple pathways involved in insulin signaling impairment by C2-ceramide treatment implies that different strategies might be needed to ameliorate insulin resistance caused by C2-ceramide.

MTORC 2 phosphorylates protein kinase Cζ to regulate its stability and activity

Protein kinase Cζ (PKCζ) is phosphorylated at the activation loop and the turn motif (TM). However, the TM kinase and functional relevance of TM phosphorylation remain largely unknown. We demonstrate that PKCζ TM is phosphorylated directly by the mTORC2 complex, and this phosphorylation is required for maintaining PKCζ kinase activity and stability. Functionally, mTORC2 regulates the activity of Rho family of GTPases, and therefore the organization of the actin cytoskeleton, through the control of PKCζ activity. Taken together, our findings identify PKCζ as a novel substrate and downstream effector of mTORC2 signaling.

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy

Aims/hypothesisDiabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.MethodsStudies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.ResultsPKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.ConclusionsIn the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.

Role of PKC-ζ in NADPH oxidase–PKCα–Giα axis dependent inhibition of β-adrenergic response by U46619 in pulmonary artery smooth muscle cells

Treatment of bovine pulmonary artery smooth muscle cells (BPASMCs) with U46619 attenuated isoproterenol caused stimulation of adenyl cyclase activity and cAMP production. Pretreatment with SQ29548 (Tp receptor antagonist), apocynin (NADPH oxidase inhibitor) and Go6976 (PKC-α inhibitor) eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity. Pretreatment with SQ29548 and apocynin prevented U46619 induced increase in NADPH oxidase activity, PKC-α activity and Giα phosphorylation. However, pretreatment with CZI, a PKC-ζ inhibitor, markedly, but not completely, inhibited U46619 induced increase in NADPH oxidase activity, PKC-α activity, Giα phosphorylation and also significantly eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976 inhibited U46619 induced increase in Giα phosphorylation, but not PKC-ζ activity and NADPH oxidase activity. Pretreatment with pertussis toxin eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity without any discernible change in PKC-ζ, NADPH oxidase and PKC-α activities. Transfection of the cells with Tp, PKC-ζ and PKC-α siRNA duplexes corroborate the findings observed with their respective pharmacological inhibitors on the responses produced by U46619. Taken together, we suggest involvement of PKC-ζ in U46619 caused attenuation of isoproterenol stimulated β-adrenergic response, which is regulated by NADPH oxidase–PKCα–Giα axis in pulmonary artery smooth muscle cells.

Transient activation of protein kinase C contributes to fluoride-induced apoptosis of rat erythrocytes

Role of PKC in fluoride-induced apoptosis of rat erythrocytes was studied in vitro and in vivo. Treatment of erythrocytes with 5mM NaF for 1–24h caused progressive accumulation of cytosolic Ca2+ and PS exposure at outer membrane surface. After 1h, these processes were suppressed by PKC inhibitors staurosporine, GF 109203X and chelerythrine, but increased by PKC activator PMA. Following 24h, NaF-induced Ca2+ uptake and PS externalization were partly prevented by PMA or staurosporine, but not by GF 109293X and chelerythrine. Application of PP inhibitor OA augmented NaF-induced cell responses within 1h, but not after 24h. Incubation of erythrocytes with 0.1–10mM NaF for 1h produced a dose-dependent PKCα translocation from cytosol to membranes with appearance of active PKM fragment. 24h NaF exposure led to complete loss of cytosolic PKCα and proteolysis of membrane PKCα. Besides, NaF weakly stimulated membrane PKCζ, although its subcellular distribution was not altered. Thus, transient PKCα activation/translocation positively contributes to NaF-induced apoptosis in vitro. Consumption of 2–20ppm fluoride by the rats for 12months also induced dose-dependent PKCα translocation to membranes and activation of membrane PKCζ, what indicates that PKC stimulation is an important physiological mechanism of fluoride toxicity.

The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

Cell polarization is essential for many biological processes, including directed cell migration, and loss of polarity contributes to pathological conditions such as cancer. The Par complex (Par3, Par6, and PKCζ) controls cell polarity in part by recruiting the Rac-specific guanine nucleotide exchange factor T-lymphoma invasion and metastasis 1 (Tiam1) to specialized cellular sites, where Tiam1 promotes local Rac1 activation and cytoskeletal remodeling. However, the mechanisms that restrict Par-Tiam1 complex activity to the leading edge to maintain cell polarity during migration remain unclear. We identify the Rac-specific GTPase-activating protein (GAP) breakpoint cluster region protein (Bcr) as a novel regulator of the Par-Tiam1 complex. We show that Bcr interacts with members of the Par complex and inhibits both Rac1 and PKCζ signaling. Loss of Bcr results in faster, more random migration and striking polarity defects in astrocytes. These polarity defects are rescued by reducing PKCζ activity or by expressing full-length Bcr, but not an N-terminal deletion mutant or the homologous Rac-GAP, Abr, both of which fail to associate with the Par complex. These results demonstrate that Bcr is an integral member of the Par-Tiam1 complex that controls polarized cell migration by locally restricting both Rac1 and PKCζ function.

Inhibition of PKC-ζ Activation Prevents Bleomycin Induce Lung Injury and Promotes Lung Repair

Inhibition of PKC-ζ Activation Prevents Bleomycin Induce Lung Injury and Promotes Lung Repair