Atypical PKC Isoform Research Articles

Loss of polarity regulators initiates gasdermin-E-mediated pyroptosis in syncytiotrophoblasts.

The syncytiotrophoblast is a human epithelial cell that is bathed in maternal blood on the maternal-facing surface of the human placenta. It therefore acts as a barrier and exchange interface between the mother and fetus. Syncytiotrophoblast dysfunction is a feature of pregnancy pathologies, like preeclampsia. Dysfunctional syncytiotrophoblasts display a loss of microvilli, which is a marker of aberrant apical-basal polarization, but little data exist about the regulation of syncytiotrophoblast polarity. Atypical PKC isoforms are conserved polarity regulators. Thus, we hypothesized that aPKC isoforms regulate syncytiotrophoblast polarity. Using human placental explant culture and primary trophoblasts, we found that loss of aPKC activity or expression induces syncytiotrophoblast gasdermin-E-dependent pyroptosis, a form of programmed necrosis. We also establish that TNF-α induces an isoform-specific decrease in aPKC expression and gasdermin-E-dependent pyroptosis. Therefore, aPKCs are homeostatic regulators of the syncytiotrophoblast function and a pathogenically relevant pro-inflammatory cytokine leads to the induction of programmed necrosis at the maternal-fetal interface. Hence, our results have important implications for the pathobiology of placental disorders like preeclampsia.

Involvement of PKMζ in Stress Response and Depression.

The stress system in the brain plays a pivotal role in keeping humans and animals from harmful stimuli. However, excessive stress will cause maladaptive changes to the stress system and lead to depression. Despite the high prevalence of depression, the treatment remains limited. PKMζ, an atypical PKC isoform, has been demonstrated to play a crucial role in maintaining long-term potentiation and memory. Recent evidence shows that PKMζ is also involved in stress response and depressive-like behavior. In particular, it was demonstrated that stress that resulted in depressive-like behavior could decrease the expression of PKMζ in the prefrontal cortex, which could be reversed by antidepressants. Importantly, modulation of PKMζ expression could regulate depressive-like behaviors and the actions of antidepressants. These data suggested that PKMζ could be a molecular target for developing novel antidepressants. Here, I review the advance on the role of PKMζ in mediating stress response and its involvement in the development of depression.

Alternative AKT2 splicing produces protein lacking the hydrophobic motif regulatory region.

Three AKT serine/threonine kinase isoforms (AKT1/AKT2/AKT3) mediate proliferation, metabolism, differentiation and anti-apoptotic signals. AKT isoforms are activated downstream of PI3-kinase and also by PI3-kinase independent mechanisms. Mutations in the lipid phosphatase PTEN and PI3-kinase that increase PIP3 levels increase AKT signaling in a large proportion of human cancers. AKT and other AGC kinases possess a regulatory mechanism that relies on a conserved hydrophobic motif (HM) C-terminal to the catalytic core. In AKT, the HM is contiguous to the serine 473 and two other newly discovered (serine 477 and tyrosine 479) regulatory phosphorylation sites. In AKT genes, this regulatory HM region is encoded in the final exon. We identified a splice variant of AKT2 (AKT2-13a), which contains an alternative final exon and lacks the HM regulatory site. We validated the presence of mRNA for this AKT2-13a splice variant in different tissues, and the presence of AKT2-13a protein in extracts from HEK293 cells. When overexpressed in HEK293 cells, AKT2-13a is phosphorylated at the activation loop and at the zipper/turn motif phosphorylation sites but has reduced specific activity. Analysis of the human transcriptome corresponding to other AGC kinases revealed that all three AKT isoforms express alternative transcripts lacking the HM regulatory motif, which was not the case for SGK1-3, S6K1-2, and classical, novel and atypical PKC isoforms. The transcripts of splice variants of Akt1-3 excluding the HM regulatory region could lead to expression of deregulated forms of AKT.

The Atypical Protein Kinase C Small Molecule Inhibitor ζ-Stat, and Its Effects on Invasion Through Decreases in PKC-ζ Protein Expression

Ovarian cancer is estimated to reach 22,530 diagnoses and cause 13,980 cancer deaths per year. The most common histology diagnosed of ovarian cancer is epithelial ovarian carcinomas (EOC). An aggressive epithelial subtype is clear cell ovarian carcinoma (CCOC) and is characterized as a non-serous ovarian cancer. Protein kinase C (PKC) is an enzymatic family of proteins that have been found to be a component in cancer progression, tissue invasion, and metastasis. The atypical PKC (aPKC) isoforms, PKC-ι and PKC-ζ, have been suggested to participate in the increased proliferation of ovarian cancers. Previous studies have indicated that novel aPKC inhibitors ICA-1S and ζ-Stat decreased the migratory behaviors of colorectal cancer cells and were selective for PKC-ι/λ and PKC-ζ, respectively. The aims of this investigation were to further determine the binding mechanisms of ζ-Stat, expand on the tissue range of these compounds, investigate the therapeutic potential of ζ-Stat in CCOC, and to illustrate the disruption of invasion via the PKC-ζ signaling cascade. The methods utilized were molecular docking and virtual target screening, Western blot analysis, end-point PCR, GST pull down, cell viability and invasion and migration assays. We discovered that the small molecule inhibitor, ζ-Stat, is a prospective drug candidate to investigate as a novel potential treatment for CCOC. We also found that the PKC-ζ/Ect2/Rac1 activation pathway was decreased by ζ-Stat, which in turn decreased invasive behavior of CCOC.

The role of PKC and PKD in CXCL12 directed prostate cancer migration

Cancer metastasis is the cause of most cancer related deaths and many cancers are becoming resistant to current therapies. An alternative approach is to investigating signalling pathways that cause cancer cell migration such as chemokine signalling pathways. Such downstream signalling proteins are PKC and PKD. Therefore, we investigated the role of these two proteins in CXCL12 mediated PC3 prostate cancer migration. Whereas PKC and PKD inhibitors do not affect the release of calcium in PC3 prostate cancer cells, both are involved in migration, particularly inhibition of the atypical PKC isoform PKCζ causes the greatest reduction in PC3 cell migration. Classical and/or Novel PKC isoform inhibition changes the shape of the PC3 cells, they show a more rounded morphology, whereas PKD inhibition causes prostate cancer cell to elongate. PKCζ inhibition causes the enlargement of PC3 area possibly due to dysregulated actin cytoskeletal control. These results highlight the importance of verifying which signalling proteins, in which cell and in which chemokine signalling cascade enable cancer cellular migration.

Metformin attenuates the effect of Staphylococcus aureus on airway tight junctions by increasing PKCζ-mediated phosphorylation of occludin.

Airway epithelial tight junction (TJ) proteins form a resistive barrier to the external environment, however, during respiratory bacterial infection TJs become disrupted compromising barrier function. This promotes glucose flux/accumulation into the lumen which acts as a nutrient source for bacterial growth. Metformin used for the treatment of diabetes increases transepithelial resistance (TEER) and partially prevents the effect of bacteria but the mechanisms of action are unclear. We investigated the effect of metformin and Staphylococcus aureus on TJ proteins, zonula occludins (ZO)‐1 and occludin in human airway epithelial cells (H441). We also explored the role of AMP‐activated protein kinase (AMPK) and PKCζ in metformin‐induced effects. Pretreatment with metformin prevented the S. aureus‐induced changes in ZO‐1 and occludin. Metformin also promoted increased abundance of full length over smaller cleaved occludin proteins. The nonspecific PKC inhibitor staurosporine reduced TEER but did not prevent the effect of metformin indicating that the pathway may involve atypical PKC isoforms. Investigation of TJ reassembly after calcium depletion showed that metformin increased TEER more rapidly and promoted the abundance and localization of occludin at the TJ. These effects were inhibited by the AMPK inhibitor, compound C and the PKCζ pseudosubstrate inhibitor (PSI). Metformin increased phosphorylation of occludin and acetyl‐coA‐carboxylase but only the former was prevented by PSI. This study demonstrates that metformin improves TJ barrier function by promoting the abundance and assembly of full length occludin at the TJ and that this process involves phosphorylation of the protein via an AMPK‐PKCζ pathway.

Persistent Increases of PKMM in Sensorimotor Cortex Maintain Procedural long-Term Memory Storage

Procedural memories, such as for riding a bicycle, can be maintained without practice for long periods of time and are thought to be supported by the persistent reorganization of sensorimotor cortices (S1/M1). Whereas enhanced synaptic strength and structural changes accompany the learning of motor tasks, the persistent molecular modifications that store long-term procedural memories within specific layers of sensorimotor cortex have not been identified. The persistent increase in the autonomously active, atypical PKC isoform, PKMζ, is a putative molecular mechanism for maintaining enhanced synaptic strength during long-term potentiation (LTP) and several forms of long-term memory. Here we examine whether persistent increases in PKMζ store long-term memory for a reaching task in rat sensorimotor cortex that could reveal the sites of procedural memory storage. Perturbing PKMζ synthesis with PKMζ – antisense oligodeoxynucleotides or blocking atypical PKC activity with zeta inhibitory peptide (ZIP) in S1/M1 disrupts and erases the maintenance of long-term motor memories. Only memories that are maintained without daily reinforcement are affected, indicating atypical PKCs (via ZIP) and PKMζ specifically (via antisense) stores consolidated long-term procedural memories. Analysis of changes in the amount of PKMζ in S1/M1 reveals PKMζ increases in layers II/III and V of both S1 and M1 cortices as performance improves to an asymptote during training. After storage for 1 month without reinforcement, the increase in M1 layer V but not other layers persists without decrement. Thus, the sustained increases in PKMζ reveal that the persistent molecular changes storing long-term procedural memory are localized to the descending output layer of primary motor cortex.

Spinal activation of protein kinase C elicits phrenic motor facilitation

The protein kinase C family regulates many cellular functions, including multiple forms of neuroplasticity. The novel PKCθ and atypical PKCζ isoforms have been implicated in distinct forms of spinal, respiratory motor plasticity, including phrenic motor facilitation (pMF) following acute intermittent hypoxia or inactivity, respectively. Although these PKC isoforms are critical in regulating spinal motor plasticity, other isoforms may be important for phrenic motor plasticity. We tested the impact of conventional/novel PKC activator, phorbol 12-myristate 13-acetate (PMA) on pMF. Rats given cervical intrathecal injections of PMA exhibited pMF, which was abolished by pretreatment of broad-spectrum PKC inhibitors bisindolymalemide 1 (BIS) or NPC-15437 (NPC). Because PMA fails to activate atypical PKC isoforms, and NPC does not block PKCθ, this finding demonstrates that classical/novel PKC isoforms besides PKCθ are sufficient to elicit pMF. These results advance our understanding of mechanisms producing respiratory motor plasticity, and may inspire new treatments for disorders that compromise breathing, such as ALS, spinal injury and obstructive sleep apnea.

Phrenic long-term facilitation requires PKCθ activity within phrenic motor neurons.

Acute intermittent hypoxia (AIH) induces a form of spinal motor plasticity known as phrenic long-term facilitation (pLTF); pLTF is a prolonged increase in phrenic motor output after AIH has ended. In anesthetized rats, we demonstrate that pLTF requires activity of the novel PKC isoform, PKCθ, and that the relevant PKCθ is within phrenic motor neurons. Whereas spinal PKCθ inhibitors block pLTF, inhibitors targeting other PKC isoforms do not. PKCθ is highly expressed in phrenic motor neurons, and PKCθ knockdown with intrapleural siRNAs abolishes pLTF. Intrapleural siRNAs targeting PKCζ, an atypical PKC isoform expressed in phrenic motor neurons that underlies a distinct form of phrenic motor plasticity, does not affect pLTF. Thus, PKCθ plays a critical role in spinal AIH-induced respiratory motor plasticity, and the relevant PKCθ is localized within phrenic motor neurons. Intrapleural siRNA delivery has considerable potential as a therapeutic tool to selectively manipulate plasticity in vital respiratory motor neurons.

Differential regulation of TROP2 release by PKC isoforms through vesicles and ADAM17

TROP2, a cancer cell surface protein with both pro-oncogenic and anti-oncogenic properties is cleaved by ADAM17. ADAM17 dependent cleavage requires novel PKC activity which is blocked by the ADAM10/ADAM17 inhibitor GW64 as well as by the PKC inhibitor Bim-1. Full length TROP2 release is induced by classical PKC activation and blocked by Gö6979, without affecting ADAM17 dependent TROP2 cleavage. Full length TROP2 is released in ectosomes, as inhibition of endocytosis did not prevent release. Inhibition of the atypical PKC isoform PKCζ stimulated metalloproteinase dependent N-terminal alternative TROP2 cleavage. The resulting alternative TROP2 cleavage product remains membrane associated via a disulphide bond, but is released in microvesicles with an average size of 107nm. Inhibition of endocytosis following PKCζ inhibition prevented alternative cleavage and release of TROP2, suggesting that these events require endocytic uptake and exosomal release of the corresponding microvesicles. The alternative TROP2 cleavage product was also found in PC3 cell lysates following deglycosylation, and may represent a novel biomarker in prostate cancer.

Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing.

Melatonin, a circadian rhythm-promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesenchymal stem cells (MSCs) has yet to be studied. In a mouse skin excisional wound model, we found that transplantation of umbilical cord blood (UCB)-MSCs pretreated with melatonin enhanced wound closure, granulation, and re-epithelialization at mouse skin wound sites, where relatively more UCB-MSCs which were engrafted onto the wound site were detected. Thus, we identified the signaling pathway of melatonin, which affects the motility of UCB-MSCs. Melatonin (1 μm) significantly increased the motility of UCB-MSCs, which had been inhibited by the knockdown of melatonin receptor 2 (MT2). We found that Gαq coupled with MT2 and that the binding of Gαq to MT2 uniquely stimulated an atypical PKC isoform, PKCζ. Melatonin induced the phosphorylation of FAK and paxillin, which were concurrently downregulated by blocking of the PKC activity. Melatonin increased the levels of active Cdc42 and Arp2/3, and it has the ability to stimulate cytoskeletal reorganization-related proteins such as profilin-1, cofilin-1, and F-actin in UCB-MSCs. Finally, a lack of MT2 expression in UCB-MSCs during a mouse skin transplantation experiment resulted in impaired wound healing and less engraftment of stem cells at the wound site. These results demonstrate that melatonin signaling via MT2 triggers FAK/paxillin phosphorylation to stimulate reorganization of the actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB-MSCs motility.

Abstract 5288: PHLPP controls cell polarity by negatively regulating the activity of atypical PKC in colon cancer cells

Abstract The proper establishment of epithelial polarity allows cells to sense and to respond signals that arise from the microenvironment in a spatiotemporally controlled manner. Increasing evidence has suggested that disruption of polarity promotes the malignant progression of cancer cells. However, the molecular mechanism underlying polarity regulation in the initiation and progression of colon cancer remains largely unknown. PHLPP belongs to a novel family of Ser/Thr protein phosphatases that consists of PHLPP1 and PHLPP2 isoforms. Previously, we have identified conventional PKC isoforms as substrates of PHLPP. Here, we demonstrated that PHLPP controls phosphorylation levels of atypical PKC (aPKC) isoforms, including PKCζ and PKCι, as well. Overexpression of either PHLPP isoform resulted in a decrease in aPKC phosphorylation at both the activation loop (Thr403/410) and the turn motif (Thr555/560) sites in colon cancer cells; conversely, knockdown of PHLPP increased aPKC phosphorylation. In addition, in vitro dephosphorylation experiments revealed that both aPKC isoforms are direct substrates of PHLPP. Since aPKC has been widely implicated in the regulation of cell polarity in intestinal epithelial cells, we determined the function of PHLPP in establishing cell polarity using a 3-D Matrigel culture model. Whereas control Caco2 cells form well polarized cysts in 2 weeks, PHLPP depletion alters the acinar structure by inducing the formation of multiple lumens. Interestingly, knockdown of PKCζ, but not PKCι, results in similar disruption of the polarized lumen structure in Caco2 cells, suggesting that PHLPP may regulate cell polarity through dephosphorylating PKCζ. Taken together, our results identify both aPKC isoforms as novel substrates of PHLPP and downregulation of PHLPP may promote tumorigenesis in colon cancer by disrupting cell polarity. Citation Format: Xiaopeng Xiong, Xin li, Tianyan Gao. PHLPP controls cell polarity by negatively regulating the activity of atypical PKC in colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5288. doi:10.1158/1538-7445.AM2014-5288

Requirements for Pseudosubstrate Arginine Residues during Autoinhibition and Phosphatidylinositol 3,4,5-(PO4)3-dependent Activation of Atypical PKC

Atypical PKC (aPKC) isoforms are activated by the phosphatidylinositol 3-kinase product phosphatidylinositol 3,4,5-(PO4)3 (PIP3). How PIP3 activates aPKC is unknown. Although Akt activation involves PIP3 binding to basic residues in the Akt pleckstrin homology domain, aPKCs lack this domain. Here we examined the role of basic arginine residues common to aPKC pseudosubstrate sequences. Replacement of all five (or certain) arginine residues in the pseudosubstrate sequence of PKC-ι by site-directed mutagenesis led to constitutive activation and unresponsiveness to PIP3 in vitro or insulin in vivo. However, with the addition of the exogenous arginine-containing pseudosubstrate tridecapeptide to inhibit this constitutively active PKC-ι, PIP3-activating effects were restored. A similar restoration of responsiveness to PIP3 was seen when exogenous pseudosubstrate was used to inhibit mouse liver PKC-λ/ζ maximally activated by insulin or ceramide and a truncated, constitutively active PKC-ζ mutant lacking all regulatory domain elements and containing "activating" glutamate residues at loop and autophosphorylation sites (Δ1-247/T410E/T560E-PKC-ζ). NMR studies suggest that PIP3 binds directly to the pseudosubstrate. The ability of PIP3 to counteract the inhibitory effects of the exogenous pseudosubstrate suggests that basic residues in the pseudosubstrate sequence are required for maintaining aPKCs in an inactive state and are targeted by PIP3 for displacement from the substrate-binding site during kinase activation.

Persistent increases of PKMζ correlate with the retention and duration of spatial long‐term memory (728.11)

PKMζ is a constitutively active and brain‐specific atypical PKC isoform that plays an important role during long‐term memory (LTM) storage. Overexpressing PKMζ enhances LTM, and inhibiting PKMζ by overexpressing a dominant negative version of PKMζ or injecting ζ inhibitory peptide (ZIP) disrupts LTM, even very long‐term spatial memory 1 month post‐training. Here we examined endogenous PKMζ expression at different time points after short‐ or long‐term memory and correlated the amount of expressed PKMζ with LTM retention.Adult male Long‐Evans rats and C57BL/6J mice were trained by massed and spaced active place avoidance, respectively. Massed training consisted of eight 10‐min‐shock trials with 10 min inter‐trial interval (ITI), and spaced training consisted of three 30‐min‐shock trials with 120 min ITI. Quantitative immunoblotting showed that dorsal hippocampal PKMζ increased by the end of the massed training phase, and 1 day after both massed and spaced training, compared to animals that had not received shock when placed in the apparatus. The increase in PKMζ remained at least 30 days after the spaced training. Thus, the PKMζ level significantly correlated with 1 day LTM, but not with the number of shocks during the training. No PKMζ increase was detected after either short‐term memory training or 1 day after the administration of the tethered shocks in which animals received the same number and timing of shocks as the avoidance‐training group, but without association with spatial information. No long‐term contextual fear conditioned behaviors, such as freezing, or locomotor activity change was observed in the shock‐training group. Immunohistochemistry demonstrated that the increased PKMζ after the spatial training localized in the strata pyramidale, radiatum, and lacunosum‐moleculare in CA1 region at both 1 day and 1 month posttraining. These results support the hypothesis that persistently increased hippocampal PKMζ might be the molecular mechanism of the spatial LTM storage.Grant Funding Source: NIH Grant R01 MH053576

Inhibition of PKC disrupts addiction-related memory

The atypical PKC isoforms, PKMζ and PKCλ have been proposed as integral substrates of long-term memory (LTM). Inhibition of these isoforms has recently been demonstrated to be sufficient for impairing the expression and maintenance of long-term potentiation. Additionally, the pseudosubstrate inhibitor, zeta inhibitory peptide (ZIP), which effectively blocks PKMζ and PKCλ, has previously been shown to disrupt associative memory; very little is known about its effects on pathological nonassociative forms of memory related to addiction. The neural and molecular substrates of memory and addiction have recently been argued to overlap. Here, we used ZIP to disrupt PKMζ and PKCλ activity to examine their role in cocaine sensitization, a nonassociative, addiction-related memory argued to underlie the transition from casual to pathological drug use. We examined the effects of both continuous and acute administration of ZIP. Even a single application of ZIP blocked the development of sensitization; sustained inhibition using osmotic pumps produced an almost complete blockade of sensitization. Further, a single application of ZIP was shown to reduce membrane-bound AMPAR expression. These results demonstrate a novel, critical role for the atypical PKC isoforms in nonassociative memory and cocaine addiction.

Structural and biochemical insights into the homotypic PB1-PB1 complex between PKCζ and p62

The atypical PKC isoforms (ζ and ı) play essential roles in regulating various cellular processes. Both the hetero-interaction between PKCζ and p62 through their N-terminal PB1 domains and the homo-oligomerization of p62 via its PB1 domain are critical for the activation of NF-κB signaling; however, the molecular mechanisms concerning the formation and regulation of these homotypic complexes remain unclear. Here we determined the crystal structure of PKCζ-PB1 in complex with a monomeric p62-PB1 mutant, where the massive electrostatic interactions between the acidic OPCA motif of PKCζ-PB1 and the basic surface of p62-PB1, as well as additional hydrogen bonds, ensure the formation of a stable and specific complex. The PKCζ-p62 interaction is interfered with the modification of a specific Cys of PKCζ by the antiarthritis drug aurothiomalate, though all four cysteine residues in the PKCζ-PB1 domain can be modified in in vitro assay. In addition, detailed structural and biochemical analyses demonstrate that the PB1 domains of aPKCs belong to the type I group, which can depolymerize the high-molecular-weight p62 aggregates into homo-oligomers of lower order. These data together unravel the molecular mechanisms of the homo-or hetero-interactions between p62 and PKCζ and provide the basis for designing inhibitors of NF-κB signaling.

Conventional protein kinase C isoforms mediate phorbol ester-induced lysophosphatidic acid LPA1 receptor phosphorylation

Using C9 cells stably expressing LPA1 receptors fused to the enhanced green fluorescent protein, it was observed that activation of protein kinase C induced a rapid and strong increase in the phosphorylation state of these receptors. Overnight incubation with phorbol esters markedly decreased the amount of conventional (α, βI, βII and γ) and novel (δ) but not atypical (ζ) immunodetected PKC isoforms, this treatment blocks the action of protein kinase on receptor function and phosphorylation. Bis-indolylmaleimide I a general, non-subtype selective protein kinase C inhibitor, and Gö 6976, selective for the isoforms α and β, were also able to block LPA1 receptor desensitization and phosphorylation; hispidin, isoform β-selective blocker partially avoided receptor desensitization.Expression of dominant-negative protein kinase C α or β II mutants and knocking down the expression of these kinase isozymes markedly decreased phorbol ester-induced LPA1 receptor phosphorylation without avoiding receptor desensitization. This effect was blocked by bis-indolyl-maleimide and Gö 6976, suggesting that these genetic interventions were not completely effective. It was also observed that protein kinase C α and β II isozymes co-immunoprecipitate with LPA1 receptors and that such an association was further increased by cell treatments with phorbol esters or lysophosphatidic acid. Our data suggest that conventional protein kinase C α and β isozymes modulate LPA1 receptor phosphorylation state. Receptor desensitization appears to be a more complex process that might involve additional elements.

Abstract 261: Differential Regulation of Apolipoprotein E Secretion from Primary Human Macrophages by Different Protein Kinase C Isoforms

Macrophage-specific apolipoprotein E (apoE) secretion plays an important role in atherosclerosis. Recently, we established that inhibition of protein kinase A, phospholipase C, protein phosphatase 2B and calcium results in a reduction in apoE secretion. Research suggests PKC may have a role in this process, but the isoforms and pathways involved in human macrophages are unknown. Here, we investigate a role for PKC in regulating apoE secretion from primary human monocyte-derived macrophages (HMDM). HMDM treated with pan-PKC inhibitors (Calphostin C, Ro-318220, Bisindolylmaleimide I) that primarily target the classical and novel PKC isoforms result in a dose-dependent decrease in apoE secretion. Further, the specific classical PKC inhibitor, Gö6976, also resulted in a reduction in apoE secretion. Metabolic labelling studies indicate that CalpC and Gö6976 have no effect on apoE synthesis or degradation but directly inhibit the secretion of apoE. This occurred post-golgi as evidenced by normal glycosylation of apoE on 2D electrophoresis. Direct visualisation of apoE-GFP containing vesicles in HMDM by live cell confocal microscopy demonstrated a marked reduction in vesicular speed from 0.42μm/s in control cells to 0.14μm/s or 0.15μm/s by CalpC and Ro-318220 respectively. Finally, siRNA knockdown (∼70%) of the classical PKC isoforms, PKCalpha and PKCbeta, resulted in decreased basal apoE secretion (∼50%), whereas knockdown of PKCdelta (novel PKC isoform) did not. Interestingly, preliminary data demonstrate that treatment of HMDM with the PKCtheta or PKCzeta pseudosubstrate inhibitor increases apoE secretion from HMDM, whereas both the PKCepsilon translocation inhibitory peptide and siRNA knockdown of PKCepsilon did not affect apoE secretion. This suggests that apoE secretion is differentially regulated by different and specific PKC isoforms. Inhibition of PKCalpha and/or PKCbeta decreases apoE secretion by targeting the apoE trafficking pathway in a post-golgi vesicular compartment, whereas inhibition of PKCzeta (atypical PKC isoform) or PKCtheta (novel PKC isoform) increases apoE secretion. Future studies will further confirm and investigate the role of these novel and atypical PKC isoforms in regulating apoE secretion.

Abstract 175: Atypical PKC knockdown alters TGFβ signaling potential

Abstract Transforming growth factor beta (TGFβ) regulates many important cellular processes including growth control and cell migration. In epithelial tumor cells, TGFβ signaling is fundamentally altered and induces epithelial to mesenchymal transition (EMT) leading to increased migratory potential. We have recently reported that the inhibition of atypical PKC isoforms (aPKC) extends TGFβ induced Smad signaling by altering receptor trafficking. Here we report that siRNA targeting of aPKC in A549 lung adenocarcinoma cells extends temporal P-Smad2 signaling but does not alter the transcript levels of Snail and Slug, as assessed by qPCR analysis. Interestingly, TGFβ was not observed to have as robust an increase in MMP9 and PAI-1 levels in aPKC-silenced cells compared to cells transfected with control siRNA. To further analyze the functional consequences, we assessed EMT by immunofluorescence microscopy analysis as well as immunoblotting for epithelial and mesenchymal cell markers. We found that TGFβ-dependent EMT was significantly reduced in aPKC silenced cells. We also uncovered that in addition to increased P-Smad2 signaling, P38 MAPK phosphorylation was increased and extended in aPKC silenced cells. Finally, we discovered that aPKC phosphorylates Par6 on a conserved serine residue that has been shown to regulate EMT and metastasis in breast cancer. Taken together, we believe that aPKC regulates several aspects of TGFβ signaling, including P-Smad2, P-P38 and P-Par6 levels. Consequently, convergence of these signals in aPKC silenced cells leads to reduced EMT and migratory potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 175. doi:1538-7445.AM2012-175

Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation

Most retinal proliferative diseases involve blood-retinal barrier (BRB) breakdown, exposing the retinal pigment epithelium (RPE) to thrombin, which triggers cell transformation, proliferation and migration through the activation of PAR-1. These processes require the assembly of contractile stress fibers containing actin and non-muscle myosin II, which allow cell movement upon phosphorylation of the myosin light chains (MLCs). PKC family of kinases promotes agonist-mediated contraction in smooth muscle and endothelial cells through the activation of its downstream target, the PKC-potentiated inhibitory protein of 17 kDa (CPI-17), which specifically inhibits MLC phosphatase. Although the participation of PKC in RPE cell transdifferentiation has been suggested, the role of PKC/CPI-17 signaling has not been investigated. The purpose of this study was to analyze the involvement of specific PKC isoenzymes and their effector protein CPI-17 in thrombin-induced MLC phosphorylation and actin stress fiber assembly in RPE cells. Rat RPE cells in primary culture were shown to respond to thrombin stimulation by activation of conventional, novel and atypical PKC isoforms and the downstream phosphorylation of CPI-17 and MLC, which in turn promoted actin stress fiber assembly. These effects were prevented by the pharmacological inhibition of conventional PKC isoenzymes (Ro-32-0432) and novel PKCδ (rottlerin and δV1-1 antagonist peptide), as well as by myristoylated pseudosubstrates specifically directed to conventional and atypical PKC isoforms. Thrombin effects were mimicked by phorbol 12-myristate 13-acetate (PMA), further confirming the involvement of diacylglycerol (DAG)-sensitive classical and novel PKC isoforms in thrombin-induced actin cytoskeleton modification. The present work shows, for the first time, the functional expression of the oncoprotein CPI-17 in RPE cells and suggests that PKC/CPI-17 signaling is involved in the control of actin cytoskeletal remodeling leading to cell motility in RPE cells exposed to thrombin, and hence could contribute to the development of proliferative eye diseases.