Domain Of Protein Kinase C Research Articles

Identification of protein kinase C domains involved in its translocation induced by propofol

Propofol is widely used for general anesthesia and sedation; however, the mechanisms of its anesthetic and adverse effects are not fully understood. We have previously shown that propofol activates protein kinase C (PKC) and induces its translocation in a subtype-specific manner. The purpose of this study was to identify the PKC domains involved in propofol-induced PKC translocation. The regulatory domains of PKC consist of C1 and C2 domains, and the C1 domain is subdivided into the C1A and C1B subdomains. Mutant PKCα and PKCδ with each domain deleted were fused with green fluorescent protein (GFP) and expressed in HeLa cells. Propofol-induced PKC translocation was observed by time-lapse imaging using a fluorescence microscope. The results showed that persistent propofol-induced PKC translocation to the plasma membrane was abolished by the deletion of both C1 and C2 domains in PKCα and by the deletion of the C1B domain in PKCδ. Therefore, propofol-induced PKC translocation involves the C1 and C2 domains of PKCα and the C1B domain of PKCδ. We also found that treatment with calphostin C, a C1 domain inhibitor, abolished propofol-induced PKCδ translocation. In addition, calphostin C inhibited the propofol-induced phosphorylation of endothelial nitric oxide synthase (eNOS). These results suggest that it may be possible to modulate the exertion of propofol effects by regulating the PKC domains involved in propofol-induced PKC translocation.

Expanding the Paradigm of Structure-Based Drug Design: Molecular Dynamics Simulations Support the Development of New Pyridine-Based Protein Kinase C-Targeted Agonists.

Protein kinase C (PKC) modulators hold therapeutic potential for various diseases, including cancer, heart failure, and Alzheimer's disease. Targeting the C1 domain of PKC represents a promising strategy; the available protein structures warrant the design of PKC-targeted ligands via a structure-based approach. However, the PKC C1 domain penetrates the lipid membrane during binding, complicating the design of drug candidates. The standard docking-scoring approach for PKC lacks information regarding the dynamics and the membrane environment. Molecular dynamics (MD) simulations with PKC, ligands, and membranes have been used to address these shortcomings. Previously, we observed that less computationally intensive simulations of just ligand-membrane interactions may help elucidate C1 domain-binding prospects. Here, we present the design, synthesis, and biological evaluation of new pyridine-based PKC agonists implementing an enhanced workflow with ligand-membrane MD simulations. This workflow holds promise to expand the approach in drug design for ligands targeted to weakly membrane-associated proteins.

Structural Basis of Agonist Capture by Regulatory C1 Domain of PKC

Diacylglycerol (DAG)‐sensing C1 domains of Protein kinase C (PKC) isoforms are targets for therapeutic intervention in cancers, neurodegenerative disorders, and HIV latency reversal. High‐resolution structural information on the C1 domains complexed to DAG or exogenous PKC agonists remained elusive for nearly 3 decades. This is attributed to the extreme hydrophobicity of the C1 complexes and the need to create an optimal membrane‐mimicking environment to facilitate the complex formation and crystallization. We overcame these challenges and obtained high‐resolution crystal structures of the C1B domain from PKCδ (C1Bδ) complexed to DAG and four potent PKC agonists of pharmacological relevance. The structures reveal precise stereospecific binding modes and hydrophobic interactions formed by the lipophilic groups of DAG and DAG lactone (AJH‐836) with C1Bδ. Comparison of the C1Bδ complexes with PDBu (a tumor‐promoting phorbol ester) and Prostratin (a candidate for the HIV latency reversal) provides a structural rationale for their distinct PKC‐mediated cellular responses. The complex with Ingenol mebutate, a drug clinically approved for the treatment of actinic keratosis, shows how its angelyl group completes the hydrophobic rim of C1Bδ. The analysis of all five complexes enabled us to assign the functional roles to residues that form the consensus sequence of DAG‐sensitive C1 domains. We conclude that the ligand promiscuity of C1 domains stems from a subset of conserved residues that can “cage” chemically diverse ligands while interfacing with the membrane environment. These structures serve as valuable guides for pushing the frontiers of PKC pharmacology and suggest a new strategy for the crystallization of peripheral membrane proteins.

Structural basis of agonist capture by PKC-regulatory C1 domain

Diacylglycerol (DAG)-binding C1 domains of Protein kinase C (PKC) isoforms are prime drug targets for intervention in cancers, neurodegenerative diseases, and HIV/AIDS progression. For nearly three decades, structural characterization of C1-DAG and exogenous agonist interactions at atomic resolution has remained a “dark side” of PKC pharmacology. This is attributed to immense technical difficulties in developing optimal hydrophobic scaffolds to facilitate the crystallization of amphipathic protein-ligand complexes.

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.

Protein kinase C fusion proteins are paradoxically loss of function in cancer

Within the AGC kinase superfamily, gene fusions resulting from chromosomal rearrangements have been most frequently described for protein kinase C (PKC), with gene fragments encoding either the C-terminal catalytic domain or the N-terminal regulatory moiety fused to other genes. Kinase fusions that eliminate regulatory domains are typically gain of function and often oncogenic. However, several quality control pathways prevent accumulation of aberrant PKC, suggesting that PKC fusions may paradoxically be loss of function. To explore this topic, we used biochemical, cellular, and genome editing approaches to investigate the function of fusions that retain the portion of the gene encoding either the catalytic domain or regulatory domain of PKC. Overexpression studies revealed that PKC catalytic domain fusions were constitutively active but vulnerable to degradation. Genome editing of endogenous genes to generate a cancer-associated PKC fusion resulted in cells with detectable levels of fusion transcript but no detectable protein. Hence, PKC catalytic domain fusions are paradoxically loss of function as a result of their instability, preventing appreciable accumulation of protein in cells. Overexpression of a PKC regulatory domain fusion suppressed both basal and agonist-induced endogenous PKC activity, acting in a dominant-negative manner by competing for diacylglycerol. For both catalytic and regulatory domain fusions, the PKC component of the fusion proteins mediated the effects of the full-length fusions on the parameters examined, suggesting that the partner protein is dispensable in these contexts. Taken together, our findings reveal that PKC gene fusions are distinct from oncogenic fusions and present a mechanism by which loss of PKC function occurs in cancer.

Structural Basis of Protein Kinase C Tumor‐Promoting Response And Membrane Recruitment

Protein Kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. The hallmark of their activation is translocation to lipid membranes. Dysregulation of PKC signaling has been implicated in cancer progression, cardiac disease, and Alzheimer’s disease. One of the major challenges in the field is to understand the structural basis of PKC activation and regulation ‐‐ at the level of atomic resolution.Here, we applied NMR and FRET spectroscopy to gain insight into the structure and protein‐membrane interactions of the regulatory domain of PKC that consist of three peripheral membrane modules: the tandem conserved homology‐1 (C1) and conserved homology‐2 (C2) domains. The C1 domains undergo membrane insertion upon binding their natural agonist, diacylglycerol, as well as tumor‐promoting phorbol esters and other small‐molecule agonists. C2 associates with anionic membranes upon binding Ca2+. Simultaneous association of all domains with membranes ensures the coincidence of four signals: Ca2+ ions, diacylglycerol, phosphatidylserine, and phosphatidylinositol‐4,5‐bisphosphate.Here, we report the first structure of the C1 domain complexed to a potent tumor‐promoting agent, phorbol 12,13‐dibutyrate (PDBu), in the membrane‐mimicking environment. The structure of the complex shows that C1 has to undergo a conformational change in order to capture the membrane‐embedded ligand. The structure also revealed the role of individual amino acids in the interactions with PDBu and membrane‐mimicking environment. To probe how different agonists modulate the interaction of C1 with membranes, we conducted NMR paramagnetic relaxation enhancement (PRE) experiments with bicelles doped with paramagnetic lipid. The PRE patterns revealed that the C1 domains adopt tilted rather than vertical orientation in the membrane. Our data on four different agonists indicate that C1 domains dictate the general orientation of the agonist complexes in the membrane, but the agonists determine the depth of insertion. This provides a plausible explanation for the differences in the potency of these ligands to achieve the acute and chronic activation response of PKC.To gain insight into the mechanism of coincidence detection, we developed protein‐to‐membrane FRET assay that we used to characterize the interplay of the C1 and C2 domains in the membrane association step. The FRET experiments revealed a clear picture of how tumor‐promoting agents can elicit constitutive activation of PKC through simultaneous recruitment of C1 domains to membranes and sensitization of the C2 domain to Ca2+.Support or Funding InformationNIH R01 GM108998; NSF CHE‐1905116; Welch Foundation A‐1784Agonist‐guided membrane interaction of C1B domain of PKCδ studied by paramagnetic NMR. (A) Schematic representation of the approach. (B) Chemical representations of the different agonists used in the study.Figure 1

Fusion Gene TANC2‐PRKCA Reveals Another Mechanism for Loss of Protein Kinase C Function in Cancer

The protein kinase C (PKC) family of serine/threonine kinases plays a critical role in signal transduction, and dysregulation of PKC activity is implicated in a variety of diseases, including cancer. Over 60 fusion genes involving PKC isozymes have been identified in an array of cancers, making PKC the most frequently fused member of the AGC kinase superfamily. With previous studies in the laboratory revealing a tumor suppressive role for PKC, we sought to characterize these PKC fusions and determine whether they may be loss‐of‐function in cancer. For our analysis, we selected TANC2‐PRKCA, a fusion gene identified in lung squamous cell carcinoma. This fusion retains the C‐terminal catalytic domain of PKC while having a truncated N‐terminal regulatory moiety due to fusion with TANC2. Overexpression in cells reveals that the fusion protein lacks the conserved priming phosphorylations necessary for proper PKC maturation yet is constitutively active, a result of the loss of the autoinhibitory pseudosubstrate from within the PKC regulatory moiety. As an additional consequence, the fusion is also markedly unstable, unable to adopt the stable, autoinhibited conformation of wild‐type PKC. We thus reasoned that TANC2‐PKCα, in an active and degradation‐sensitive conformation, would be too unstable to significantly accumulate in cells. To address this, we utilized CRISPR/Cas9 gene‐editing to express the TANC2‐PRKCA fusion in cells. While the fusion mRNA was detected in the CRISPR‐edited clones, the fusion protein was not, consistent with the protein's inherent instability. Thus, while the loss of autoinhibition renders the fusion constitutively active, the resulting instability leads to a dramatic reduction of its steady‐state levels, making it paradoxically loss‐of‐function. While overexpression studies showed that the fusion protein is more ubiquitinated compared to wild‐type protein, inhibition of proteasomal, lysosomal, and endosomal degradative pathways in the CRISPR‐edited cells did not result in accumulation of the fusion protein. Knockdown of RINCK (TRIM41), an E3 ligase for PKC, and CHIP (STUB1), an E3 ligase for misfolded proteins, by siRNA similarly did not result in accumulation of the fusion protein. Taken together, our analyses show that the TANC2‐PKCα fusion protein is constitutively active but locked in an open and unstable conformation, resulting in its downregulation. Fusion of PKC's catalytic domain to other genes thus presents another mechanism by which loss of PKC activity can occur in cancer, supporting a tumor suppressive role for PKC.Support or Funding InformationThis work was supported by NIH R35 GM122523 (ACN). ANV, TRB, and CEA were supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), and CEA was supported by an NSF Graduate Research Fellowship (DGE1144086).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C.

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain–targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

Cancer‐Associated Fusions of the Protein Kinase C Kinase Domain are Loss‐of‐Function

Gain‐of‐function fusion proteins have emerged as novel cancer drivers and potential therapeutic targets, exemplified by the successful targeting of the oncogenic Bcr‐Abl gene fusion that drives chronic myelogenous leukemia. Over 60 fusions of protein kinase C (PKC) family members with other genes have been identified in diverse cancers, accounting for the most abundant fusions in the AGC family of kinases. Given our recent findings that PKC isozymes generally function as tumor suppressors, we explored whether such fusions may provide another mechanism for loss of PKC function in cancer. Specifically, we addressed the cellular activity of fusions in which the C‐terminal catalytic domain of PKC was retained, but the N‐terminal regulatory domain was truncated by fusion to the N‐terminus of unrelated genes: these fusions are TANC2‐PRKCA, which encodes a protein containing the first 46 residues of the adaptor protein TANC2 fused to the N‐terminus of the C2 domain of PKCα, and GGA2‐PRKCB, which encodes a protein containing the first 158 residues of the Golgi trafficking protein GGA2 fused to the C1A domain of PKCβ. Overexpression in COS7 cells revealed that both fusion proteins are constitutively active as assessed by the FRET‐based C Kinase Activity Reporter (CKAR). This constitutive activity is not a result of the various proteins to which PKC is fused, but rather is a consequence of loss of the autoinhibitory pseudosubstrate from within the PKC regulatory moiety; constructs of PKC deleted in the pseudosubstrate are also constitutively active in cells. However, the fusion proteins are unstable as assessed by their accelerated turnover compared to wild‐type PKC following inhibition of protein synthesis. Because active PKC is in a degradation‐sensitive conformation, we reasoned that PKC fusion proteins would be too unstable for significant levels to accumulate in cancer cells. To test this, we used CRISPR/Cas9 to edit one allele of PKCα to express the TANC2‐PRKCA fusion observed in cancer. Western blot analysis of these clones revealed readily detectable PKCα from the intact allele but no detectable fusion protein, verifying that the PKC fusion is indeed too unstable to accumulate in cells. Thus, while the fusion proteins are rendered constitutively active due to a loss of autoinhibitory constraints, their inherent instability results in a dramatic reduction of their steady‐state levels, effectively making them loss‐of‐function. PKC fusion proteins therefore present another mechanism by which loss of PKC activity can occur in the context of cancer, supporting a tumor suppressive role for PKC.Support or Funding InformationThis work was supported by NIH R35 GM122523 (ACN). ANV, TRB, and CEA were supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), and CEA was supported by an NSF Graduate Research Fellowship (DGE1144086).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biochemical Studies and Virtual Screening of Phytochemical Reservoir from Northeastern Indian Plants to Identify Anti-Cancer Agents

In the present study, medicinal phytochemicals found in plants of north-eastern India were virtually screened against the C1b regulatory domain of Protein Kinase C (PKC), an important enzyme in cancer cell-biology. Amongst the few top hits, the molecules Gallic acid (GA) and Epigallocatechin gallate (EGCG) were identified through an agonist competition assay. Both phytochemical are non-toxic and non-mutagenic. An in vitro PKC binding assay confirmed that GA and EGCG were strong ligands of PKC. Both the phytochemicals formed numerous hydrogen bonding and hydrophobic interactions with the amino acids lining the ligand binding pocket of the C1b domain of PKC. They were able to induce translocation of PKC to the plasma membrane in MDAMB-231 breast cancer cells. Furthermore, GA and EGCG dose-dependently reduced the viability of breast cancer cells by arresting the cell-cycle. Eventually, mitochondrial pathway of apoptosis was identified as the mechanism of loss of breast cancer cellular viability. Thus, our study has revealed that tea phytochemicals GA and EGCG exhibit anti-cancer activity by acting as strong ligands of PKC. This study encourages the further identification of newer novel and even more potent such PKC-directed phytochemicals from natural herbs & beverages as a part of herbal anti-cancer therapy.

Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation.

Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the role of this tryptophan, we generated W22A, W22K, W22D, W22Y, and W22F substitutions in the full-length Munc13-1, expressed the GFP-tagged constructs in Neuro-2a cells, and measured their membrane translocation in response to phorbol ester treatment by imaging of the live cells using confocal microscopy. The extent of membrane translocation followed the order, wild-type > W22K > W22F > W22Y > W22A > W22D. The phorbol ester binding affinity of the wild-type Munc13-1C1 domain and its mutants was phosphatidylserine (PS)-dependent following the order, wild-type > W22K > W22A ≫ W22D in both 20% and 100% PS. Phorbol ester affinity was higher for Munc13-1 than the C1 domain. While Munc13-1 translocated to the plasma membrane, the C1 domain translocated to internal membranes in response to phorbol ester. Molecular dynamics (80 ns) studies reveal that Trp-22 is relatively less flexible than the homologous Trp-22 of PKCδ and PKCθ. Results are discussed in terms of the overall negative charge state of the Munc13-1C1 domain and its possible interaction with the PS-rich plasma membrane. This study shows that Trp-588 is an important structural element for ligand binding and membrane translocation in Munc13-1.

The C1 domain of Vav3, a novel potential therapeutic target

Vav1/2/3 comprise a protein family with guanyl nucleotide exchange activity for Rho and Rac as well as with motifs conferring adapter activity. Biologically, Vav1 plays a critical role in hematologic cell signaling, whereas Vav2/3 have a wider tissue distribution, but all 3 Vav proteins are implicated in cancer development. A structural feature of Vav1/2/3 is the presence of an atypical C1 domain, which possesses close structural homology to the typical C1 domains of protein kinase C but which fails to bind the second messenger diacylglycerol or the potent analogs, the phorbol esters. Previously, we have shown that five residues in the Vav1 C1 domain are responsible for its lack of phorbol ester binding. Here, we show that the lack of phorbol ester binding of Vav3 has a similar basis. We then explore the consequences of phorbol ester binding to a modified Vav3 in which the C1 domain has been altered to allow phorbol ester binding. We find both disruption of the guanyl nucleotide exchange activity of the modified Vav 3 as well as a shift in localization to the membrane upon phorbol ester treatment. This change in localization is associated with altered interactions with other signaling proteins. The studies provide a first step in assessing the potential for the design of custom C1 domain targeted molecules selective for the atypical C1 domains of Vav family proteins.

Phorbol esters dPPA/dPA promote furin expression involving transcription factor CEBPβ in neuronal cells.

Using high-throughput small molecule screening targeting furin gene, we identified that phorbol esters dPPA (12-Deoxyphorbol 13-phenylacetate 20-acetate) and dPA (12-Deoxyphorbol 13-acetate) significantly increased furin protein and mRNA expression in SH-SY5Y cells. This effect was prevented by PKC (protein kinase C) inhibitor calphostin C but not Ro318220, suggesting that the C1 domain, rather than the catalytic domain of PKC plays an important role. Luciferase assay revealed that nucleotides −7925 to −7426 were sufficient to mediate dPPA/dPA enhancement of furin P1 promoter activity. RNA interference of transcriptional factors CEBPβ (CCAAT/enhancer-binding protein β) and GATA1 revealed that knockdown of CEBPβ significantly attenuated the effect of dPPA on furin expression. Pharmacological inhibition of ERK and PI3K but not TGFβ receptor diminished the up-regulation of furin by dPPA. These results suggested that in neuronal cells, transcriptional activation of furin by dPPA/dPA may be initiated by C1 domain containing proteins including PKC; the intracellular signaling involves ERK and PI3K and transcription factor CEBPβ.

Beyond the affinity for protein kinase C: exploring 2-phenyl-3-hydroxypropyl pivalate analogues as C1 domain-targeting ligands

Over the past fifteen years, we reported the design and synthesis of different series of compounds targeting the C1 domain of protein kinase C (PKC) that were based on various templates.

Diacylglycerol lactones targeting the structural features that distinguish the atypical C1 domains of protein kinase C ζ and ι from typical C1 domains.

To explore the feasibility of developing ligands targeted to the atypical C1 domains of protein kinase C ζ and ι, we have prepared diacylglycerol lactones substituted with hydrophilic groups on their side chains, which potentially could interact with the arginine residues that distinguish the atypical C1 domains of PKCζ and PKCι from typical C1 domains, and we have measured their binding to mutated versions of the C1b domain of PKCδ that incorporate one or more of these arginine residues. The most selective of the diacylglycerol lactones showed only a 10-fold reduction in binding affinity with the triple arginine mutant (N7R/S10R/L20R) compared to the wild-type, whereas phorbol 12,13-dibutyrate showed a 6000-fold loss of affinity. Molecular modeling confirms that these ligands are indeed able to interact with the arginine residues. Our results show that dramatic changes in selectivity can be obtained through appropriate substitution of diacylglycerol lactones.

Fusions involving protein kinase C and membrane-associated proteins in benign fibrous histiocytoma

Benign fibrous histiocytoma (BFH) is a mesenchymal tumor that most often occurs in the skin (so-called dermatofibroma), but may also appear in soft tissues (so-called deep BFH) and in the skeleton (so-called non-ossifying fibroma). The origin of BFH is unknown, and it has been questioned whether it is a true neoplasm. Chromosome banding, fluorescence in situ hybridization, single nucleotide polymorphism arrays, RNA sequencing, RT-PCR and quantitative real-time PCR were used to search for recurrent somatic mutations in a series of BFH. BFHs were found to harbor recurrent fusions of genes encoding membrane-associated proteins (podoplanin, CD63 and LAMTOR1) with genes encoding protein kinase C (PKC) isoforms PRKCB and PRKCD. PKCs are serine–threonine kinases that through their many phosphorylation targets are implicated in a variety of cellular processes, as well as tumor development. When inactive, the amino-terminal, regulatory domain of PKCs suppresses the activity of their catalytic domain. Upon activation, which requires several steps, they typically translocate to cell membranes, where they interact with different signaling pathways. The detected PDPN-PRKCB, CD63-PRKCD and LAMTOR1-PRKCD gene fusions are all predicted to result in chimeric proteins consisting of the membrane-binding part of PDPN, CD63 or LAMTOR1 and the entire catalytic domain of the PKC. This novel pathogenetic mechanism should result in constitutive kinase activity at an ectopic location. The results show that BFH indeed is a true neoplasm, and that distorted PKC activity is essential for tumorigenesis. The findings also provide means to differentiate BFH from other skin and soft tissue tumors.This article is part of a Directed Issue entitled: Rare cancers.

PKCη Regulates the TGFβ3-induced Chondevrepogenic Differentiation of Human Mesenchymal Stem Cell.

Transforming growth factor (TGF) family is well known to induce the chondevrepogenic differentiation of mesenchymal stem cells (MSC). However, the precise signal transduction pathways and underlying factors are not well known. Thus the present study aims to evaluate the possible role of C2 domain in the chondevrepogenic differentiation of human mesenchymal stem cells. To this end, 145 C2 domains in the adenovirus were individually transfected to hMSC, and morphological changes were examined. Among 145 C2 domains, C2 domain of protein kinase C eta (PKCη) was selected as a possible chondevrepogenic differentiation factor for hMSC. To confirm this possibility, we treated TGFβ3, a well known chondevrepogenic differentiation factor of hMSC, and examined the increased-expression of glycosaminoglycan (GAG), collagen type II (COL II) as well as PKCη using PT-PCR, immunocytochemistry and Western blot analysis. To further evaluation of C2 domain of PKCη, we examined morphological changes, expressions of GAG and COL II after transfection of PKCη -C2 domain in hMSC. Overexpression of PKCη-C2 domain induced morphological change and increased GAG and COL II expressions. The present results demonstrate that PKCη involves in the TGF-β3-induced chondevrepogenic differentiation of hMSC, and C2 domain of PKCη has important role in this process.

The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells

Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20μM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10μM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that activation of the PKCα and ERK1/2 pathways participate in regulating neuronal differentiation. Furthermore, since PKC has been classified as one of the cognitive kinases, and activation of PKC is considered a potential therapeutic strategy for the treatment of cognitive disorders, our findings suggest that HMI-1b11 represents a promising lead compound in research aimed to prevent or counteract memory impairment.

A novel mutation in the C2 domain of protein kinase C gamma associated with spinocerebellar ataxia type 14

Spinocerebellar ataxia type 14 (SCA14; Online Mendelian Inheritance in Man, OMIM #605361) is an autosomal dominant neurodegenerative disorder characterized by mild and slowly progressive cerebellar ataxia. Additional symptoms, including myoclonus, spasticity, dystonia, disturbance of deep sensation, and cognitive impairment, are observed in some patients [1, 2]. The SCA14 is caused by mutations in the protein kinase C gamma (PRKCG) gene (OMIM *176980), which encodes protein kinase C gamma (PKCc). PRKCG contains 18 exons, and the PKCc protein is composed of regulatory C1 and C2 domains and catalytic C3 and C4 domains. Most PRKCG mutations described till this date lie in the C1 domain [3, 4], while no mutations have been found in the C2 domain so far. Here we describe, for the first time, a mutation in the C2 domain in a patient with a typical SCA14 phenotype. A 58-year-old Japanese woman visited our hospital with gait disturbance and lisping, the latter being noticed by her husband. At the age of 40, she frequently experienced unsteadiness. At the age of 55, she visited a clinic after experiencing a fall, and brain imaging revealed cerebellar atrophy. Her father suffered from gait disturbance and dysphagia in his late 70s, and her father’s brother (the patient’s uncle) and his nephew (the patient’s cousin) had an unsteady gait. This family history indicated autosomal dominant inheritance (Fig. 1a). Neurological examination revealed cerebellar ataxia, including slurred speech, saccadic eye movement, and limb and truncal ataxia. Although there was a mild decrease in the deep sensations in her lower limbs, her gait disturbance was mostly consistent with ataxic gait. At her first visit to our institution, she had a score of 27/100 on the International Cooperative Ataxia Rating Scale. Blood, respiratory function, and cerebrospinal fluid tests yielded no remarkable findings. Brain magnetic resonance imaging demonstrated cerebellar atrophy, particularly in the upper part (Fig. 1b), and N-isopropyl-p-[123I]-iodoamphetamine single-photon emission computed tomography revealed decreased blood flow only in the upper cerebellum. She had no autonomic failure or cognitive dysfunction. Clinically, her symptoms corresponded with typical SCA14 features. While she had a family history of autosomal dominant inheritance, genetic testing for SCA types 1, 2, 3, 6, 7, 8, and 31 and dentatorubral–pallidoluysian atrophy, genes relatively frequent in Japan, did not show the presence of abnormal alleles. Next, to test for mutations in PRKCG, we performed genomic PCR followed by a direct sequencing analysis. This revealed the presence of the single-base substitution c.518T[G in exon five involving amino acid change p.Ile173Ser (Fig. 1c). This substitution has not been reported, either as a single-nucleotide polymorphism or as a mutation. The amino acid p.Ile173 residue is conserved among PRKCG proteins from mammals and zebrafish (Fig. 1d). To ascertain whether this was a disease-associated mutation, we first performed direct sequencing of this region in 200 Japanese healthy controls and found that none of them carried this alternative allele. Next, we employed three computer algorithms to predict the effect of this novel mutation: Sorting Intolerant From Tolerant (SIFT, http://sift.bii.a-star.edu.sg/), Polymorphism Phenotyping v2 T. Ueda (&) T. Seki K. Katanazaka K. Sekiguchi K. Kobayashi F. Kanda T. Toda Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, Chuo-ku, Kobe 650-0017, Japan e-mail: taueda@med.kobe-u.ac.jp