Loss Of Protein Kinase C Activity Research Articles

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.

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.

Involvement of desmoplakin phosphorylation in the regulation of desmosomes by protein kinase C, in HeLa cells.

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.

An early loss in membrane protein kinase C activity precedes the excitatory amino acid-induced death of primary cortical neurons.

Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons following cerebral ischemia. However, in primary neuronal cultures, cytotoxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This apparent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on membrane PKC activity was determined in cortical neurons using an assay that measures only PKC that is active in isolated membranes, which can be used to differentiate active enzyme from that associated with membranes in an inactive state. A 15-min exposure of day 14-18 cortical neurons to 100 microM glutamate, AMPA, or NMDA caused a rapid and persistent loss in membrane PKC activity, which by 4 h fell to 30-50% of that in control cultures. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphorylation of the MARCKS protein, a PKC-selective substrate, was reduced in intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inactivation by NMDA and AMPA was dependent on extracellular Ca2+, but less so on Na+, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca2+ entry through specific routes because the bulk increase in intracellular free [Ca2+] effected by the Ca2+ ionophore ionomycin did not cause the inactivation of PKC. The results indicate that the pattern of PKC activity in neurons killed by glutamate, NMDA, and AMPA in vitro is consistent with that observed in neurons injured by cerebral Ischemia in vivo.

Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields.

We examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5-1,000 mV/cm p-p) were applied for 1 h to cells (10(7)/ml) in Teflon chambers at 37 degrees C in the presence or absence of 2 microM phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100-1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA, EFs (33-330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents.

The expression of 80K/MARCKS, a major substrate of protein kinase C (PKC), is down-regulated through both PKC-dependent and -independent pathways. Effects of bombesin, platelet-derived growth factor, and cAMP.

We have examined the regulation of expression of 80K/MARCKS, a major and specific protein kinase C (PKC) substrate of Swiss 3T3 fibroblasts. Addition of bombesin (10 nM) to confluent quiescent cultures of these cells induced a dramatic and sustained down-regulation of 80-kDa mRNA and protein levels to a minimum of 5% of control within 8 and 48 h, respectively, without depletion of PKC activity. In contrast, the effect of phorbol 12,13-dibutyrate on 80K/MARCKS mRNA levels was transient, and recovery of these transcripts correlated with the loss of PKC activity. The ability of bombesin to down-regulate 80K/MARCKS mRNA levels was dose-dependent (ED50 0.5 nM) and was abolished by both the specific bombesin antagonist [Leu13 psi (CH2NH),Leu14]bombesin and by prior depletion of PKC. Of a range of agents tested, platelet-derived growth factor (PDGF), but not insulin or Ca2+ ionophore, also down-regulated 80K/MARCKS mRNA to 24% of control within 5 h. Prior down-regulation of PKC abolished the effect of PDGF at a concentration of 7 ng/ml. Surprisingly, at higher doses (25 ng/ml), PDGF induced the down-regulation of 80K/MARCKS mRNA in a PKC-independent manner. Furthermore, elevation of cAMP, either through receptor-mediated mechanisms (e.g. prostaglandin E1) or by direct stimulation of adenylate cyclase (e.g. forskolin), also caused a marked dose-dependent depletion of 80K/MARCKS mRNA levels, which were further reduced by co-administration with cAMP-phosphodiesterase inhibitors. The rate of transcription of the 80K/MARCKS gene was unaltered by treatment of cells with either bombesin, PDGF, or forskolin/1-methyl-3-isobutylxanthine. These results indicate a role for both PKC-dependent and -independent pathways in growth factor-induced down-regulation of 80K/MARCKS expression, through a post-transcriptional mechanism.

Insulin, oxytocin, and vasopressin stimulate protein kinase C activity in adipocyte plasma membranes.

Incubation of isolated rat adipocytes with insulin, vasopressin, or oxytocin increased plasma membrane-bound protein kinase C (PKC) activity by 100-400%. PKC activity was assayed by a procedure that is virtually background-free, thus permitting assay of protein kinase activity in highly diluted samples of solubilized membranes. Hormone-dependent increases in PKC activity were limited to plasma membranes. Stimulation of the kinase was half-maximal with 70 pM insulin, and the hormone effect was rapid. Oxytocin and vasopressin produced effects on PKC similar to insulin, but the magnitude of the vasopressin stimulation exhibited seasonal variations. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a loss of PKC activity from the cytosol and a gain in plasma membrane activity, indicative of translocation of the enzyme. With activity measurements it was not possible to determine if insulin stimulated a translocation of the kinase. However, Western blot analysis of plasma membranes with polyclonal antibodies directed against PKC suggest that at least some of the insulin-stimulated PKC activity resulted from enzyme translocation.

Alteration in protein kinase C activity and subcellular distribution in sickle erythrocytes

In agreement with previous data, membrane protein phosphorylation was found to be altered in intact sickle cells (SS) relative to intact normal erythrocytes (AA). Similar changes were observed in their isolated membranes. The involvement of protein kinase C (PKC) in this process was investigated. The membrane PKC content in SS cells, measured by [3H]phorbol ester binding, was about 6-times higher than in AA cells. In addition, the activity of the enzyme, measured by histone phosphorylation was also found to be increased in SS cell membranes but decreased in their cytosol compared to the activity in AA cell membranes and cytosol. The increase in membrane PKC activity was observed mostly in the light fraction of SS cells, fractionated by density gradient, whereas the decrease in cytosolic activity was only observed in the dense fraction. PKC activity, measured in cells from the blood of reticulocyte-rich patients, exhibited an increase in both membranes and cytosol, thus explaining some of the effects observed in the SS cell light fraction, which is enriched in reticulocytes. The increase in PKC activity in the membranes of SS cells is partly explained by their young age but the loss of PKC activity in their cytosol, particularly in that of the dense fraction, seems to be specific to SS erythrocytes. The relative decrease in membrane PKC activity between the dense and the light fractions of SS cells might be related to oxidative inactivation of the enzyme.

Role of substrate in imparting calcium and phospholipid requirements to protein kinase C activation.

The role of substrate in influencing the cofactor requirements of the phospholipid- and Ca2+-dependent protein kinase C (PKC) was investigated by using several substrates. All of the substrates tested, including histone, troponin I, myosin light chain, protamine, poly(arginine, serine) (PAS), poly(lysine, serine) (PLS), and myelin basic protein (MBP), were found to interact with and aggregate phospholipid vesicles as well as phosphatidylserine (PS)-Triton mixed micelles. Phosphorylation of these different substrates by PKC indicated the presence of three distinct substrate categories: substrates such as protamine requiring no cofactors; substrates such as PLS, PAS, and MBP requiring only the presence of phospholipid; and substrates such as histone, myosin light chain, and troponin I requiring the presence of Ca2+ and phospholipid. Diacylglycerol was a major cofactor only with category C substrates. These different requirements correlated with the interaction of the substrate with phospholipid and/or enzyme. The substrates in category A interacted strongly with and aggregated PKC in a binary mixture. In the absence of Ca2+, PKC bound to substrates of category B directly but not to substrates in category C. Thus, substrate-enzyme binding eliminated the Ca2+ requirement of phosphorylation, and aggregation of substrate-enzyme complex eliminated the phospholipid requirements as well. Substrate-phospholipid interaction and substrate phosphorylation were inhibited by increasing salt concentrations, but the amount needed depended upon the substrate. Loss of PKC activity appeared to coincide with loss of substrate-PS aggregation while dissociation of PKC from the membranes required much higher salt concentrations. Poly(L-lysine) and poly(L-arginine), two potent inhibitors of PKC, also showed substrate-dependent inhibition characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol 12-myristate 13-acetate induces prolactin secretion from rat anterior pituitary gland by the activation of protein kinase-C

The purpose of this study was to characterize the action of phorbol 12-myristate 13-acetate (PMA), a tumor-promoting agent, on rat anterior pituitary gland, focusing the attention on prolactin secretion. PMA elicited a significant increase in prolactin secretion without affecting phosphatidylinositol turnover, considered as an early post-receptor event controlling PRL secretion. However incubation of anterior pituitary glands with PMA caused a loss of protein kinase-C activity in cytoplasm concomitant with an increased enzyme activity in the membrane. The action of PMA on prolactin secretion seems to be mainly dependent from the redistribution and activation of protein kinase-C. In fact, the phorbol ester did not affect pituitary cAMP and cGMP metabolism either in basal conditions or after theophylline.