Etta Livneh - Academia.edu (original) (raw)
Papers by Etta Livneh
Journal of Biological Chemistry, 1993
Release of large soluble NH2-terminal fragments of the amyloid precursor protein (APP) of Alzheim... more Release of large soluble NH2-terminal fragments of the amyloid precursor protein (APP) of Alzheimer's disease was measured in two Swiss 3T3 fibroblast cell lines (designated SF1.4 and SF3.2), overexpressing the a subtype of protein kinase C, and in two control cell lines (SC1 and SC2) (Eldar, H., Zisman, Y., Ullrich, A,, and Livneh, E. (1990) J. Bioi. Chem. 265, 13290-13296). Basal release of APP was significantly increased in SF1.4 cells, but not in S F 3 2 cells, relative to controls. Phorbol 12-myristate 13-acetate, an activator of protein kinase C, elicited a concentrationdependent increase in APP release in all four cell lines. However, the estimated ECao for this effect was lower in the two cell lines overexpressing protein kinase C-a (7 and 6 nM, in SF1.4 and SF3.2 cells, respectively) than in control SC1 and SC2 cells (56 and 22 nM, respectively). The absolute amount of A P P released by maximal concentrations of phorbol ester was not altered by overexpression of protein kinase Ca. The protein kinase C inhibitor H-7 (l-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) significantly reduced the response to phorbol esters in control (SC1) cells but not in cells (SF1.4) that overexpress protein kinase Ca. Levels of cell-associated APP were slightly elevated, and rates of APP turnover were unchanged, in SF1.4 cells relative to controls. However, cell-associated A P P levels were lower in SF3.2 cells than in controls. The results demonstrate that protein kinase Ca regulates APP release in Swiss 3T3 fibroblasts, and perhaps in other tissues, including brain, and may be the isozyme that mediates receptor-evoked release of APP. The amyloid precursor protein (APP)' of Alzheimer's disease (AD) is an integral membrane glycoprotein (Kang et al., 1987; Dyrks et al., 1988; Weidemann et al., 1989) found in a wide variety of cell types including brain neurons and glia, cultured neuronal cells, platelets, fibroblasts, and embryonic kidney cells. Proteolytic processing of APP generates a 39-42143-amino acid peptide, the amyloid p peptide (A/?), that is the principal component of the amyloid which accumulates in
Journal of Biological Chemistry, 1990
Journal of Biological Chemistry, 1986
Journal of Biological Chemistry, 1993
Incumbent of the Helena Rubinstein Career Development Chair. 1 Recipient of an Yigal Allon Fellow... more Incumbent of the Helena Rubinstein Career Development Chair. 1 Recipient of an Yigal Allon Fellowship and the incumbent of the Shloimo and Michla Tomarin Career Development Chair in Membrane Physiology. To whom reprint requests should be addressed.
The EMBO Journal, 1986
Communicated by J.Schlessinger Binding of epidermal growth factor (EGF) to its receptor results i... more Communicated by J.Schlessinger Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affimity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor.
Journal of lipid mediators, 1993
Studies carried out in many laboratories have demonstrated the activation of phospholipase D (PLD... more Studies carried out in many laboratories have demonstrated the activation of phospholipase D (PLD) by a variety of receptor agonists and in many cell types. The signal-dependent formation of phosphatidic acid (PA), by PLD-catalyzed hydrolysis of phosphatidylcholine (PC), may represent a novel and ubiquitous signal transduction pathway in mammalian cells. The mode(s) of coupling between agonist receptors and PLD activation are not well understood. Studies utilizing NIH-3T3 fibroblasts indicated that PLD activation by different mitogens involves distinct mechanisms. Protein kinase C (PKC) seems to play a role both as a mediator and as a modulator of PLD activation. The role of PKC was further examined in Swiss/3T3-derived fibroblasts which stably overexpress PKC-alpha. In these cells, both basal and agonist-stimulated PLD activity are higher than in control cells. In vitro analysis of PLD activity in detergent-solubilized cell membranes, utilizing exogenous C6-NBD-PC as fluorescent su...
Cell Death & Disease, 2014
Senescence is characterized by permanent cell-cycle arrest despite continued viability and metabo... more Senescence is characterized by permanent cell-cycle arrest despite continued viability and metabolic activity, in conjunction with the secretion of a complex mixture of extracellular proteins and soluble factors known as the senescence-associated secretory phenotype (SASP). Cellular senescence has been shown to prevent the proliferation of potentially tumorigenic cells, and is thus generally considered a tumor suppressive process. However, some SASP components may act as pro-tumorigenic mediators on premalignant cells in the microenvironment. A limited number of studies indicated that protein kinase C (PKC) has a role in senescence, with different isoforms having opposing effects. It is therefore important to elucidate the functional role of specific PKCs in senescence. Here we show that PKCη, an epithelial specific and anti-apoptotic kinase, promotes senescence induced by oxidative stress and DNA damage. We further demonstrate that PKCη promotes senescence through its ability to upregulate the expression of the cell cycle inhibitors p21 Cip1 and p27 Kip1 and enhance transcription and secretion of interleukin-6 (IL-6). Moreover, we demonstrate that PKCη creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor, whereas the expression of IL-8 is specifically suppressed by PKCη. Thus, the presence/absence of PKCη modulates major components of SASP. Furthermore, we show that the human polymorphic variant of PKCη, 374I, that exhibits higher kinase activity in comparison to WT-374V, is also more effective in IL-6 secretion, p21 Cip1 expression and the promotion of senescence, further supporting a role for PKCη in senescence. As there is now considerable interest in senescence activation/elimination to control tumor progression, it is first crucial to reveal the molecular regulators of senescence. This will improve our ability to develop new strategies to harness senescence as a potential cancer therapy in the future.
The Journal of cell biology, 1986
The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with... more The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.
Frontiers in Immunology
Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and pred... more Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished t...
Molecular and Cellular Biology, 1991
We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the ... more We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing th...
Journal of Cell Biology, 1988
Interactions between membrane proteins are believed to be important for the induction of trans- m... more Interactions between membrane proteins are believed to be important for the induction of trans- membrane signaling. Endocytosis is one of the re- sponses which is regulated by both intracellular and extracellular signals. To study such interactions, we have measured the lateral mobility and rate of endocy- tosis of epidermal growth factor receptor in three transfected NIH-3T3 cell lines (HER84, HER22,
The emergence of chemoresistance in neoplastic cells is one of the major obstacles in cancer ther... more The emergence of chemoresistance in neoplastic cells is one of the major obstacles in cancer therapy. Autophagy was recently reported as one of the mechanisms that promote chemoresistance in cancer cells by protecting from apoptosis and driving senescence. Thus, understanding the role of autophagy and its underlying signaling pathways is crucial for the development of new therapeutic strategies to overcome chemoresistance. We have previously reported that PKCη is a stress-induced kinase that confers resistance in breast cancer cells against chemotherapy by inducing senescence. Here we show that PKCη promotes autophagy induced by ER and oxidative stress and facilitates the transition from autophagy to senescence. We demonstrate that PKCη knockdown reduces both the autophagic flux and markers of senescence. Additionally, using autophagy inhibitors, such as chloroquine and 3-methyladenine, we show that PKCη and autophagy are required for establishing senescence in MCF-7 in response to ...
Biochemical Journal, 1992
Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PK... more Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PKC-L), only PKC-epsilon has been characterized in great detail. In order to compare the regulatory and catalytic properties of these new kinases, we have expressed PKC-delta, -epsilon, -zeta and PKC-L as recombinant proteins from their cDNAs in insect cells via baculovirus vectors and in mammalian COS-1 cells. After expression in insect cells, phorbol ester binding and kinase activities of the group B enzymes were compared with the respective activities of a member of group A, PKC-gamma. Although PKC-delta and PKC-L(eta) bind phorbol ester to a similar or the same extent as PKC-gamma, they show a distinctively different behaviour towards conventional PKC substrates such as histone, myelin basic protein, protamine and protamine sulphate, suggesting either that phorbol esters are not able to fully activate these enzymes or that their substrate specificities are very different from those of ...
The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to the... more The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of the ZAP70 protein tyrosine kinase, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here we demonstrate that ZAP70 in activated T cells is regulated by the cyclophilin A (CypA) peptidyl-prolyl cis-trans isomerase (PPIase), and that this regulation is blocked by the CypA inhibitor, cyclosporin A (CsA). We found that TCR crosslinking promoted the rapid and transient formation of cyclophilin A (CypA)-ZAP70 complexes, which were dependent on Lck-mediated phosphorylation of ZAP70. Cyclosporin A (CsA) inhibited CypA binding to ZAP70 and prevented CypA-ZAP70 colocalization at the cell membrane. In addition, fluorescent cell staining and imaging analyses rev...
Anticancer Research, 2012
Novel biomarkers which may serve as therapeutic targets are essential for lung cancer treatment. ... more Novel biomarkers which may serve as therapeutic targets are essential for lung cancer treatment. Here we investigated the prognostic significance of protein kinase Cη (PKCη), a cell cycle regulator involved in tumorigenesis and chemotherapy resistance, in patients diagnosed with non-small cell lung cancer (NSCLC). Sixty-three chemotherapy-naïve patients were examined for PKCη by immunohistochemistry and divided into PKCη H-Score tertiles (low, intermediate and high). Time until event (relapse or mortality) within one year was determined using Cochran-Armitage test and Cox proportional hazards regression model. The distribution of patients according to clinical stage 1-4 was: 27%, 5%, 26% and 42%, respectively. PKCη overexpression was associated with advanced stage (p=0.03) and the risk for an event (p=0.045). Patients of the lowest tertile were less likely to experience an event. PKCη is a novel prognostic marker in NSCLC that may predict poor prognosis. The use of PKCη-specific inh...
Molecules, 2021
Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to the... more 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 PK...
Phorbol 12-myristate 13-acetate (PMA) stimulated radiolabelled choline uptake and incorporation i... more Phorbol 12-myristate 13-acetate (PMA) stimulated radiolabelled choline uptake and incorporation into phosphatidylcholine (PtdCho) in a timeand concentration-dependent manner in wild-type NIH 3T3 fibroblasts. The accumulation of labelled choline induced by PMA was paralled by an increase in choline mass. The results implicate protein kinase C (PKC) in the regulation of choline uptake. In order to address the PKCsubtype specificity of this response, a study was undertaken in Swiss 3T3 fibroblast cells, which normally express very low levels of PKCa. A retroviral expression system was used to introduce the genes for PKCa and neomycin resistance (used for selection) into the cells. Two resulting lines expressed PKCa at levels that were 20-fold higher than those found in the control (neomycinresistant) line, or in the wild-type cells. In control Swiss 3T3
Proceedings of the National Academy of Sciences
Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in... more Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cance...
Glia
Alzheimer's disease (AD) is the primary cause of age‐related dementia. Pathologically, AD is ... more Alzheimer's disease (AD) is the primary cause of age‐related dementia. Pathologically, AD is characterized by synaptic loss, the accumulation of β‐amyloid peptides and neurofibrillary tangles, glial activation, and neuroinflammation. Whereas extensive studies focused on neurons and activation of microglia in AD, the role of astrocytes has not been well‐characterized. Protein kinase C (PKC) was also implicated in AD; however, its role in astrocyte activation was not elucidated. Using the 5XFAD mouse model of AD, we show that PKC‐eta (PKCη), an astrocyte‐specific stress‐activated and anti‐apoptotic kinase, plays a role in reactive astrocytes. We demonstrate that PKCη staining is highly enriched in cortical astrocytes in a disease‐dependent manner and in the vicinity of amyloid‐β peptides plaques. Moreover, activation of PKCη, as indicated by its increased phosphorylation levels, is exhibited mainly in cortical astrocytes derived from adult 5XFAD mice. PKCη activation was associated with elevated levels of reactive astrocytic markers and upregulation of the pro‐inflammatory cytokine interleukin 6 (IL‐6) compared to littermate controls. Notably, inhibiting the kinase activity of PKCη in 5XFAD astrocyte cultures markedly increased the levels of secreted IL‐6—a phenomenon that was also observed in wild‐type astrocytes stimulated by inflammatory cytokines (e.g., TNFα, IL‐1). Similar increase in the release of IL‐6 was also observed upon inhibition of either the mammalian target of rapamycin (mTOR) or the protein phosphatase 2A (PP2A). Our findings suggest that the mTOR‐PKCη‐PP2A signaling cascade functions as a negative feedback loop of NF‐κB‐induced IL‐6 release in astrocytes. Thus, we identify PKCη as a regulator of neuroinflammation in AD.
Journal of Biological Chemistry, 1993
Release of large soluble NH2-terminal fragments of the amyloid precursor protein (APP) of Alzheim... more Release of large soluble NH2-terminal fragments of the amyloid precursor protein (APP) of Alzheimer's disease was measured in two Swiss 3T3 fibroblast cell lines (designated SF1.4 and SF3.2), overexpressing the a subtype of protein kinase C, and in two control cell lines (SC1 and SC2) (Eldar, H., Zisman, Y., Ullrich, A,, and Livneh, E. (1990) J. Bioi. Chem. 265, 13290-13296). Basal release of APP was significantly increased in SF1.4 cells, but not in S F 3 2 cells, relative to controls. Phorbol 12-myristate 13-acetate, an activator of protein kinase C, elicited a concentrationdependent increase in APP release in all four cell lines. However, the estimated ECao for this effect was lower in the two cell lines overexpressing protein kinase C-a (7 and 6 nM, in SF1.4 and SF3.2 cells, respectively) than in control SC1 and SC2 cells (56 and 22 nM, respectively). The absolute amount of A P P released by maximal concentrations of phorbol ester was not altered by overexpression of protein kinase Ca. The protein kinase C inhibitor H-7 (l-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) significantly reduced the response to phorbol esters in control (SC1) cells but not in cells (SF1.4) that overexpress protein kinase Ca. Levels of cell-associated APP were slightly elevated, and rates of APP turnover were unchanged, in SF1.4 cells relative to controls. However, cell-associated A P P levels were lower in SF3.2 cells than in controls. The results demonstrate that protein kinase Ca regulates APP release in Swiss 3T3 fibroblasts, and perhaps in other tissues, including brain, and may be the isozyme that mediates receptor-evoked release of APP. The amyloid precursor protein (APP)' of Alzheimer's disease (AD) is an integral membrane glycoprotein (Kang et al., 1987; Dyrks et al., 1988; Weidemann et al., 1989) found in a wide variety of cell types including brain neurons and glia, cultured neuronal cells, platelets, fibroblasts, and embryonic kidney cells. Proteolytic processing of APP generates a 39-42143-amino acid peptide, the amyloid p peptide (A/?), that is the principal component of the amyloid which accumulates in
Journal of Biological Chemistry, 1990
Journal of Biological Chemistry, 1986
Journal of Biological Chemistry, 1993
Incumbent of the Helena Rubinstein Career Development Chair. 1 Recipient of an Yigal Allon Fellow... more Incumbent of the Helena Rubinstein Career Development Chair. 1 Recipient of an Yigal Allon Fellowship and the incumbent of the Shloimo and Michla Tomarin Career Development Chair in Membrane Physiology. To whom reprint requests should be addressed.
The EMBO Journal, 1986
Communicated by J.Schlessinger Binding of epidermal growth factor (EGF) to its receptor results i... more Communicated by J.Schlessinger Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affimity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor.
Journal of lipid mediators, 1993
Studies carried out in many laboratories have demonstrated the activation of phospholipase D (PLD... more Studies carried out in many laboratories have demonstrated the activation of phospholipase D (PLD) by a variety of receptor agonists and in many cell types. The signal-dependent formation of phosphatidic acid (PA), by PLD-catalyzed hydrolysis of phosphatidylcholine (PC), may represent a novel and ubiquitous signal transduction pathway in mammalian cells. The mode(s) of coupling between agonist receptors and PLD activation are not well understood. Studies utilizing NIH-3T3 fibroblasts indicated that PLD activation by different mitogens involves distinct mechanisms. Protein kinase C (PKC) seems to play a role both as a mediator and as a modulator of PLD activation. The role of PKC was further examined in Swiss/3T3-derived fibroblasts which stably overexpress PKC-alpha. In these cells, both basal and agonist-stimulated PLD activity are higher than in control cells. In vitro analysis of PLD activity in detergent-solubilized cell membranes, utilizing exogenous C6-NBD-PC as fluorescent su...
Cell Death & Disease, 2014
Senescence is characterized by permanent cell-cycle arrest despite continued viability and metabo... more Senescence is characterized by permanent cell-cycle arrest despite continued viability and metabolic activity, in conjunction with the secretion of a complex mixture of extracellular proteins and soluble factors known as the senescence-associated secretory phenotype (SASP). Cellular senescence has been shown to prevent the proliferation of potentially tumorigenic cells, and is thus generally considered a tumor suppressive process. However, some SASP components may act as pro-tumorigenic mediators on premalignant cells in the microenvironment. A limited number of studies indicated that protein kinase C (PKC) has a role in senescence, with different isoforms having opposing effects. It is therefore important to elucidate the functional role of specific PKCs in senescence. Here we show that PKCη, an epithelial specific and anti-apoptotic kinase, promotes senescence induced by oxidative stress and DNA damage. We further demonstrate that PKCη promotes senescence through its ability to upregulate the expression of the cell cycle inhibitors p21 Cip1 and p27 Kip1 and enhance transcription and secretion of interleukin-6 (IL-6). Moreover, we demonstrate that PKCη creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor, whereas the expression of IL-8 is specifically suppressed by PKCη. Thus, the presence/absence of PKCη modulates major components of SASP. Furthermore, we show that the human polymorphic variant of PKCη, 374I, that exhibits higher kinase activity in comparison to WT-374V, is also more effective in IL-6 secretion, p21 Cip1 expression and the promotion of senescence, further supporting a role for PKCη in senescence. As there is now considerable interest in senescence activation/elimination to control tumor progression, it is first crucial to reveal the molecular regulators of senescence. This will improve our ability to develop new strategies to harness senescence as a potential cancer therapy in the future.
The Journal of cell biology, 1986
The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with... more The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.
Frontiers in Immunology
Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and pred... more Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished t...
Molecular and Cellular Biology, 1991
We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the ... more We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing th...
Journal of Cell Biology, 1988
Interactions between membrane proteins are believed to be important for the induction of trans- m... more Interactions between membrane proteins are believed to be important for the induction of trans- membrane signaling. Endocytosis is one of the re- sponses which is regulated by both intracellular and extracellular signals. To study such interactions, we have measured the lateral mobility and rate of endocy- tosis of epidermal growth factor receptor in three transfected NIH-3T3 cell lines (HER84, HER22,
The emergence of chemoresistance in neoplastic cells is one of the major obstacles in cancer ther... more The emergence of chemoresistance in neoplastic cells is one of the major obstacles in cancer therapy. Autophagy was recently reported as one of the mechanisms that promote chemoresistance in cancer cells by protecting from apoptosis and driving senescence. Thus, understanding the role of autophagy and its underlying signaling pathways is crucial for the development of new therapeutic strategies to overcome chemoresistance. We have previously reported that PKCη is a stress-induced kinase that confers resistance in breast cancer cells against chemotherapy by inducing senescence. Here we show that PKCη promotes autophagy induced by ER and oxidative stress and facilitates the transition from autophagy to senescence. We demonstrate that PKCη knockdown reduces both the autophagic flux and markers of senescence. Additionally, using autophagy inhibitors, such as chloroquine and 3-methyladenine, we show that PKCη and autophagy are required for establishing senescence in MCF-7 in response to ...
Biochemical Journal, 1992
Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PK... more Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PKC-L), only PKC-epsilon has been characterized in great detail. In order to compare the regulatory and catalytic properties of these new kinases, we have expressed PKC-delta, -epsilon, -zeta and PKC-L as recombinant proteins from their cDNAs in insect cells via baculovirus vectors and in mammalian COS-1 cells. After expression in insect cells, phorbol ester binding and kinase activities of the group B enzymes were compared with the respective activities of a member of group A, PKC-gamma. Although PKC-delta and PKC-L(eta) bind phorbol ester to a similar or the same extent as PKC-gamma, they show a distinctively different behaviour towards conventional PKC substrates such as histone, myelin basic protein, protamine and protamine sulphate, suggesting either that phorbol esters are not able to fully activate these enzymes or that their substrate specificities are very different from those of ...
The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to the... more The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of the ZAP70 protein tyrosine kinase, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here we demonstrate that ZAP70 in activated T cells is regulated by the cyclophilin A (CypA) peptidyl-prolyl cis-trans isomerase (PPIase), and that this regulation is blocked by the CypA inhibitor, cyclosporin A (CsA). We found that TCR crosslinking promoted the rapid and transient formation of cyclophilin A (CypA)-ZAP70 complexes, which were dependent on Lck-mediated phosphorylation of ZAP70. Cyclosporin A (CsA) inhibited CypA binding to ZAP70 and prevented CypA-ZAP70 colocalization at the cell membrane. In addition, fluorescent cell staining and imaging analyses rev...
Anticancer Research, 2012
Novel biomarkers which may serve as therapeutic targets are essential for lung cancer treatment. ... more Novel biomarkers which may serve as therapeutic targets are essential for lung cancer treatment. Here we investigated the prognostic significance of protein kinase Cη (PKCη), a cell cycle regulator involved in tumorigenesis and chemotherapy resistance, in patients diagnosed with non-small cell lung cancer (NSCLC). Sixty-three chemotherapy-naïve patients were examined for PKCη by immunohistochemistry and divided into PKCη H-Score tertiles (low, intermediate and high). Time until event (relapse or mortality) within one year was determined using Cochran-Armitage test and Cox proportional hazards regression model. The distribution of patients according to clinical stage 1-4 was: 27%, 5%, 26% and 42%, respectively. PKCη overexpression was associated with advanced stage (p=0.03) and the risk for an event (p=0.045). Patients of the lowest tertile were less likely to experience an event. PKCη is a novel prognostic marker in NSCLC that may predict poor prognosis. The use of PKCη-specific inh...
Molecules, 2021
Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to the... more 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 PK...
Phorbol 12-myristate 13-acetate (PMA) stimulated radiolabelled choline uptake and incorporation i... more Phorbol 12-myristate 13-acetate (PMA) stimulated radiolabelled choline uptake and incorporation into phosphatidylcholine (PtdCho) in a timeand concentration-dependent manner in wild-type NIH 3T3 fibroblasts. The accumulation of labelled choline induced by PMA was paralled by an increase in choline mass. The results implicate protein kinase C (PKC) in the regulation of choline uptake. In order to address the PKCsubtype specificity of this response, a study was undertaken in Swiss 3T3 fibroblast cells, which normally express very low levels of PKCa. A retroviral expression system was used to introduce the genes for PKCa and neomycin resistance (used for selection) into the cells. Two resulting lines expressed PKCa at levels that were 20-fold higher than those found in the control (neomycinresistant) line, or in the wild-type cells. In control Swiss 3T3
Proceedings of the National Academy of Sciences
Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in... more Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cance...
Glia
Alzheimer's disease (AD) is the primary cause of age‐related dementia. Pathologically, AD is ... more Alzheimer's disease (AD) is the primary cause of age‐related dementia. Pathologically, AD is characterized by synaptic loss, the accumulation of β‐amyloid peptides and neurofibrillary tangles, glial activation, and neuroinflammation. Whereas extensive studies focused on neurons and activation of microglia in AD, the role of astrocytes has not been well‐characterized. Protein kinase C (PKC) was also implicated in AD; however, its role in astrocyte activation was not elucidated. Using the 5XFAD mouse model of AD, we show that PKC‐eta (PKCη), an astrocyte‐specific stress‐activated and anti‐apoptotic kinase, plays a role in reactive astrocytes. We demonstrate that PKCη staining is highly enriched in cortical astrocytes in a disease‐dependent manner and in the vicinity of amyloid‐β peptides plaques. Moreover, activation of PKCη, as indicated by its increased phosphorylation levels, is exhibited mainly in cortical astrocytes derived from adult 5XFAD mice. PKCη activation was associated with elevated levels of reactive astrocytic markers and upregulation of the pro‐inflammatory cytokine interleukin 6 (IL‐6) compared to littermate controls. Notably, inhibiting the kinase activity of PKCη in 5XFAD astrocyte cultures markedly increased the levels of secreted IL‐6—a phenomenon that was also observed in wild‐type astrocytes stimulated by inflammatory cytokines (e.g., TNFα, IL‐1). Similar increase in the release of IL‐6 was also observed upon inhibition of either the mammalian target of rapamycin (mTOR) or the protein phosphatase 2A (PP2A). Our findings suggest that the mTOR‐PKCη‐PP2A signaling cascade functions as a negative feedback loop of NF‐κB‐induced IL‐6 release in astrocytes. Thus, we identify PKCη as a regulator of neuroinflammation in AD.