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Author: An-Angela Ngoc Van
Publisher:
ISBN:
Category :
Languages : en
Pages : 152
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Book Description
Protein kinase C (PKC) plays a critical role in cell signaling and homeostasis, regulating biological processes such as proliferation, differentiation, and apoptosis. Its dysregulation is associated with a multitude of pathophysiological states, with recent analyses of disease-associated mutations indicating that loss of PKC function is generally associated with cancer and gain of function with degenerative diseases. This dissertation expands on the mechanisms of PKC dysregulation in cancer, focusing on how gene fusions or mutations that disrupt autoinhibition cause loss of PKC. In the first part of the dissertation, newly identified PKC gene fusions in cancer encoding proteins that retain either the PKC catalytic or regulatory domain were characterized. Overexpression of catalytic domain fusions in cells revealed that they are constitutively active, as assessed using biosensors and other cellular assays. However, their inability to adopt an autoinhibited conformation resulted in their marked stability compared to full-length PKC. To assess whether these fusions were too unstable to accumulate at endogenous levels, CRISPR/Cas9-mediated gene editing was used to engineer a fusion of TANC2 with PRKCA. While the fusion mRNA was detected in the engineered cells, no detectable protein was expressed. Thus, the catalytic domain fusions are paradoxically loss-of-function. Characterization of a regulatory domain fusion revealed a dominant-negative role for the protein, suppressing the activity of wild-type PKC. The second part of the dissertation focused on additional mechanisms by which PKC is dysregulated in cancer. This work showed that [1] PKC that cannot be autoinhibited is subject to dephosphorylation by the phosphatase PHLPP and unphosphorylated PKC is sensitive to degradation; [2] inactivating mutants of PKC can be dominant-negative by sequestering common titratable components; and [3] impairment of the regulatory domains causes mislocalization of PKC. Taken together, these studies illustrate the diverse ways in which PKC function is lost in cancer, allowing cancer cells to overcome this cellular brake to tumor growth.
Author: An-Angela Ngoc Van
Publisher:
ISBN:
Category :
Languages : en
Pages : 152
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Book Description
Protein kinase C (PKC) plays a critical role in cell signaling and homeostasis, regulating biological processes such as proliferation, differentiation, and apoptosis. Its dysregulation is associated with a multitude of pathophysiological states, with recent analyses of disease-associated mutations indicating that loss of PKC function is generally associated with cancer and gain of function with degenerative diseases. This dissertation expands on the mechanisms of PKC dysregulation in cancer, focusing on how gene fusions or mutations that disrupt autoinhibition cause loss of PKC. In the first part of the dissertation, newly identified PKC gene fusions in cancer encoding proteins that retain either the PKC catalytic or regulatory domain were characterized. Overexpression of catalytic domain fusions in cells revealed that they are constitutively active, as assessed using biosensors and other cellular assays. However, their inability to adopt an autoinhibited conformation resulted in their marked stability compared to full-length PKC. To assess whether these fusions were too unstable to accumulate at endogenous levels, CRISPR/Cas9-mediated gene editing was used to engineer a fusion of TANC2 with PRKCA. While the fusion mRNA was detected in the engineered cells, no detectable protein was expressed. Thus, the catalytic domain fusions are paradoxically loss-of-function. Characterization of a regulatory domain fusion revealed a dominant-negative role for the protein, suppressing the activity of wild-type PKC. The second part of the dissertation focused on additional mechanisms by which PKC is dysregulated in cancer. This work showed that [1] PKC that cannot be autoinhibited is subject to dephosphorylation by the phosphatase PHLPP and unphosphorylated PKC is sensitive to degradation; [2] inactivating mutants of PKC can be dominant-negative by sequestering common titratable components; and [3] impairment of the regulatory domains causes mislocalization of PKC. Taken together, these studies illustrate the diverse ways in which PKC function is lost in cancer, allowing cancer cells to overcome this cellular brake to tumor growth.
Author: Marcelo G. Kazanietz
Publisher: Springer Science & Business Media
ISBN: 1607615436
Category : Medical
Languages : en
Pages : 492
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Book Description
Protein kinase C (PKC), a family of serine-threonine kinases, rocketed to the forefront of the cancer research field in the early 1980’s with its identification as an effector of phorbol esters, natural products with tumor promoting activity. Phorbol esters had long been of interest to the cancer research field due to early studies in the mouse skin carcinogenesis model, which showed that prolonged topical application of phorbol esters promoted the formation of skin tumors on mice previously treated with mutagenic agents. Research in the last years has established key roles for PKC isozymes in the control of cell proliferation, migration, adhesion, and malignant transformation. In addition, there is a large body of evidence linking PKC to invasion and cancer cell metastasis. Moreover, it is now well established that the expression of PKC isozymes is altered in various types of cancers. More importantly, small molecule inhibitors have been developed with significant anti-cancer activity. The relevance of PKC isozymes in cancer signaling is therefore remarkable. This book will have 4 sections. There will be 23 chapters. Each section will have a brief introduction by an expert in the field (~ 1-2 pages).
Author: Corina Elena Antal
Publisher:
ISBN: 9781321900941
Category :
Languages : en
Pages : 147
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Book Description
The serine/threonine protein kinase C (PKC) family has been extensively studied over the last 35 years, yet fundamental questions regarding the regulation of its signaling and its dysregulation in disease remain unanswered. PKC is involved in a multitude of cellular processes and precise control of the amplitude of PKC signaling is essential for maintaining cellular homeostasis. This thesis expands on the knowledge of PKC at the molecular level by unveiling how intramolecular conformational changes tune the affinity of PKC for its ligands, and at the pathophysiological level by overturning a 30-year-old scientific dogma on the role of PKC in cancer. First, mechanistic studies reveal that processing phosphorylations promote intramolecular interactions that clamp PKC in a closed conformation to prevent signaling in the absence of agonists, but allow efficient activation in response to small changes in agonist levels. These studies offer novel means of therapeutically targeting PKC with molecules or peptides that can either disrupt these interactions to activate PKC or maintain them closed to inhibit PKC activity. Second, analysis of PKC gene family mutations in human cancers reveals that they are loss-of-function, and that this loss-of-function confers a growth advantage, both in vitro and in vivo. These data suggest that therapies should focus on restoring, not inhibiting, PKC activity in the treatment of cancer. Taken together, this thesis identifies novel strategies to modulate PKC activity in therapies and, most importantly, establishes that PKC is a tumor suppressor.
Author: Timothy R. Baffi
Publisher:
ISBN:
Category :
Languages : en
Pages : 212
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Book Description
Protein kinase C (PKC) isozymes transduce the myriad of signals downstream of phospholipid hydrolysis that potentiate an array of cellular processes including proliferation, differentiation, migration, and memory. PKC function is dysregulated in a variety of pathological states, including cancer and neurodegenerative disease. To maintain signaling fidelity, PKCs rely upon precise regulatory mechanisms that orchestrate the phosphorylations and conformational transitions that specify their signaling output. This thesis describes the molecular mechanisms by which PKC phosphorylation and autoinhibition depends upon the kinases PDK1 and mTORC2, and is opposed by PHLPP phosphatases, to produce a primed enzyme that is appropriately tuned to respond to activating signals. Specifically, we uncover the molecular basis for the controversial role of mTORC2 in AGC kinase activation by identifying a novel and conserved mTOR phosphorylation site in the C-terminal tail. Phosphorylation of this, which we term the TOR-Interaction Motif (TIM), promotes PDK1 phosphorylation of the activation loop and intramolecular autophosphorylation of the hydrophobic motif to control activation of PKC and related AGC kianse Akt. Examination of the interrelated processes of phosphorylation and autoinhibition unveils a critical role for the pseudosubstrate in protecting PKC from dephosphorylation by phosphatase PHLPP1, which selectively promotes the dephosphorylation and degradation of aberrantly active PKCs to provide a PKC quality control mechanism. High-throughput protein-level analysis from patient samples reveals that PKC quality control is a critical signaling node that sets PKC expression levels and serves as a prominent loss-of-function mechanism to impair PKC tumor-suppressive function in cancer. Critically, diseases driven by PKC dysregulation rely upon impaired PKC quality control. LOF PKC mutations in chordoid glioma act in a dominant-negative fashion to globally suppress PKC output; whereas, GOF PKC mutations in spinocerebellar ataxia drive phosphoproteome-wide changes in the cerebellum. Taken together, this thesis expands upon biochemical mechanisms of PKC maturation to identify the structural and molecular determinants of PKC phosphorylation and implicates PHLPP1 as the master regulator of PKC signaling fidelity through PKC quality control. This work is not only relevant to the pathology of disease-associated mutations in cancer and neurodegenerative disease, but also to the development of therapeutics that attempt to modulate PKC activity by targeting these regulatory mechanisms.
Author: Marcelo G. Kazanietz
Publisher: Humana Press
ISBN: 9781607615453
Category : Medical
Languages : en
Pages : 494
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Book Description
Protein kinase C (PKC), a family of serine-threonine kinases, rocketed to the forefront of the cancer research field in the early 1980’s with its identification as an effector of phorbol esters, natural products with tumor promoting activity. Phorbol esters had long been of interest to the cancer research field due to early studies in the mouse skin carcinogenesis model, which showed that prolonged topical application of phorbol esters promoted the formation of skin tumors on mice previously treated with mutagenic agents. Research in the last years has established key roles for PKC isozymes in the control of cell proliferation, migration, adhesion, and malignant transformation. In addition, there is a large body of evidence linking PKC to invasion and cancer cell metastasis. Moreover, it is now well established that the expression of PKC isozymes is altered in various types of cancers. More importantly, small molecule inhibitors have been developed with significant anti-cancer activity. The relevance of PKC isozymes in cancer signaling is therefore remarkable. This book will have 4 sections. There will be 23 chapters. Each section will have a brief introduction by an expert in the field (~ 1-2 pages).
Author: Ayse Basak Engin
Publisher: Springer Nature
ISBN: 3030498441
Category : Medical
Languages : en
Pages : 415
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Book Description
Protein phosphorylation via protein kinases is an inevitable process that alters physiological and pathological functions of the cells. Thus, protein kinases play key roles in the regulation of cell life or death decisions. Protein kinases are frequently a driving factor in a variety of human diseases including aging and cellular senescence, immune system and endothelial dysfunctions, cancers, insulin resistance, cholestasis and neurodegenerative diseases, as well as bacterial resistance in persistent infections. Recent developments in quantitative proteomics provide important opinions on kinase inhibitor selectivity and their modes of action in the biological context. Protein Kinase-mediated Decisions Between Life and Death aims to have the reader catch insights about up-to-date opinions on “Protein Kinases” related pathways that threaten human health and life. As “Protein Kinases” are related to many health problems, clinicians, basic science researchers and students need this information. Chapter “Signal Transduction in Immune Cells and Protein Kinases” is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
Author: Alexandra C. Newton
Publisher: The Rosen Publishing Group
ISBN: 9781588290687
Category : Medical
Languages : en
Pages : 608
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Book Description
The first compilation of classic and emerging laboratory techniques for the study of the biochemistry and cell biology of protein kinase C (PKC). Described in step-by-step detail, these methods can be easily used to explore the structure, function, regulation, subcellular localization, and macromolecular interactions of protein kinase C.. Each protocol is introduced in the context of PKC function and regulation and contains many notes on how best to deal with the problems that may occur. Comprehensive and authoritative, Protein Kinase C Protocols is a timely compilation of biophysical, biochemical, cell biological, and molecular biological approaches that brings protein kinase C research into any laboratory interested in studying it.
![Deregulation of Protein Kinase C[gamma] in Disease PDF](https://books.google.com/books/content/images/frontcover/vAxRzwEACAAJ?fife=w80-h100)
Author: Caila Ann Pilo
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
This thesis aims to elucidate the mechanisms governing protein kinase C[gamma] (PKC[gamma]) autoinhibition and activity and how impairing these mechanisms in different ways leads to pathogenesis in the context of both neurodegeneration and cancer. The family of serine/threonine protein kinase C (PKC) isozymes transduce a multitude of signals within the cell in response to the generation of second messengers from membrane phospholipids. The conventional isozyme PKC[gamma] is reversibly activated by Ca2+ and diacylglycerol, which allows the enzyme to adopt an open state in which downstream signaling can occur. Here, we show how impairing autoinhibition can result in either gain or loss of PKC[gamma] activity. First, we use a variety of biochemical assays to show that PKC[gamma] variants linked to the neurodegenerative disorder spinocerebellar ataxia type 14 (SCA14) enhance basal activity by impacting C1 domain autoinhibitory constraints, while evading quality control degradation mechanisms mediated by the phosphatase PHLPP. We also use a transgenic mutant mouse model of ataxia to establish that mice with enhanced PKC[gamma] basal activity exhibit significant changes in their cerebellar phosphoproteome. Additionally, we show an inverse correlation between level of mutant biochemical defect and average age of symptom onset in patients, establishing that impaired PKC[gamma] autoinhibition is a main driver of SCA14. Lastly, we use a variety of FRET-based approaches to examine a number of cancer-associated PKC[gamma] mutants, all of which result in loss of PKC function by a variety of mechanisms. We also show that PKC[gamma] expressed in cancer cells is not granted a stability advantage as it is with SCA14-associated mutants, and thus likely results in downregulation of PKC activity. Taken together, the work described herein serves to clarify the mechanisms by which PKC[gamma] can become deregulated and provide insight into how to better target this unique enzyme in disease.
Author: Dean J. Pierce
Publisher:
ISBN: 9781536132106
Category : Protein kinase C
Languages : en
Pages : 0
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Book Description
In this compilation, the authors review the structural basis of PKC isozymes and focus on the C1 domain, as well as the plausible binding mechanisms of its activators. Additionally, the recent molecular dynamics simulation studies of how phorbol esters or bryostatin bind to the activator pocket are described and some of the key amino acid residues recently identified as important for activator binding are investigated. The following chapter focuses on the expression pattern and function of PKCα in cancer cells, and newly emerging PKCα-targeted cancer therapies. PKCα acts directly and/or indirectly in various signaling mechanisms in cancer cells, including proliferation, survival, invasion, migration, apoptosis and drug resistance. A final review is provided which dissects the crosstalk between p53 and PKCδ in the context of apoptotic cell death and cancer therapy. PKCδ is implicated in a transcriptional regulation of the p53 tumor suppressor that is critical for cell cycle arrest and apoptosis in response to DNA damage.
Author: Michelle Lum
Publisher:
ISBN:
Category :
Languages : en
Pages : 188
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Book Description
Protein Kinase C (PKC) is a family of AGC kinases (a family including the A, G, and C protein kinases) that play critical roles in regulation of cell growth and cell cycle progression, differentiation, cell survival and apoptosis, gene expression, receptor trafficking and desensitization, and cell transformation. As regulators of fundamental cellular processes, the activity of members of this family is tightly controlled. While mechanisms of activation of these proteins have been extensively characterized, the pathways underlying signal termination are less well understood. PKCs are divided into 3 classes based on structure and coactivator requirements. Classical PKC (cPKC: PKC alpha, PKC beta I, PKC beta II and PKC gamma) are activated by diacylglycerol (DAG), Ca2+ and phosphatidylserine, while novel PKC (nPKC) do not require Ca2+ and atypical PKC (aPKC) are independent of both DAG and Ca2+.^The difference in cofactor requirements for these isozymes is a result of differences in their C1 and C2 regulatory domain structures. The C1 domain is involved in DAG binding, while the C2 domain is involved in Ca2+ binding. Ligand binding by a number of growth factor and cytokine receptors leads to activation of phospholipase C and subsequent generation of DAG and inositide trisphosphate (which mobilizes intracellular Ca2+). The accumulation of DAG leads to recruitment of cPKCs and nPKCs to the plasma membrane where they are activated due to displacement of a pseudosubstrate domain from the active site. In addition to activation by physiological signals, cPKCs and nPKCs can also be activated by pharmacological agonists that bind to the same site as DAG. These agonists include phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), and bryostatins, such as bryostatin-1 (bryo). Newly synthesized PKCs are not competent for activation but require priming phosphorylation on three conserved sites on the activation loop, turn motif and hydrophobic motif. These priming phosphorylations, which occur in an ordered manner, render the kinase competent for activation and stabilize it against denaturation and degradation. Acute termination of signaling is achieved by the rapid metabolism of DAG (by e. g., diacylglycerol kinase). This loss of signal leads to "reverse translocation" of membrane bound PKC to the cytoplasm in a process that is dependent on kinase activity. Pharmacological agonists are not subject to rapid metabolism and thus elicit a sustained activation of PKCs. In addition to acute termination of signal, it has long been recognized that prolonged activation of PKCs (either by pharmacological agonists or sustained physiological stimuli) leads to desensitization of this signaling system via degradation of the protein. Although activation-induced loss of PKC expression has been observed in many systems, there is considerable confusion regarding the mechanism(s) underlying agonist-induced desensitization of PKC signaling. Part of this confusion may stem from the extensive use of overexpression systems to analyze aspects of PKC regulation. While these systems can yield valuable information, our analysis indicates that overexpression can drive activated PKC isozymes into alternate pathways of degradation, which do not reflect those utilized by the endogenous proteins. Studies detailed herein analyze various mechanisms of desensitization of PKC signaling that are relevant to the endogenous protein by examining the effects of various agonists on the localization and processing of cellular PKC alpha.^The first part of this work used a combination of biochemical and immunolocalization studies to examine the mechanisms underlying a novel non-proteasomal pathway of degradation that is induced by prolonged bryo treatment in various cell types. Bryo initially induces translocation of endogenous PKC alpha; to the plasma membrane, where a pool of the protein is subjected to proteasomal degradation. A second subpopulation is internalized through a clathrin-independent, but cholesterol- and genistein-sensitive pathway, which involves trafficking through EEA1-positive early endosomes and Rab7-positive late endosomes/multivesicular bodies. The ultimate fate of internalized PKC alpha is degradation by lysosomal proteases in the perinuclear region. Analysis of the effect of endolysosomal disrupting agents in multiple cell lines points to lysosomal processing of activated PKC alpha as a common mechanism for its desensitization. The second part of this thesis explored the role of dephosphorylation and heat shock proteins (Hsps) in agonist-induced proteasomal degradation of endogenous PKC alpha. A widely accepted mechanism for stimulus-induced downregulation of PKCs involves priming site dephosphorylation, which targets the protein for proteasomal degradation. However, studies described here demonstrate that PKC agonists induce downregulation of endogenous PKCalpha with minimal accumulation of non-phosphorylated enzyme in multiple cell types. Furthermore, all or most of the non-phosphorylated enzyme detected in agonist-treated cells results from delayed maturation rather than dephosphorylation of the protein. Thus, PKC agonists induce at most low levels of dephosphorylation of endogenous PKC alpha;, and dephosphorylation is not a prerequisite for enzyme degradation. Analysis of the functions of Hsp90 and Hsp70/Hsc70 revealed distinct roles for these chaperones in regulating agonist-induced PKC alpha; degradation. Hsp90 prevents dephosphorylation of the activated enzyme and protects the mature, phosphorylated form of protein from proteasomal clearance following activation. In contrast, while Hsp70/Hsc70 also protects PKC alpha; from dephosphorylation, it enhances degradation of activated PKC alpha; by facilitating proteasomal processing of mature phosphorylated protein. Notably, downregulation of non-phosphorylated enzyme showed little dependence on Hsp70/Hsc70, suggesting that mature and non-phosphorylated species are targeted for proteasomal degradation via different pathways. Finally, lysosomal degradation of PKC alpha is not dependent on Hsps or the phosphorylation state of the enzyme. The third part of this work examines desensitization of PKC signaling following stimulation by DAG, the major physiological activator of cPKC and nPKC isozymes.^Analysis of the effects of a single addition of various DAGs (which are rapidly metabolized), and of short term pulse treatment with phorbol esters, confirmed that acute reversal of PKC alpha signaling following loss of signal involves dissociation of fully phosphorylated PKC alpha from the plasma membrane and cytosolic accumulation of the enzyme, via a mechanism that is dependent on PKC alpha activity. In contrast, repeated addition of DAGs resulted in sustained association of PKC alpha with the plasma membrane, in a pattern comparable with that induced by the phorbol ester PMA. Unlike the effects of PMA, however, chronic DAG stimulation failed to promote degradation/downregulation of PKC alpha, although downregulation of PKC delta and epsilon was readily apparent. Priming site dephosphorylation of PKC alpha was also not observed and Hsp70/Hsc70 and Hsp90 were excluded as regulators of PKC alpha phosphorylation and stability in this context. Importantly, while long-term activation of PKC alpha by DAG did not lead to degradation of the enzyme, it did induce desensitization of PKC alpha signaling, as seen by reversal of PKC alpha mediated effects on ERK activation, p21Waf1/Cip1 induction, and Id1 and cyclin D1 downregulation. These findings point to a previously undefined mechanisms for desensitization of PKC alpha, which can be attributed either to direct effects on PKC alpha function or to alterations in its downstream signaling pathways. Through analysis of the endogenous protein, each of these studies has identified a novel mechanism for desensitization of PKC alpha signaling. Collectively, the data confirm that the phosphorylated species of PKC alpha is a direct target for proteasomal and lysosomal degradation, and highlight the existence of multiple mechanisms for processing activated PKCs in cells.
