Current Search:  protein kinase (x)

View All Items

  • CSV Spreadsheet
(1 - 6 of 6)
Proteomic Analysis Delineates the Signaling Networks of Plasmodium falciparum
Title
Proteomic Analysis Delineates the Signaling Networks of Plasmodium falciparum.
Creator
Pease, Brittany, Chakrabarti, Debopam, Khaled, Annette, Jewett, Mollie, Chakrabarti, Ratna, Cole, Alexander, University of Central Florida
Abstract / Description
Malaria is a life-threatening disease caused by Plasmodium parasites that are spread through the bites of infected mosquito vectors. It is a worldwide pandemic that threatens 3.4 billion people annually. Currently, there are only a few validated Plasmodium drug targets, while drug resistance continues to rise. This marks the urgency for the development of novel parasite-specific therapeutics. Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycle by undergoing multiple...
Show moreMalaria is a life-threatening disease caused by Plasmodium parasites that are spread through the bites of infected mosquito vectors. It is a worldwide pandemic that threatens 3.4 billion people annually. Currently, there are only a few validated Plasmodium drug targets, while drug resistance continues to rise. This marks the urgency for the development of novel parasite-specific therapeutics. Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycle by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for the design of rational intervention strategies. To achieve this goal, we performed an isobaric tag-based approach for a system-wide quantitative analysis of protein expression and site-specific phosphorylation events of the Plasmodium asexual developmental cycle in the red blood cells. This study identified 2,767 proteins, 1,337 phosphoproteins, and 6,293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Because the links between Plasmodium protein kinases as key cell cycle regulators to cellular events are largely unknown, it is of importance to define their cognate physiological substrates. To test the hypothesis that genetic screening would be a useful approach for discovery of candidate substrates of a protein kinase, we used the orphan kinase PfPK7 as a model. Our comparison of the phosphoproteome profiles between the wild-type 3D7 and PfPK7- parasites identified 146 proteins with 239 phosphorylation sites exhibiting decreased phosphorylation in the absence of PfPK7 at the developmental stages where nuclear division and merozoite formation occur. Further analysis of the decreased phosphorylated events revealed three motifs that are enriched among phosphorylated sites in proteins that are down regulated. In vitro kinase assays were done to validate the potential substrates of PfPK7 and to elucidate the signaling events that are regulated by PfPK7. In parallel to our experimental analysis, we used a computational approach for substrate prediction from our phosphoproteome dataset. This analysis identified 43 distinct phosphorylation motifs and a range of proline-directed potential MAPK/CDK substrates. To identify substrates/ interactors of Plasmodium CDK-like kinases, we also used HA-tagged CDK-like kinases, PfPK6 and Pfmrk lines. Co-immunoprecipitation of the HA-tagged PfPK6 and Pfmrk baits, followed by mass spectrometric analyses, identified the components of the protein interaction complexes of these kinases. Our analyses of HA-PfPK6 and HA-Pfmrk immunoprecipitates identified 15 and 21 proteins in the interaction complex, respectively. The ability of recombinant PfPK6 and Pfmrk to interact and/or utilize any of the proteins identified in the interaction complex as substrates was verified through in vitro kinase assays and pull-down analysis. This study is the most comprehensive definition of the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, and offered an insight into the dynamics of phosphorylation during the asexual cycle progression [1]. In summary, this study has 1) defined the constitutive and regulated expression of the Plasmodium proteome during its asexual life cycle, 2) demonstrated that fluctuation and reversible phosphorylation is important for the regulation of P. falciparum's unique cell cycle, 3) provided the foundation for quantitative phosphoproteomic analysis of kinase negative mutants to understand their function, 4) provided a major step towards defining kinase-substrate pairs operative within parasite's signaling networks, and 5) generated a preliminary interactome for PfPK6.
Show less
Date Issued
2015
Identifier
CFE0005863, ucf:50898
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005863
A MEMBER OF THE NOVEL FIKK FAMILY OF PLASMODIUM FALCIPARUM PUTATIVE PROTEIN KINASES EXHIBITS DIACYLGLYCEROL KINASE ACTIVITY AND IS EXPORTED TO THE HOST ERYTHROCYTE
Title
A MEMBER OF THE NOVEL FIKK FAMILY OF PLASMODIUM FALCIPARUM PUTATIVE PROTEIN KINASES EXHIBITS DIACYLGLYCEROL KINASE ACTIVITY AND IS EXPORTED TO THE HOST ERYTHROCYTE.
Creator
Curtis, David, Chakrabarti, Debopam, University of Central Florida
Abstract / Description
Plasmodium falciparum is one of four species known to cause malaria in humans and is the species that is associated with the most virulent form of the disease. Malaria causes nearly two million deaths each year, many of these occurring among children in under-developed countries of the world. One reason for this is the prevalence of drug resistant strains of malaria that mitigate the efficacy of existing drugs. Hence, the identification of a new generation of pharmacological agents for...
Show morePlasmodium falciparum is one of four species known to cause malaria in humans and is the species that is associated with the most virulent form of the disease. Malaria causes nearly two million deaths each year, many of these occurring among children in under-developed countries of the world. One reason for this is the prevalence of drug resistant strains of malaria that mitigate the efficacy of existing drugs. Hence, the identification of a new generation of pharmacological agents for malaria is extremely urgent. The recent identification of a group of novel protein kinases within the Plasmodium falciparum genome has provided researchers with a basis for what many hope to be new potential drug targets for malaria. Identified within the Plasmodium genome and a few select apicomplexans, these novel proteins have been predicted to be protein kinases based solely on certain sequence features shared with other eukaryotic protein kinases (ePKs). However, to date, no significant studies to determine the function of these novel kinases have been performed. Termed FIKKs, these proteins all possess a non-conserved N-terminal sequence that contains a Plasmodium export element (Pexel) which may target the proteins for export from the parasite and a conserved C-terminal catalytic domain containing a FIKK sequence common to all twenty members of this family. We analyzed the localization of one of the FIKK proteins, FIKK11, encoded by the PF11_0510 locus, during intraerythrocyte differentiation of P. falciparum by Western blot analysis and indirect immunofluorescence assay. Western blot analysis demonstrated that FIKK 11 is expressed within the parasite at all stages of its erythrocytic life cycle with its highest expression occurring during the schizont stage. Immunofluorescence assays showed that this protein is exported from the Plasmodium parasite into the host erythrocyte cytosol which is consistent with studies on other Plasmodium proteins that also have the Pexel motif. To determine the enzymatic activity of FIKK11, we overexpressed the recombinant protein in E. coli and then purified it. However, no protein kinase activity was detected using several commonly used protein kinase substrates including histone H1, myelin basic protein, or dephosphorylated casein. We also did not detect any kinase activity of the native enzyme using pull-down assays of the Plasmodium falciparum cell extract against those same substrates. In addition, kinase substrate peptide array analysis of FIKK11 showed no evidence of protein kinase activity either for FIKK11. Interestingly, however, we were able to detect some kinase activity using the recombinant protein alone with no substrate. The lack of the glycine triad within subdomain I of these FIKK kinases as compared with most traditional eukaryotic protein kinases may explain why we were unable to find any interactions between FIKK11 and other commonly protein kinase substrates. Of interest was the observation that the protein reproducibly exhibited what appeared to be an autophosphorylation activity when using the standard protein kinase assay. Further analyses, however, showed that FIKK11 actually possesses diacylglycerol kinase activity utilizing 1-Stearoyl-2-arachidonoyl-sn-glycerol as a substrate. This is the first evidence of diacylglycerol kinase activity in Plasmodium falciparum. Because FIKK11 is exported into the host cell and is localized on the erythrocyte membrane, its enzymatic activity may potentially have relevance in the pathophysiology of the disease.
Show less
Date Issued
2007
Identifier
CFE0001879, ucf:47407
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001879
IDENTIFICATION AND CHARACTERIZATION OF INTERACTORS OF PLASMODIUM FALCIPARUM PFPK6, AN ATYPICAL PROTEIN KINASE
Title
IDENTIFICATION AND CHARACTERIZATION OF INTERACTORS OF PLASMODIUM FALCIPARUM PFPK6, AN ATYPICAL PROTEIN KINASE.
Creator
Cummins, Andi J, Chakrabarti, Debopam, University of Central Florida
Abstract / Description
Plasmodium falciparum, the organism that causes the most prevalent and most virulent cases of malaria in humans, poses a major health burden on the developing world, especially in the tropical regions of Sub-Saharan Africa, Southeast Asia, and Latin America. The burden of the disease is intensified by the fact that the parasite has developed widespread resistance to all current antimalarial therapies, such as chloroquine. This drug resistance underscores the need to develop novel therapeutics...
Show morePlasmodium falciparum, the organism that causes the most prevalent and most virulent cases of malaria in humans, poses a major health burden on the developing world, especially in the tropical regions of Sub-Saharan Africa, Southeast Asia, and Latin America. The burden of the disease is intensified by the fact that the parasite has developed widespread resistance to all current antimalarial therapies, such as chloroquine. This drug resistance underscores the need to develop novel therapeutics that target the parasite, but show low toxicity in the human host. Protein kinases, because of their integral roles in cell signaling networks, are considered to be attractive drug targets. Cyclin dependent kinases, or CDKs, and Mitogen-Activated Protein kinases, or MAPKs, are common to eukaryotes and regulate cellular processes of growth and proliferation. Plasmodium falciparum Protein Kinase 6, or PfPK6, is an atypical protein kinase that shows similarities to both MAPKs and CDKs. PfPK6 is expected to have an important role in the intraerythrocytic cell cycle progression and growth in the malaria organism, as it has been found to be essential in the parasite. In order to better understand the function of PfPK6 within Plasmodium, we have identified serveral potential substrates and interactors of the kinase using co-immunoprecipitation with an HA epitope-tagged cell line of PfPK6, as well as phosphoproteomic analysis. These methods resulted identification of 15 novel protein interactors, with 4 being studied for further investigation, and 45 putative substrates after strict peptide filtering, five of which are used in this study. In order to verify putative substrates and interactors, both in vitro and in vivo methods were used. In vitro kinase assays using GST-PfPK6 with 5 recombinant substrates confirmed direct phosphorylation of two novel substrates: MAL7P1.38, a regulator of chromosome condensation, and PF10_0047, a putative RNA binding protein. After attempts to generate bacterial constructs of several putative interactors and a global failure of a usable amount of protein to express under IPTG induction conditions, an alternative form of expression using a cell free Transcription and Translation reaction (TNT) with Wheat Germ Extract was used to generate radiolabeled PF11_0154, PFF0625w, and PF11_0305. Pull down analysis using GST-PfPK6 showed the kinase�s ability to �pull� the interactors out of solution, confirming the interactions defined by the initial epitope tagged Co-Immunoprecipitation. Additionally, for in vivo analysis, parasites were transfected with RFP- PFF_0695w, an uncharacterized Plasmodium protein, in order to cellular localization of this interactors. Immunofluorescence assays of transfected lines showed punctate forms of PFF_0695w in the host erythrocyte in the late trophozoite and schizont stages of the parasite development, suggesting this interactor is a previously undiscovered protein in the Plasmodium secretome. The research presented here is an initial step to defining the interactome of PfPK6.
Show less
Date Issued
2016
Identifier
CFH2000041, ucf:45517
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000041
Diabetes Phenotypes in Transgenic Pancreatic Cancer Mouse Models
Title
Diabetes Phenotypes in Transgenic Pancreatic Cancer Mouse Models.
Creator
Albury, Toya, Altomare, Deborah, Zhao, Jihe, Masternak, Michal, Khaled, Annette, University of Central Florida
Abstract / Description
Protein Kinase B/AKT, a serine/threonine kinase with three isoforms (AKT1-3), is downstream of phosphatidylinositol 3-kinase (PI3K), and signals through the phosphorylation and subsequent activation or inhibition of downstream substrates, such as mammalian target of rapamycin complex 1 (mTORC1) or glycogen synthase kinase 3 beta (GSK-3?), respectively. The AKT1 isoform is predominantly recognized for regulation of cell survival, growth, and proliferation, due to its constitutive activation in...
Show moreProtein Kinase B/AKT, a serine/threonine kinase with three isoforms (AKT1-3), is downstream of phosphatidylinositol 3-kinase (PI3K), and signals through the phosphorylation and subsequent activation or inhibition of downstream substrates, such as mammalian target of rapamycin complex 1 (mTORC1) or glycogen synthase kinase 3 beta (GSK-3?), respectively. The AKT1 isoform is predominantly recognized for regulation of cell survival, growth, and proliferation, due to its constitutive activation in pancreatic cancers (e.g., islet cell carcinoma and pancreatic adenocarcinoma). The progression of pancreatic ductal adenocarcinoma (PDAC), the most lethal common cancer, is initiated by activation mutations of the KRas oncogene. This leads to additional molecular changes, such as activation of the AKT1 oncogene, which drives PDAC progression and tumor formation. By mating transgenic mice with activation of KRas (Pdx- Cre;LSL-KRasG12D) and mice with activation of AKT1 (Pdx- Tta;TetO-MyrAKT1) we were able to produce mice with two activated oncogenes (AKT1Myr/KRasG12D) for comparative studies. Kaplan-Meier survival curves, histology, and genomic/proteomic analysis were used to characterize the incidence and frequency of histological (e.g. presence of mucin-4 in pancreatic intraepithelial neoplasms) and genetic (e.g. loss of tumor suppressors p16Ink4a and p19Arf) alterations known to commonly occur in human pancreatic cancer, as well as delineate the role of AKT1 in accelerating pancreatic tumor progression and metastasis. We determined that AKT1Myr/KRasG12D mice, unlike other PDAC mouse models, accurately mimic the human PDAC progression molecularly, structurally, and temporally. Interestingly, the AKT1Myr and AKT1Myr/KRasG12D models both exhibit a pre-tumor, diabetic phenotype. While, AKT1 hyperactivation in various cancers has been thoroughly studied, its role in glucose metabolism has been noted, but comparatively overlooked. As early as the 1900s a relationship between diabetes and pancreatic cancer has been proposed. With 80% of PDAC patients suffering from hyperglycemia or diabetes prior to diagnosis, one prevailing theory is that new onset diabetes is an early marker for pancreatic cancer. This is also supported by experimental and clinical studies, such as the resolution of diabetes with tumor removal and the induction of hyperglycemia with the implantation of cancer cell lines. To better understand the role of AKT1 and its hyperactivation in glucose metabolism, AKT1Myr mice were characterized via metabolic (e.g. glucose/insulin tolerance test) and histological (e.g. immunohistochemistry) studies. Beginning at weaning, 3 weeks of age, the glucose intolerant AKT1Myr mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. Additionally, AKT1Myr/KRasG12D mice currently being studied, appear to display a more severe diabetic phenotype, with fasted hyperglycemia noticeable at an earlier age, fasted hyperglucagonemia, polyuria, muscle wasting, and bloating. Treatment of both models with doxycycline diet, to turn-off the transgene, caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. These newly revealed roles of AKT1, along with future studies of these mouse models, will better delineate the molecular mechanisms responsible for the individual and joint roles of AKT1 and KRas in pancreatic cancer oncogenesis, the initiation of cancer associated diabetes, and the association of these two diseases.
Show less
Date Issued
2015
Identifier
CFE0006245, ucf:51081
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006245
IDENTIFICATION OF PLASMODIUM FALCIPARUM PROTEIN KINASE SUBSTRATES AND INTERACTING PROTEINS
Title
IDENTIFICATION OF PLASMODIUM FALCIPARUM PROTEIN KINASE SUBSTRATES AND INTERACTING PROTEINS.
Creator
Yap, Jessica, Chakrabarti, Debopam, University of Central Florida
Abstract / Description
Malaria is a devastating disease that results in almost one million deaths annually. Most of the victims are children under the age of five in Sub-Saharan Africa. Malaria parasite strains throughout developing countries are continually building resistance to available drugs. Current therapies such as mefloquine, chloroquine, as well as artemisinin are becoming less effective, and this underscores the urgency for therapeutics directed against novel drug targets. In order to identify new drug...
Show moreMalaria is a devastating disease that results in almost one million deaths annually. Most of the victims are children under the age of five in Sub-Saharan Africa. Malaria parasite strains throughout developing countries are continually building resistance to available drugs. Current therapies such as mefloquine, chloroquine, as well as artemisinin are becoming less effective, and this underscores the urgency for therapeutics directed against novel drug targets. In order to identify new drug targets, the molecular biology of the malaria parasite Plasmodium needs to be elucidated. Plasmodium exhibits a unique cell cycle in which it undergoes multiple rounds of DNA synthesis and mitosis without cytokinesis. Thus, cell cycle regulatory proteins are likely to be promising pathogen-specific drug targets. It is expected that fluctuating activity of key proteins, such as protein kinases, play an essential role in regulating the noncanonical life cycle of Plasmodium. Consequently, malarial kinases are a prime target for therapy. One way to better understand the role of malarial kinases in Plasmodium cell cycle regulation is to identify putative protein kinase substrates and interacting proteins. Two malarial kinases that have been implicated in regulating malaria parasite cell cycle stages were investigated in this study: P. falciparum CDK-like Protein Kinase 5 (PfPK5) and cAMP-Dependent Protein Kinase A (PfPKA). A transgenic P. falciparum line was created for the expression of epitope-tagged PfPK5 for pull-down analysis. Phospho-substrate antibodies were used to identify physiological substrates of both PfPK5 and PfPKA. Immunoblotting with these antibodies identified several potential substrates. Identities of the PfPKA physiological substrates were determined from the global P. falciparum phosphoproteome dataset that has recently been generated in our laboratory. Characterization of PfPKA and PfPK5 substrates, as well as the proteins they interact with, will help us to develop innovative therapies targeting binding sites.
Show less
Date Issued
2012
Identifier
CFH0004157, ucf:44829
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004157
The Effects of Phosphatidylserine on Reaction Time and Cognitive Function Following an Exercise Stress
Title
The Effects of Phosphatidylserine on Reaction Time and Cognitive Function Following an Exercise Stress.
Creator
Wells, Adam, Hoffman, Jay, Fragala, Maren, Stout, Jeffrey, University of Central Florida
Abstract / Description
Phosphatidylserine (PS) is an endogenously occurring phospholipid that has been shown to have cognition and mood enhancing properties in humans, possibly through its role as an enzyme co-factor in cellular signal transduction. Specifically, PS has been identified as activator of classical isoforms of protein kinase C, an enzyme known to be involved in the growth and differentiation of neural cells, and is therefore thought to play a role in the protection of neurons.The purpose of this study...
Show morePhosphatidylserine (PS) is an endogenously occurring phospholipid that has been shown to have cognition and mood enhancing properties in humans, possibly through its role as an enzyme co-factor in cellular signal transduction. Specifically, PS has been identified as activator of classical isoforms of protein kinase C, an enzyme known to be involved in the growth and differentiation of neural cells, and is therefore thought to play a role in the protection of neurons.The purpose of this study was to examine the effects of supplementation with PS and caffeine on measures of cognition, reaction time and mood prior to and following an exercise stress. Twenty, healthy, resistance trained males (17) and females (3) (mean (&)#177; SD; age: 22.75 (&)#177; 3.27 yrs; height: 177.03 (&)#177; 8.44cm; weight: 78.98 (&)#177; 11.24kg; body fat%: 14.28 (&)#177; 6.6), volunteered to participate in this randomized, double-blind, placebo-controlled study. Participants were assigned to a PS group (400mg/day PS; 100mg/day caffeine, N=9) or PL (16g/day Carbs, N=11) delivered in the form of 4 candy chews identical in size, shape and color. Subjects performed an acute bout of full body resistance exercise, prior to (T1) and following 14 days of supplementation (T2). Measures of reaction time (Dynavision(&)#174; D2 Visuomotor Training Device), cognition (Serial Subtraction Test, SST), and mood (Profile of Mood States, POMS) were assessed immediately before and following resistance exercise in both T1 and T2. Data was analyzed using two-way ANCOVA and repeated measures ANOVA.Supplementation with 400mg PS and 100mg caffeine did not have a significant impact upon measures of reaction time or cognition between groups at baseline or following acute resistance exercise. However, there was a non-significant trend to the attenuation of fatigue between groups, following acute resistance exercise (p = 0.071). Interestingly, our data suggests that acute resistance exercise alone may improve cognitive function.Although more research is necessary regarding optimal dosage and supplementation duration, the current findings suggest that supplementation 400mg/day PS with 100mg/day caffeine may attenuate fatigue following acute resistance exercise. It is possible that the lack of significance may be the result of both an inhibition of the PS activated pathway and a withdrawal effect from caffeine.
Show less
Date Issued
2012
Identifier
CFE0004457, ucf:49325
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004457