scholarly journals Regulation of the tumour suppressor Fbw7α by PKC-dependent phosphorylation and cancer-associated mutations

2010 ◽  
Vol 432 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Joanne Durgan ◽  
Peter J. Parker
Keyword(s):  
Mammalian Cells ◽  
Tumour Suppressor ◽  
Dependent Manner ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Pkc Protein Kinase C ◽  
Specific Regulation ◽  
The Relationship ◽  
Substrate Binding Domain

Fbw7 (F-box WD40 protein 7) is a major tumour suppressor, which mediates the degradation of several potent oncogenes. PKC (protein kinase C) comprises a serine/threonine kinase family that can promote transformation when dysregulated. In the present study, we investigated the relationship between Fbw7 and PKC. Multiple members of the PKC superfamily interact with the substrate-binding domain of Fbw7. However, we find no evidence for Fbw7-mediated degradation of PKC. Instead, we demonstrate that Fbw7 is a novel substrate for PKC. Two residues within the isoform-specific N-terminus of Fbw7α are phosphorylated in a PKC-dependent manner, both in vitro and in mammalian cells (Ser10 and Ser18). Mutational analyses reveal that phosphorylation of Fbw7α at Ser10 can regulate its nuclear localization. Cancer-associated mutations in nearby residues (K11R and the addition of a proline residue at position 16) influence Fbw7α localization in a comparable manner, suggesting that mislocalization of this protein may be of pathological significance. Together these results provide evidence for both physical and functional interactions between the PKC and Fbw7 families, and yield insights into the isoform-specific regulation of Fbw7α.

scholarly journals Inhibition of ULK2 Autophagic Activity by Its Association with YAP

2016 ◽  
Vol 8 (1) ◽  
pp. 12 ◽  
Author(s):  
Sung Hwa Shin ◽  
Eun Jeoung Lee ◽  
Sunghee Hyun ◽  
Dowonkyoung Park ◽  
Sang Sun Kang
Keyword(s):  
Mammalian Cells ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Wild Type ◽  
Therapeutic Approaches ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Py Motif

Uncoordinated 51-like kinase 2 (ULK2) is a member of the serine/threonine kinase family that functions an essential role regulating autophagy in mammalian cells. As autophagy is implicated in normal cellular homeostasis and multiple diseases, better mechanisticinsight will drive thedevelopment of novel therapeutic approaches. Here, we present evidence that ULK2 interacts withYAP for its degradation and subcellular localization. A potential PPxY motif (328PPnY331) was identified, which is similar with the consensus PPxY motif in ULK2 S/P domain. TheP329A (PA) mutation in the PY motif of ULK2 abolished the YAP-ULK2 association.At first, we observed that ULK2 physically interacted to YAP in vivo and in vitro, using a pull-down approach. Secondly, ULK2 and YAP co- localized at the apical (or tight junction) membrane as visualized by confocal microscopy. Furthermore, the PA mutant substantially increased during autophagy than that of wild-type ULK2 or the P242A mutant in transient transfection assays. Thus, the association between ULK2 and YAP through the WW domain links autophagy and the Hippo signal transduction pathway.

scholarly journals Insulin-Induced Phosphorylation and Activation of Cyclic Nucleotide Phosphodiesterase 3B by the Serine-Threonine Kinase Akt

1999 ◽  
Vol 19 (9) ◽  
pp. 6286-6296 ◽  
Author(s):  
Tadahiro Kitamura ◽  
Yukari Kitamura ◽  
Shoji Kuroda ◽  
Yasuhisa Hino ◽  
Miwa Ando ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Second Messengers ◽  
Dominant Negative ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Serine Threonine Kinase ◽  
Intact Cells ◽  
Threonine Kinase

ABSTRACT Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

scholarly journals Ferroptosis contributes to isoflurane-induced neurotoxicity and learning and memory impairment

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Pengfei Liu ◽  
Jing Yuan ◽  
Yetong Feng ◽  
Xin Chen ◽  
Guangsuo Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Learning And Memory ◽  
Memory Impairment ◽  
Close Association ◽  
Dimethyl Fumarate ◽  
Dependent Manner ◽  
Transport Chain ◽  
The Relationship

AbstractFerroptosis is a novel type of programmed cell death, which is different from apoptosis and autophagic cell death. Recently, ferroptosis has been indicated to contribute to the in vitro neurotoxicity induced by isoflurane, which is one of the most common anesthetics in clinic. However, the in vivo position of ferroptosis in isoflurane-induced neurotoxicity as well as learning and memory impairment remains unclear. In this study, we mainly explored the relationship between ferroptosis and isoflurane-induced learning and memory, as well as the therapeutic methods in mouse model. Our results indicated that isoflurane induced the ferroptosis in a dose-dependent and time-dependent manner in hippocampus, the organ related with learning and memory ability. In addition, the activity of cytochrome c oxidase/Complex IV in mitochondrial electron transport chain (ETC) was increased by isoflurane, which might further contributed to cysteine deprivation-induced ferroptosis caused by isoflurane exposure. More importantly, isoflurane-induced ferroptosis could be rescued by both ferroptosis inhibitor (ferrostatin-1) and mitochondria activator (dimethyl fumarate), which also showed effective therapeutic action against isoflurane-induced learning and memory impairment. Taken together, our data indicate the close association among ferroptosis, mitochondria and isoflurane, and provide a novel insight into the therapy mode against isoflurane-induced learning and memory impairment.

scholarly journals WNK4 Kinase: from structure to physiology

2021 ◽  
Author(s):  
Adrian Rafael Murillo-de-Ozores ◽  
Alejandro Rodriguez-Gama ◽  
Hector Carbajal-Contreras ◽  
Gerardo Gamba ◽  
Maria Castaneda-Bueno
Keyword(s):  
Oxidative Stress ◽  
Mouse Models ◽  
Serine Threonine Kinase ◽  
Gene Encoding ◽  
Threonine Kinase ◽  
Physiological Conditions ◽  
Different Levels ◽  
Familial Hyperkalemic Hypertension

With No Lysine (K) kinase 4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that cause Familial Hyperkalemic Hypertension (FHHt). This disease is mainly driven by increased downstream activation of the Ste20-related Proline Alanine Rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1)-NCC pathway, which increases salt reabsorption in the DCT and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

Arabidopsis MLK3, a Plant-specific Casein Kinase 1, Negatively Regulated Flowering and Phosphorylated Histone H3 in vivo

2019 ◽  
Author(s):  
Zhen Wang ◽  
Junmei Kang ◽  
Shangang Jia ◽  
Tiejun Zhang ◽  
Zhihai Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Histone H3 ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Altered Expression ◽  
Casein Kinase 1 ◽  
Threonine Kinase

Abstract Background: Casein kinase 1 (CK1) family members are highly conserved serine/threonine kinase present in most eukaryotes with multiple biological functions. Arabidopsis MUT9-like kinases ( MLKs ) belong to a clade CK1 specific to the plant kingdom and have been implicated collectively in modulating flowering related processes. Three of the four MLKs ( MLK1/2/4 ) have been characterized, however, little is known about MLK3 , the most divergent MLKs. Results: We demonstrated that compared with wild type, mlk3 , a truncated MLK3 , flowered slightly early under long day conditions and ectopic expression of MLK3 rescued the morphological defects of mlk3 , indicating that MLK3 negatively regulates flowering. GA 3 application accelerated flowering of both wild type and mlk3 , suggesting that mlk3 had normal GA response. The recombinant MLK3-GFP was localized in the nucleus exclusively. In vitro kinase assay revealed that the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3ph). Mutation of a conserved catalytic residue (Lysine 175) abolished the kinase activity and resulted in failure to complement the early flowering phenotype of mlk3 . Interestingly, the global level of H3T3 phosphorylation in mlk3 did not differ significantly from wild type, suggesting the redundant roles of MLKs in flowering regulation. The transcriptomic analysis demonstrated that 425 genes significantly altered expression level in mlk3 relative to wild type. The mlk3 mlk4 double mutant generated by crossing mlk3 with mlk4 , a loss-of-function mutant of MLK4 showing late flowering, flowered between the two parental lines, suggesting that MLK3 played an antagonistic role to MLK4 in plant transition to flowering. Conclusions: A serine/threonine kinase encoding gene MLK3 is a casein kinase 1 specific to the plant species and represses flowering slightly. MLK3 located in nucleus catalyzes the phosphorylation of histone H3 at threonine 3 in vitro and an intact lysine residue (K175) is indispensible for the kinase activity. This study sheds new light on the delicate control of flowering by the plant-specific CK1 in Arabidopsis.

scholarly journals The Pseudomonas aeruginosa product pyochelin interferes with Trypanosoma cruzi infection and multiplication in vitro

2020 ◽  
Vol 114 (7) ◽  
pp. 492-498 ◽  
Author(s):  
Gabriele Sass ◽  
Laura C Miller Conrad ◽  
Terrence-Thang H Nguyen ◽  
David A Stevens
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Iron Acquisition ◽  
Vero Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Current Standard ◽  
Cruzi Infection

Abstract Background Bacteria are sources of numerous molecules used in treatment of infectious diseases. We investigated effects of molecules produced by 26 Pseudomonas aeruginosa strains against infection of mammalian cell cultures with Trypanosoma cruzi, the aetiological agent of Chagas disease. Methods Vero cells were infected with T. cruzi in the presence of wild-type P. aeruginosa supernatants or supernatants of mutants with defects in the production of various virulence, quorum sensing and iron acquisition factors. Quantification of T. cruzi infection (percentage of infected cells) and multiplication (number of amastigotes per infected cell) was performed and cell viability was determined. Results Wild-type P. aeruginosa products negatively affected T. cruzi infection and multiplication in a dose-dependent manner, without evident toxicity for mammalian cells. PvdD/pchE mutation (loss of the P. aeruginosa siderophores pyoverdine and pyochelin) had the greatest impact on anti–T. cruzi activity. Negative effects on T. cruzi infection by pure pyochelin, but not pyoverdine, or other P. aeruginosa exoproducts studied, were quantitatively similar to the effects of benznidazole, the current standard therapy against T. cruzi. Conclusions The P. aeruginosa product pyochelin showed promising activity against T. cruzi and might become a new lead molecule for therapy development.

scholarly journals Motility patterns of Trypanosoma cruzi trypomastigotes correlate with the efficiency of parasite invasion in vitro

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jorge A. Arias-del-Angel ◽  
Jesús Santana-Solano ◽  
Moisés Santillán ◽  
Rebeca G. Manning-Cela
Keyword(s):  
Trypanosoma Cruzi ◽  
Cell Line ◽  
Mammalian Cells ◽  
Dependent Manner ◽  
Parasite Invasion ◽  
Mammalian Cell Cultures ◽  
A Cell ◽  
Parasite Replication ◽  
Parasite Motility

Abstract Numerous works have demonstrated that trypanosomatid motility is relevant for parasite replication and sensitivity. Nonetheless, although some findings indirectly suggest that motility also plays an important role during infection, this has not been extensively investigated. This work is aimed at partially filling this void for the case of Trypanosoma cruzi. After recording swimming T. cruzi trypomastigotes (CL Brener strain) and recovering their individual trajectories, we statistically analyzed parasite motility patterns. We did this with parasites that swim alone or above monolayer cultures of different cell lines. Our results indicate that T. cruzi trypomastigotes change their motility patterns when they are in the presence of mammalian cells, in a cell-line dependent manner. We further performed infection experiments in which each of the mammalian cell cultures were incubated for 2 h together with trypomastigotes, and measured the corresponding invasion efficiency. Not only this parameter varied from cell line to cell line, but it resulted to be positively correlated with the corresponding intensity of the motility pattern changes. Together, these results suggest that T. cruzi trypomastigotes are capable of sensing the presence of mammalian cells and of changing their motility patterns accordingly, and that this might increase their invasion efficiency.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

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