The identification and characterization of novel PKCϵ phosphorylation sites provide evidence for functional cross-talk within the PKC superfamily

2008 ◽  
Vol 411 (2) ◽  
pp. 319-331 ◽  
Author(s):  
Joanne Durgan ◽  
Angus J. Cameron ◽  
Adrian T. Saurin ◽  
Sarah Hanrahan ◽  
Nick Totty ◽  
...  
Keyword(s):  
Cross Talk ◽  
Phosphorylation Sites ◽  
Serine Threonine Kinase ◽  
Cellular Processes ◽  
Mammalian Cell Lines ◽  
Pkc Isoforms ◽  
Broad Array ◽  
Cellular Context ◽  
In Trans

PKCϵ (protein kinase Cϵ) is a phospholipid-dependent serine/threonine kinase that has been implicated in a broad array of cellular processes, including proliferation, survival, migration, invasion and transformation. Here we demonstrate that, in vitro, PKCϵ undergoes autophosphorylation at three novel sites, Ser234, Ser316 and Ser368, each of which is unique to this PKC isoform and is evolutionarily conserved. We show that these sites are phosphorylated over a range of mammalian cell lines in response to a number of different stimuli. Unexpectedly, we find that, in a cellular context, these phosphorylation events can be mediated in-trans by cPKC (classical PKC) isoforms. The functional significance of this cross-talk is illustrated through the observation that the cPKC-mediated phosphorylation of PKCϵ at residue Ser368 controls an established PKCϵ scaffold interaction. Thus our current findings identify three new phosphorylation sites that contribute to the isoform-specific function of PKCϵ and highlight a novel and direct means of cross-talk between different members of the PKC superfamily.

New insights into PKC family affairs: three novel phosphorylation sites in PKCϵ and at least one is regulated by PKCα

2008 ◽  
Vol 411 (2) ◽  
pp. e15-e16 ◽  
Author(s):  
Christer Larsson
Keyword(s):  
Phosphorylation Sites ◽  
Functional Importance ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Cellular Processes ◽  
Biochemical Journal ◽  
Pkc Isoforms ◽  
Evolutionarily Conserved ◽  
The Individual

PKCϵ (protein kinase Cϵ) is a serine/threonine kinase, and a member of the PKC family of isoforms. The different PKC isoforms regulate many cellular processes of importance for disease. It is therefore desirable to obtain tools to specifically modulate the activity of the individual isoforms and to develop markers of PKC activity. The paper by Durgan et al. in this issue of the Biochemical Journal has taken us some steps further towards these goals. In the paper they identify three previously unknown phosphorylation sites in PKCϵ. All of them are specific for the ϵ isoform, evolutionarily conserved and tightly regulated. The phosphorylation of one site is critical for the binding of PKCϵ to 14-3-3β, suggesting it is of functional importance. The results provide important novel findings that uncover new aspects of PKCϵ regulation and reveal new possibilities for detecting PKCϵ activity in situ.

scholarly journals Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+

2003 ◽  
Vol 374 (2) ◽  
pp. 281-296 ◽  
Author(s):  
Tim D. WERRY ◽  
Graeme F. WILKINSON ◽  
Gary B. WILLARS
Keyword(s):  
G Protein ◽  
Cross Talk ◽  
Cell Function ◽  
Feedback Regulation ◽  
G Protein Coupled Receptors ◽  
Negative Feedback Regulation ◽  
Cellular Processes ◽  
Heterologous Desensitization ◽  
G Protein Coupled

Alteration in [Ca2+]i (the intracellular concentration of Ca2+) is a key regulator of many cellular processes. To allow precise regulation of [Ca2+]i and a diversity of signalling by this ion, cells possess many mechanisms by which they are able to control [Ca2+]i both globally and at the subcellular level. Among these are many members of the superfamily of GPCRs (G-protein-coupled receptors), which are characterized by the presence of seven transmembrane domains. Typically, those receptors able to activate PLC (phospholipase C) enzymes cause release of Ca2+ from intracellular stores and influence Ca2+ entry across the plasma membrane. It has been well documented that Ca2+ signalling by one type of GPCR can be influenced by stimulation of a different type of GPCR. Indeed, many studies have demonstrated heterologous desensitization between two different PLC-coupled GPCRs. This is not surprising, given our current understanding of negative-feedback regulation and the likely shared components of the signalling pathway. However, there are also many documented examples of interactions between GPCRs, often coupling preferentially to different signalling pathways, which result in a potentiation of Ca2+ signalling. Such interactions have important implications for both the control of cell function and the interpretation of in vitro cell-based assays. However, there is currently no single mechanism that adequately accounts for all examples of this type of cross-talk. Indeed, many studies either have not addressed this issue or have been unable to determine the mechanism(s) involved. This review seeks to explore a range of possible mechanisms to convey their potential diversity and to provide a basis for further experimental investigation.

scholarly journals A search for the in trans role of GraL, an Escherichia coli small RNA*

2018 ◽  
Vol 65 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Maciej Dylewski ◽  
Monika Ćwiklińska ◽  
Katarzyna Potrykus
Keyword(s):  
Small Rna ◽  
Enteric Bacteria ◽  
Clear Correlation ◽  
Synthetic Lethal ◽  
Cellular Processes ◽  
In Trans ◽  
Regulatory Loop

Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL’s targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed. Only one target's activity (nudE) decreased under those conditions and was thus selected for further analysis. In the absence of GraL and greA, the nudE::lacZ fusion's β-galactosidase activity was increased. However, a similar effect was also visible in the strain deleted only for greA. Furthermore, overproduction of GreA alone increased the nudE::lacZ fusion’s activity as well. This suggests existence of complex regulatory loop-like interactions between GreA, GraL and nudE mRNA. To further dissect this relationship, we performed in vitro EMSA experiments employing GraL and nudE mRNA. However, stable GraL-nudE complexes were not detected, even though the detectable amount of unbound GraL decreased as increasing amounts of nudE mRNA were added. Interestingly, GraL is being bound by Hfq, but nudE easily displaces it.  We also conducted a search for genes that are synthetic lethal when deleted along with GraL. This revealed 40 genes that are rendered essential by GraL deletion, however, they are involved in many different cellular processes and no clear correlation was found. The obtained data suggest that GraL's mechanism of action is non-canonical, unique and requires further research.

scholarly journals Phosphorylation of the NBDY Microprotein Promotes Dissociation of Biomolecular Condensates

2021 ◽  
Author(s):  
Zhenkun Na ◽  
Yang Luo ◽  
Danica S. Cui ◽  
Alexandra Khitun ◽  
Stephanie Smelyansky ◽  
...  
Keyword(s):  
Liquid Phase ◽  
Fluorescence Microscopy ◽  
Phosphorylation Sites ◽  
Growth Factor Signaling ◽  
Form Complex ◽  
Reading Frame ◽  
P Bodies ◽  
Cellular Processes ◽  
Cytoplasmic Rna

This study reports detection of specific phosphorylation sites installed on a small open reading frame-encoded polypeptide or microprotein called NBDY. NBDY phosphorylation sites were mapped using phosphoproteomics and antibody-based validation. NBDY is phosphorylated during growth factor signaling and cell division, and quantitative fluorescence microscopy was used to show that NBDY phosphorylation is required for disappearance of cytoplasmic RNA-protein granules called P-bodies during these cellular processes. Because P-bodies have properties of liquid-liquid phase separated membraneless orgaenelles, reductionist system to investigate NBDY phase separation. Purified NBDY was shown to form complex coacervates in the presence of RNA via fluorescence microscopy and turbidity measurements, and phosphorylation by a kinase in vitro promotes liquid phase remixing.

scholarly journals Phosphorylation of the NBDY Microprotein Promotes Dissociation of Biomolecular Condensates

2021 ◽  
Author(s):  
Zhenkun Na ◽  
Yang Luo ◽  
Danica S. Cui ◽  
Alexandra Khitun ◽  
Stephanie Smelyansky ◽  
...  
Keyword(s):  
Liquid Phase ◽  
Fluorescence Microscopy ◽  
Phosphorylation Sites ◽  
Growth Factor Signaling ◽  
Form Complex ◽  
Reading Frame ◽  
P Bodies ◽  
Cellular Processes ◽  
Cytoplasmic Rna

This study reports detection of specific phosphorylation sites installed on a small open reading frame-encoded polypeptide or microprotein called NBDY. NBDY phosphorylation sites were mapped using phosphoproteomics and antibody-based validation. NBDY is phosphorylated during growth factor signaling and cell division, and quantitative fluorescence microscopy was used to show that NBDY phosphorylation is required for disappearance of cytoplasmic RNA-protein granules called P-bodies during these cellular processes. Because P-bodies have properties of liquid-liquid phase separated membraneless orgaenelles, reductionist system to investigate NBDY phase separation. Purified NBDY was shown to form complex coacervates in the presence of RNA via fluorescence microscopy and turbidity measurements, and phosphorylation by a kinase in vitro promotes liquid phase remixing.

scholarly journals Dynamic modelling of the PI3K/MTOR signalling network uncovers biphasic dependence of mTORC1 activity on the mTORC2 subunit SIN1

2021 ◽  
Vol 17 (9) ◽  
pp. e1008513
Author(s):  
Milad Ghomlaghi ◽  
Guang Yang ◽  
Sungyoung Shin ◽  
David E. James ◽  
Lan K. Nguyen
Keyword(s):  
Mechanistic Model ◽  
Experimental Studies ◽  
Dynamic Modelling ◽  
Loop Model ◽  
Network Modelling ◽  
Cellular Processes ◽  
Signalling Network ◽  
Broad Array ◽  
Mtor Signalling

The PI3K/MTOR signalling network regulates a broad array of critical cellular processes, including cell growth, metabolism and autophagy. The mechanistic target of rapamycin (MTOR) kinase functions as a core catalytic subunit in two physically and functionally distinct complexes mTORC1 and mTORC2, which also share other common components including MLST8 (also known as GβL) and DEPTOR. Despite intensive research, how mTORC1 and 2 assembly and activity are coordinated, and how they are functionally linked remain to be fully characterized. This is due in part to the complex network wiring, featuring multiple feedback loops and intricate post-translational modifications. Here, we integrate predictive network modelling, in vitro experiments and -omics data analysis to elucidate the emergent dynamic behaviour of the PI3K/MTOR network. We construct new mechanistic models that encapsulate critical mechanistic details, including mTORC1/2 coordination by MLST8 (de)ubiquitination and the Akt-to-mTORC2 positive feedback loop. Model simulations validated by experimental studies revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit SIN1, which is robust against cell-to-cell variation in protein expression. In addition, our integrative analysis demonstrates that ubiquitination of MLST8, which is reversed by OTUD7B, is regulated by IRS1/2. Our results further support the essential role of MLST8 in enabling both mTORC1 and 2’s activity and suggest MLST8 as a viable therapeutic target in breast cancer. Overall, our study reports a new mechanistic model of PI3K/MTOR signalling incorporating MLST8-mediated mTORC1/2 formation and unveils a novel regulatory linkage between mTORC1 and mTORC2.

scholarly journals Bbs5 functions as the docking compartment of Bbsome binding to Klc3 regulated by Bbs5-Ser246 PKC-phosphorylation during mouse spermatogenesis

2020 ◽  
Author(s):  
Dahai Yu ◽  
Lixia He ◽  
Xin Zhou ◽  
Xiuxia Wang ◽  
Bingzhi Yu
Keyword(s):  
Phosphorylation Sites ◽  
Yeast Two Hybrid ◽  
Fibrous Sheath ◽  
Bardet Biedl Syndrome ◽  
Kinesin Light Chain ◽  
Mammalian Sperm ◽  
Pkc Isoforms ◽  
Cilia And Flagella ◽  
Two Hybrid

AbstractA mitochondrial and a fibrous sheath form the midpiece of the mammalian sperm flagellum encircling most of the axoneme. It has been documented that Kinesin light chain 3 (KLC3) was involved although the formation procedure remains unclear. Yeast-two-hybrid dataset showed an interaction between Klc3 and Bardet-Biedl Syndrome 5 (BBS5) Protein, another molecular associated with cilia and flagella forming. In this study, we presumed that the most conserved IFT complex BBsome was involved in spermatogenesis via the interaction of one of its subunits, Bbs5 with Klc3. Firstly, the interaction between Klc3 and Bbs5 was confirmed with Co-IP. Secondly, we identified PKC phosphorylation sites in vitro by LC-MS/MS, Ser19 and Ser246 of Bbs5, examined the phosphorylation status of Bbs5 Ser19 and Ser246 in mouse testis. Co-IP was performed to find which PKC isoforms phosphorylate Bbs5. In addition, we tried to discuss the roles of Ser19 and Ser246 of Bbs5 in the Klc3-bbs5 interaction and in mouse spermatogenesis based on our early findings.

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 EXTH-46. ARTIFICIAL INTELLIGENCE-BASED IDENTIFICATION OF COMBINED VANDETANIB AND EVEROLIMUS IN THE TREATMENT OF ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii97-ii97
Author(s):  
Diana Carvalho ◽  
Peter Richardson ◽  
Nagore Gene Olaciregui ◽  
Reda Stankunaite ◽  
Cinzia Emilia Lavarino ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Cell Viability ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Serine Threonine Kinase ◽  
Orthotopic Xenografts ◽  
Extended Survival

Abstract Somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2 receptor, are found in a quarter of children with the currently incurable brain tumour diffuse intrinsic pontine glioma (DIPG). Treatment of ACVR1-mutant DIPG patient-derived models with multiple inhibitor chemotypes leads to a reduction in cell viability in vitro and extended survival in orthotopic xenografts in vivo, though there are currently no specific ACVR1 inhibitors licensed for DIPG. Using an Artificial Intelligence-based platform to search for approved compounds which could be used to treat ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an approved inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd=150nM) and reduce DIPG cell viability in vitro, but has been trialed in DIPG patients with limited success, in part due to an inability to cross the blood-brain-barrier. In addition to mTOR, everolimus inhibits both ABCG2 (BCRP) and ABCB1 (P-gp) transporter, and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination is well-tolerated in vivo, and significantly extended survival and reduced tumour burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Based on these preclinical data, three patients with ACVR1-mutant DIPG were treated with vandetanib and everolimus. These cases may inform on the dosing and the toxicity profile of this combination for future clinical studies. This bench-to-bedside approach represents a rapidly translatable therapeutic strategy in children with ACVR1 mutant DIPG.

Sign in / Sign up

Export Citation Format

Share Document