-
Microbiology and Molecular Biology... Jun 2014The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and... (Review)
Review
The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions.
The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.
Topics: Bacteria; Bacterial Proteins; Biological Transport; Carbohydrate Metabolism; Phosphoenolpyruvate; Phosphorylation; Phosphotransferases; Protein Binding
PubMed: 24847021
DOI: 10.1128/MMBR.00001-14 -
ENeuro 2021SHANK3 is a large scaffolding protein in the postsynaptic density (PSD) that organizes protein networks, which are critical for synaptic structure and function. The...
SHANK3 is a large scaffolding protein in the postsynaptic density (PSD) that organizes protein networks, which are critical for synaptic structure and function. The strong genetic association of with autism spectrum disorder (ASD) emphasizes the importance of SHANK3 in neuronal development. SHANK3 has a critical role in organizing excitatory synapses and is tightly regulated by alternative splicing and posttranslational modifications. In this study, we examined basal and activity-dependent phosphorylation of Shank3 using mass spectrometry (MS) analysis from phosphorylation assays, experiments, and studies with cultured neurons. We found that Shank3 is highly phosphorylated, and we identified serine 782 (S782) as a potent CaMKII phosphorylation site. Using a phosphorylation state-specific antibody, we demonstrate that CaMKII can phosphorylate Shank3 S782 and in heterologous cells on cotransfection with CaMKII. We also observed an effect of a nearby ASD-associated variant (Shank3 S685I), which increased S782 phosphorylation. Notably, eliminating phosphorylation of Shank3 with a S782A mutation increased Shank3 and PSD-95 synaptic puncta size without affecting Shank3 colocalization with PSD-95 in cultured hippocampal neurons. Taken together, our study revealed that CaMKII phosphorylates Shank3 S782 and that the phosphorylation affects Shank3 synaptic properties.
Topics: Autism Spectrum Disorder; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Humans; Nerve Tissue Proteins; Phosphorylation; Synapses
PubMed: 33568460
DOI: 10.1523/ENEURO.0481-20.2021 -
Nuclear Receptor Signaling May 2007Although the primary signal for the activation of steroid hormone receptors is binding of hormone, there is increasing evidence that the activities of cell signaling... (Review)
Review
Although the primary signal for the activation of steroid hormone receptors is binding of hormone, there is increasing evidence that the activities of cell signaling pathways and the phosphorylation status of these transcription factors and their coregulators determine the overall response to the hormone. In some cases, enhanced cell signaling is sufficient to cause activation of receptors in medium depleted of steroids. Steroid receptors are targets for multiple kinases. Many of the phosphorylation sites contain Ser/Thr-Pro motifs implicating proline-directed kinases such as the cyclin-dependent kinases and the mitogen-activated kinases (MAPK) in receptor phosphorylation. Although some sites are constitutively phosphorylated, others are phosphorylated in response to hormone. Still others are only phosphorylated in response to specific cell signaling pathways. Phosphorylation of specific sites has been implicated not only in overall transcriptional activity, but also in nuclear localization, protein stability, and DNA binding. The studies of the roles of phosphorylation in coregulator function are more limited, but it is now well established that many of them are highly phosphorylated and that phosphorylation regulates their function. There is good evidence that some of the phosphorylation sites in the receptors and coregulators are targets of multiple signaling pathways. Individual sites have been associated both with functions that enhance the activity of the receptor, as well as with functions that inhibit activity. Thus, the specific combinations of phosphorylations of the steroid receptor combined with the expression levels and phosphorylation status of coregulators will determine the genes regulated and the biological response.
Topics: Cell Nucleus; Feedback; Gonadal Steroid Hormones; Models, Biological; Nuclear Proteins; Phosphorylation; Phosphotransferases; Receptors, Cytoplasmic and Nuclear; Signal Transduction
PubMed: 17525795
DOI: 10.1621/nrs.05005 -
Traffic (Copenhagen, Denmark) Nov 2021Phosphorylation of the central adaptor protein complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). Here, we uncover the role of an uncharacterized kinase...
Phosphorylation of the central adaptor protein complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). Here, we uncover the role of an uncharacterized kinase (BMP-2 inducible kinase-BMP2K) in AP-2 phosphorylation. We demonstrate that BMP2K can phosphorylate AP-2 in vitro and in vivo. Functional impairment of BMP2K impedes AP-2 phosphorylation leading to defects in clathrin-coated pit (CCP) morphology and cargo internalization. BMP2K engages AP-2 via its extended C-terminus and this interaction is important for its CCP localization and function. Notably, endogenous BMP2K levels decline upon functional impairment of AP-2 indicating AP-2 dependent BMP2K stabilization in cells. Further, functional inactivation of BMP2K in zebrafish embryos yields gastrulation phenotypes which mirror AP-2 loss-of-function suggesting physiological relevance of BMP2K in vertebrates. Together, our findings propose involvement of a novel kinase in AP-2 phosphorylation and in the operation of CME.
Topics: Adaptor Protein Complex 2; Animals; Clathrin; Endocytosis; Phosphorylation; Zebrafish
PubMed: 34480404
DOI: 10.1111/tra.12814 -
ELife May 2023Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated...
Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated by autophosphorylation in the Aurora B activation loop and in IN-box, but it is not clear how these phosphorylations activate the enzyme. We used a combination of experimental and computational studies to investigate the effects of phosphorylation on the molecular dynamics and structure of [Aurora B/IN-box]. In addition, we generated partially phosphorylated intermediates to analyze the contribution of each phosphorylation independently. We found that the dynamics of Aurora and IN-box are interconnected, and IN-box plays both positive and negative regulatory roles depending on the phosphorylation status of the enzyme complex. Phosphorylation in the activation loop of Aurora B occurs intramolecularly and prepares the enzyme complex for activation, but two phosphorylated sites are synergistically responsible for full enzyme activity.
Topics: Aurora Kinase B; Allosteric Regulation; Amino Acid Sequence; Cell Division; Phosphorylation; Mitosis
PubMed: 37227118
DOI: 10.7554/eLife.85328 -
Cellular Signalling Jan 2014Annexin A1 (ANXA1) is the first characterized member of the annexins superfamily. It binds the cellular membrane phospholipids in Ca(2+) regulated manner. Annexin A1 has... (Review)
Review
Annexin A1 (ANXA1) is the first characterized member of the annexins superfamily. It binds the cellular membrane phospholipids in Ca(2+) regulated manner. Annexin A1 has been found in several tissues and many physiological roles as hormones secretion, vesiculation, inflammatory response, apoptosis and differentiation have been shown. Its subcellular localization and binding with many partner proteins are altered accordingly with its physiological role. The Annexin A1 membrane localization is crucial for binding to receptors, suggesting a paracrine and juxtacrine extracellular action. Annexin A1 is subjected to several post-translational modifications. In particular the protein is phosphorylated on several residues both on the N-terminal functional domain and on the C-terminus core. Different kinases have been identified as responsible for the phosphorylation status of selective residues. The specific change in the phosphorylation status on the different sites alters ANXA1 localization, binding properties and functions. This review shows the physiological relevance of the ANXA1 phosphorylation leading to the conclusion that numerous and different roles of Annexin A1 could be associated with different phosphorylations to alter not only intracellular localization and bindings to its partners but also the extracellular receptor interactions.
Topics: Amino Acids; Animals; Annexin A1; Cell Membrane; Humans; Models, Biological; Phosphorylation; Protein Transport
PubMed: 24103589
DOI: 10.1016/j.cellsig.2013.09.020 -
Trends in Endocrinology and Metabolism:... Oct 2013Steroid hormone receptors (SHRs) are hormone-activated transcription factors involved in numerous cellular functions and in health and disease. Their activities depend... (Review)
Review
Steroid hormone receptors (SHRs) are hormone-activated transcription factors involved in numerous cellular functions and in health and disease. Their activities depend on the cellular level of the receptor, the presence of coregulator proteins, and the cell signaling pathways that are active in the cell. SHRs and their coregulators are phosphorylated on multiple sites by a wide variety of kinases. Each site may contribute to multiple functions and the net effect of an individual phosphorylation depends on the activating kinase. Here we discuss functions of known SHR phosphorylation sites, kinase regulation, evidence of translational relevance, and crosstalk between SHRs and cell signaling pathways. Understanding how cell signaling pathways regulate SHRs might yield novel therapeutic targets for multiple human diseases.
Topics: Animals; Humans; Phosphorylation; Receptors, Steroid; Signal Transduction
PubMed: 23838532
DOI: 10.1016/j.tem.2013.05.008 -
Cell Cycle (Georgetown, Tex.) 2015Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show...
Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.
Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin-Dependent Kinase 5; DNA Damage; DNA Replication; Female; HeLa Cells; Humans; Hydroxyurea; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Radiation, Ionizing
PubMed: 26237679
DOI: 10.1080/15384101.2015.1078020 -
Biochimica Et Biophysica Acta Apr 2015Yeast cells have developed a variety of mechanisms to regulate the activity of metabolic enzymes in order to adjust their metabolism in response to genetic and... (Review)
Review
BACKGROUND
Yeast cells have developed a variety of mechanisms to regulate the activity of metabolic enzymes in order to adjust their metabolism in response to genetic and environmental perturbations. This can be achieved by a massive reprogramming of gene expression. However, the transcriptional response cannot explain the complexity of metabolic regulation, and mRNA stability regulation, non-covalent binding of allosteric effectors and post-translational modifications of enzymes (such as phosphorylation, acetylation and ubiquitination) are also involved, especially as short term responses, all converging in modulating enzyme activity.
SCOPE OF REVIEW
The functional significance of post-translational modifications (PTMs) to the regulation of the central carbon metabolism is the subject of this review.
MAJOR CONCLUSIONS
A genome wide analysis of PTMs indicates that several metabolic enzymes are subjected to multiple PTMs, suggesting that yeast cells can use different modifications and/or combinations of them to specifically respond to environmental changes. Glycolysis and fermentation are the pathways where phosphorylation, acetylation and ubiquitination are most frequent, while enzymes of storage carbohydrate metabolism are especially phosphorylated. Interestingly, some enzymes, such as the 6-phosphofructo-2-kinase Pfk26, the phosphofructokinases Pfk1 and Pfk2 and the pyruvate kinase Cdc19, are hubs of PTMs, thus representing central key regulation nodes. For the functionally better characterized enzymes, the role of phosphorylations and lysine modifications is discussed.
GENERAL SIGNIFICANCE
This review focuses on the regulatory mechanisms of yeast carbon metabolism, highlighting the requirement of quantitative, systematical studies to better understand PTM contribution to metabolic regulation.
Topics: Allosteric Regulation; Carbon; Fermentation; Gluconeogenesis; Glycolysis; Phosphorylation; Protein Processing, Post-Translational; RNA Stability; Saccharomyces cerevisiae; Transcription, Genetic
PubMed: 25512067
DOI: 10.1016/j.bbagen.2014.12.010 -
Microbiology (Reading, England) Sep 2015Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for... (Review)
Review
Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein kinases and bacterial protein tyrosine kinases) are also much more promiscuous than the TCS kinases, i.e. each of them can phosphorylate several substrate proteins. As a consequence, the dynamics and topology of the signal transduction networks depending on these kinases differ significantly from the TCSs. Here, we present an overview of different classes of bacterial TR phosphorylated and regulated by serine/threonine and tyrosine kinases. Particular attention is given to examples when serine/threonine and tyrosine kinases interact with TCSs, phosphorylating either the histidine kinases or the response regulators. We argue that these promiscuous kinases connect several signal transduction pathways and serve the role of signal integration.
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Histidine Kinase; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Serine; Signal Transduction; Threonine; Transcription Factors; Tyrosine
PubMed: 26220449
DOI: 10.1099/mic.0.000148