Phosphorylation-dependent Switch Research Articles

PATH-65. MONITORING MENINGIOMA CELL CYCLE ACTIVITY THROUGH DREAM-MMB ACTIVITY SWITCHING

Abstract Meningiomas account for over 40% of all primary intracranial brain tumors, with approximately one-fifth of these tumors recurring. Our lab has previously used molecular techniques to identify three subgroups: MenG A, B, and C, which have been independently validated. Interestingly, only MenG C tumors harbor recurring patients, making it an aggressive subtype of meningioma. One hallmark of MenG C tumors is the overexpression of a distinct set of genes that harbor in their promoter the CDE-CHR DNA-binding element, used by a transcriptional complex that in dormant cells functions as a repressor (DREAM). Before phosphorylation, this complex functions as an activator (MBB) in cycling cells. Upon a phosphorylation-dependent switch, the dual-specificity tyrosine kinase DYRK1A phosphorylates S28 on LIN52, which itself is dimerized with LIN9 of the core complex. Phosphorylation shifts the complex to DREAM, while lack thereof allows the core complex to interact with MYBL2 (now MBB) and activate the same genes repressed in quiescent cells. We seek to quantify this switch activity using an in vivo luciferase sensor using NanoBiT technology. Our biosensor is based on a split nanoluciferase, where one half is fused to the LIN52/LIN9 dimer and the other is fused to MYBL2. When MYBL2 binds to the LIN52/LIN9 dimer, a functional nanoluciferase is formed, and its activity can be measured using a luciferase-specific substrate. As proof of principle, we have increased cell proliferation by increasing FBS concentration in cultured cells as well as by blocking S28 phosphorylation using the DYRK1A-specific inhibitor Harmine. We screened all possible combinations (alternating NanoBiT fragments) for the highest luciferase activity after stimulation. In the future, this biosensor can be used to monitor cell cycle activity in vitro as well as in vivo using low-cost Luciferase reporter assays to better understand the regulation of specific genes in MenG C tumors.

De novo design of a reversible phosphorylation-dependent switch for membrane targeting

Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

Adaptable and Multifunctional Ion-Conducting Aquaporins.

Aquaporins function as water and neutral solute channels, signaling hubs, disease virulence factors, and metabolon components. We consider plant aquaporins that transport ions compared to some animal counterparts. These are candidates for important, as yet unidentified, cation and anion channels in plasma, tonoplast, and symbiotic membranes. For those individual isoforms that transport ions, water, and gases, the permeability spans 12 orders of magnitude. This requires tight regulation of selectivity via protein interactions and posttranslational modifications. A phosphorylation-dependent switch between ion and water permeation in AtPIP2;1 might be explained by coupling between the gates of the four monomer water channels and the central pore of the tetramer. We consider the potential for coupling between ion and water fluxes that could form the basis of an electroosmotic transducer. A grand challenge in understanding the roles of ion transporting aquaporins is their multifunctional modes that are dependent on location, stress, time, and development.

A phosphorylation-dependent switch in the disordered p53 transactivation domain regulates DNA binding

The tumor-suppressor p53 is a critical regulator of the cellular response to DNA damage and is tightly regulated by posttranslational modifications. Thr55 in the AD2 interaction motif of the N-terminal transactivation domain functions as a phosphorylation-dependent regulatory switch that modulates p53 activity. Thr55 is constitutively phosphorylated, becomes dephosphorylated upon DNA damage, and is subsequently rephosphorylated to facilitate dissociation of p53 from promoters and inactivate p53-mediated transcription. Using NMR and fluorescence spectroscopy, we show that Thr55 phosphorylation inhibits DNA-binding by enhancing competitive interactions between the disordered AD2 motif and the structured DNA-binding domain (DBD). Nonphosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer. Upon phosphorylation of Thr55, cooperativity is abolished and p53 binds initially to cognate DNA sites as a dimer. As the concentration of phosphorylated p53 is further increased, a second dimer binds and causes p53 to dissociate from the DNA, resulting in a bell-shaped binding curve. This autoinhibition is driven by favorable interactions between the DNA-binding surface of the DBD and the multiple phosphorylated AD2 motifs within the tetramer. These interactions are augmented by additional phosphorylation of Ser46 and are fine-tuned by the proline-rich domain (PRD). Removal of the PRD strengthens the AD2-DBD interaction and leads to autoinhibition of DNA binding even in the absence of Thr55 phosphorylation. This study reveals the molecular mechanism by which the phosphorylation status of Thr55 modulates DNA binding and controls both activation and termination of p53-mediated transcriptional programs at different stages of the cellular DNA damage response.

N-terminal Huntingtin (Htt) phosphorylation is a molecular switch regulating Htt aggregation, helical conformation, internalization, and nuclear targeting

Huntington's disease is a fatal neurodegenerative disorder resulting from a CAG repeat expansion in the first exon of the gene encoding the Huntingtin protein (Htt). Phosphorylation of this protein region (Httex1) has been shown to play important roles in regulating the structure, toxicity, and cellular properties of N-terminal fragments and full-length Htt. However, increasing evidence suggests that phosphomimetic substitutions in Htt result in inconsistent findings and do not reproduce all aspects of true phosphorylation. Here, we investigated the effects of bona fide phosphorylation at Ser-13 or Ser-16 on the structure, aggregation, membrane binding, and subcellular properties of the Httex1-Q18A variant and compared these effects with those of phosphomimetic substitutions. We show that phosphorylation at either Ser-13 and/or Ser-16 or phosphomimetic substitutions at both these residues inhibit the aggregation of mutant Httex1, but that only phosphorylation strongly disrupts the amphipathic α-helix of the N terminus and prompts the internalization and nuclear targeting of preformed Httex1 aggregates. In synthetic peptides, phosphorylation at Ser-13, Ser-16, or both residues strongly disrupted the amphipathic α-helix of the N-terminal 17 residues (Nt17) of Httex1 and Nt17 membrane binding. Experiments with peptides bearing different combinations of phosphorylation sites within Nt17 revealed a phosphorylation-dependent switch that regulates the Httex1 structure, involving cross-talk between phosphorylation at Thr-3 and Ser-13 or Ser-16. Our results provide crucial insights into the role of phosphorylation in regulating Httex1 structure and function, and underscore the critical importance of identifying the enzymes responsible for regulating Htt phosphorylation, and their potential as therapeutic targets for managing Huntington's disease.

Postsynaptic density 95 (PSD-95) serine 561 phosphorylation regulates a conformational switch and bidirectional dendritic spine structural plasticity

Postsynaptic density 95 (PSD-95) is a major synaptic scaffolding protein that plays a key role in bidirectional synaptic plasticity, which is a process important for learning and memory. It is known that PSD-95 shows increased dynamics upon induction of plasticity. However, the underlying structural and functional changes in PSD-95 that mediate its role in plasticity remain unclear. Here we show that phosphorylation of PSD-95 at Ser-561 in its guanylate kinase (GK) domain, which is mediated by the partitioning-defective 1 (Par1) kinases, regulates a conformational switch and is important for bidirectional plasticity. Using a fluorescence resonance energy transfer (FRET) biosensor, we show that a phosphomimetic mutation of Ser-561 promotes an intramolecular interaction between GK and the nearby Src homology 3 (SH3) domain, leading to a closed conformation, whereas a non-phosphorylatable S561A mutation or inhibition of Par1 kinase activity decreases SH3-GK interaction, causing PSD-95 to adopt an open conformation. In addition, S561A mutation facilitates the interaction between PSD-95 and its binding partners. Fluorescence recovery after photobleaching imaging reveals that the S561A mutant shows increased stability, whereas the phosphomimetic S561D mutation increases PSD-95 dynamics at the synapse. Moreover, molecular replacement of endogenous PSD-95 with the S561A mutant blocks dendritic spine structural plasticity during chemical long-term potentiation and long-term depression. Endogenous Ser-561 phosphorylation is induced by synaptic NMDA receptor activation, and the SH3-GK domains exhibit a Ser-561 phosphorylation-dependent switch to a closed conformation during synaptic plasticity. Our results provide novel mechanistic insight into the regulation of PSD-95 in dendritic spine structural plasticity through phosphorylation-mediated regulation of protein dynamics and conformation.

The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation.

Decreased tyrosine phosphorylation of nephrin in rat and human nephrosis

Phosphorylation of tyrosine residue (Y1204) of rat nephrin by Fyn kinase allows Nck adaptor protein binding to nephrin motifs, which include the phosphorylated tyrosine. This phosphorylation-dependent switch induces actin polymerization in a cell culture system. Here, we generated an antibody recognizing phosphorylated nephrin at the Nck binding sites pY1204 and pY1228 to determine the phosphorylation status of nephrin using a rat model of puromycin aminonucleoside-induced nephrosis. Changes in globular actin (G-actin) and filamentous actin (F-actin) contents in isolated glomeruli were measured by western blot. Before experimental nephrosis, both Y1204 and Y1228 were phosphorylated, and most of the actin was filamentous. Before the onset of overt proteinuria, however, phosphorylation of both Y1204 and Y1228 rapidly decreased and became almost undetectable. During this period, the amount of F-actin in glomeruli began to decrease, whereas G-actin increased. Phosphorylation of nephrin at Y1228 in glomeruli of patients with minimal change nephrosis was significantly decreased compared with that in normal glomeruli. Our study suggests that tyrosine phosphorylation of nephrin by regulating F-actin formation may be important for the maintenance of normal podocyte morphology and function.

Interferon Regulatory Factor 3 Is Regulated by a Dual Phosphorylation-dependent Switch

The transcription factor interferon regulatory factor 3 (IRF-3) regulates genes in the innate immune response. IRF-3 is activated through phosphorylation by the kinases IKK epsilon and/or TBK1. Phosphorylation results in IRF-3 dimerization and removal of an autoinhibitory structure to allow interaction with the coactivators CBP/p300. The precise role of the different phosphorylation sites has remained controversial. Using purified proteins we show that TBK1 can directly phosphorylate full-length IRF-3 in vitro. Phosphorylation at residues in site 2 (Ser(396)-Ser(405)) alleviates autoinhibition to allow interaction with CBP (CREB-binding protein) and facilitates phosphorylation at site 1 (Ser(385) or Ser(386)). Phosphorylation at site 1 is, in turn, required for IRF-3 dimerization. The data support a two-step phosphorylation model for IRF-3 activation mediated by TBK1.

Doublecortin Kinase-2, a Novel Doublecortin-related Protein Kinase Associated with Terminal Segments of Axons and Dendrites

The microtubule (MT)-associated DCX protein plays an essential role in the development of the mammalian cerebral cortex. We report on the identification of a protein kinase, doublecortin kinase-2 (DCK2), with a domain (DC) highly homologous to DCX. DCK2 has MT binding activity associated with its DC domain and protein kinase activity mediated by a kinase domain, organized in a structure in which the two domains are functionally independent. Overexpression of DCK2 stabilizes the MT cytoskeleton against cold-induced depolymerization. Autophosphorylation of DCK2 strongly reduces its affinity for MTs. DCK2 and DCX mRNAs are nervous system-specific and are expressed during the period of cerebrocortical lamination. DCX is down-regulated postnatally, whereas DCK2 persists in abundance into adulthood, suggesting that the DC sequence has previously unrecognized functions in the mature nervous system. In sympathetic neurons, DCK2 is localized to the cell body and to the terminal segments of axons and dendrites. DCK2 may represent a phosphorylation-dependent switch for the reversible control of MT dynamics in the vicinity of neuronal growth cones.

Dephosphorylated C/EBPα Accelerates Cell Proliferation through Sequestering Retinoblastoma Protein

CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been previously considered a strong inhibitor of cell proliferation which uses multiple pathways to cause growth arrest. In this paper, we describe a new function of C/EBPalpha, which is an acceleration of cell proliferation. This new function of C/EBPalpha is created in proliferating livers by protein phosphatase 2A-mediated dephosphorylation of C/EBPalpha at Ser193. The Ser193-dephosphorylated C/EBPalpha interacts with retinoblastoma protein (Rb) independently on E2Fs and sequesters Rb, leading to a reduction of E2F-Rb repressors and to acceleration of proliferation. This new function of C/EBPalpha requires Rb, since the dephosphorylated C/EBPalpha does not promote proliferation in Rb-negative cells. We also show that a balance of Rb and Ser193-dephosphorylated C/EBPalpha determines if the cells are growth arrested or have an increased rate of proliferation. Consistently with these findings, a significant portion of Rb is sequestered into Rb-C/EBPalpha complexes in proliferating livers, and E2F-Rb complexes are not detectable in these livers. Our data demonstrate a new pathway by which the phosphorylation-dependent switch of biological functions of C/EBPalpha promotes liver proliferation.

Identification of the binding interfaces on CheY for two of its targets the phosphatase CheZ and the flagellar switch protein FliM

CheY is the response regulator protein serving as a phosphorylation-dependent switch in the bacterial chemotaxis signal transduction pathway. CheY has a number of proteins with which it interacts during the course of the signal transduction pathway. In the phosphorylated state, it interacts strongly with the phosphatase CheZ, and also the components of the flagellar motor switch complex, specifically with FliM. Previous work has characterized peptides consisting of small regions of CheZ and FliM which interact specifically with CheY. We have quantitatively measured the binding of these peptides to both unphosphorylated and phosphorylated CheY using fluorescence spectroscopy. There is a significant enhancement of the binding of these peptides to the phosphorylated form of CheY, suggesting that these peptides share much of the binding specificity of the intact targets of the phosphorylated form of CheY. We also have used modern nuclear magnetic resonance methods to characterize the sites of interaction of these peptides on CheY. We have found that the binding sites are overlapping and primarily consist of residues in the C-terminal portion of CheY. Both peptides affect the resonances of residues at the active site, indicating that the peptides may either bind directly at the active site or exert conformational influences that reach to the active site. The binding sites for the CheZ and FliM peptides also overlap with the previously characterized CheA binding interface. These results suggest that interaction with these three proteins of the signal transduction pathway are mutually exclusive. In addition, since these three proteins are sensitive to the phosphorylation state of CheY, it may be that the C-terminal region of CheY is most sensitive for the conformational changes occurring upon phosphorylation.

Ménage à quatre: profilin regulates axon pathfinding in vivo

How an extracellular signal is decoded by the axonal growth cone and transformed to an intracellular signal leading to the appropriate turning or growth response is largely unknown. The highly motile membrane structures (filopodia and lamellipodia) at the tip of the growth cone are supported by a complex cytoskeletal architecture comprising actin and associated proteins. A powerful way to regulate assembly of actin polymers is through proteins, such as profilin, that can sequester monomeric G-actin. Dominant–negative profilin blocks neurite formation in vitro, and Drosophila has proven useful to identify and characterize proteins in vivo that are required for axon pathfinding.Wills et al.1xProfilin and the Abl tyrosine kinase are required for motor axon outgrowth in the Drosophila embryo. Wills, Z., Marr, L., Zinn, K., Goodman, C.S., and Van Vactor, D. Neuron. 1999; 22: 291–299Abstract | Full Text | Full Text PDF | PubMedSee all References1 show that stranded, a previously identified zygotic-lethal mutation, corresponds to profilin/chickadee; motor axon extension and pathfinding are impaired in profilin mutants both in vivo and in vitro. Profilin binds to proteins of the Enabled (Ena) family, which are known to be phosphorylated by the tyrosine kinase Abl. Mutants of profilin and kinase-deficient Abl display an identical axon growth-arrest phenotype of the intersegmental motor neuron b (ISNb), suggesting that Abl and profilin act in the same process; this was further demonstrated by a dose-sensitive genetic analysis. Moreover, inappropriate crossings of axons at the central nervous system midline were observed1xProfilin and the Abl tyrosine kinase are required for motor axon outgrowth in the Drosophila embryo. Wills, Z., Marr, L., Zinn, K., Goodman, C.S., and Van Vactor, D. Neuron. 1999; 22: 291–299Abstract | Full Text | Full Text PDF | PubMedSee all

Phosducin, Potential Role in Modulation of Olfactory Signaling

Phosducin, which tightly binds betagamma-subunits of heterotrimeric G-proteins, has been conjectured to play a role in regulating second messenger signaling cascades, but to date its specific function has not been elucidated. Here we demonstrate a potential role for phosducin in regulating olfactory signal transduction. In isolated olfactory cilia certain odorants elicit a rapid and transient cAMP response, terminated by a concerted process which requires the action of two protein kinases, protein kinase A (PKA) and a receptor-specific kinase (GRK3) (Schleicher, S., Boekhoff, I. Arriza, J., Lefkowitz, R. J., and Breer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1420-1424). The mechanism of action of GRK3 involves a Gbetagamma-mediated translocation of the kinase to the plasma membrane bound receptors (Pitcher, J. A., Inglese, J., Higgins, J. B. , Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M. , Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267). A protein with a molecular mass of 33 kDa that comigrates on SDS gels with recombinant phosducin and which is immunoreactive with phosducin antibodies is present in olfactory cilia. Recombinant phosducin added to permeabilized olfactory cilia preparations strongly inhibits termination of odorant-induced cAMP response and odorant-induced membrane translocation of GRK3. In addition, the cAMP analogue dibutyryl cAMP stimulates membrane targeting of the receptor kinase. This effect is presumably due to PKA-mediated phosphorylation of phosducin, which diminishes its affinity for binding to the Gbetagamma-subunit, thereby making Gbetagamma available to function as a membrane anchor for GRK3. A specific PKA inhibitor blocks the odorant-induced translocation of the receptor kinase. Consistent with this formulation, a non-phosphorylatable mutant of phosducin (phosducin Ser-73 --> Ala) is an even more effective inhibitor of desensitization and membrane targeting of GRK3 than the wild-type protein. A phosducin mutant that mimics phosphorylated phosducin (phosducin Ser-73 --> Asp) lacks this property and in fact recruits GRK3 to the membrane and potentiates desensitization. These results suggest that phosducin may act as a phosphorylation-dependent switch in second messenger signaling cascades, regulating the kinetics of desensitization processes by controlling the activity of Gbetagamma-dependent GRKs.