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Brain Pathology (Zurich, Switzerland) Jan 2021Deposition of extensively hyperphosphorylated tau in specific brain cells is a clear pathological hallmark in Alzheimer's disease and a number of other neurodegenerative...
Deposition of extensively hyperphosphorylated tau in specific brain cells is a clear pathological hallmark in Alzheimer's disease and a number of other neurodegenerative disorders, collectively termed the tauopathies. Furthermore, hyperphosphorylation of tau prevents it from fulfilling its physiological role as a microtubule-stabilizing protein and leaves it increasingly vulnerable to self-assembly, suggestive of a central underlying role of hyperphosphorylation as a contributing factor in the etiology of these diseases. Via in vitro phosphorylation and regulation of kinase activity within cells and acute brain tissue, we reveal that the inflammation associated kinase, protein kinase R (PKR), directly phosphorylates numerous abnormal and disease-modifying residues within tau including Thr181, Ser199/202, Thr231, Ser262, Ser396, Ser404 and Ser409. Similar to disease processes, these PKR-mediated phosphorylations actively displace tau from microtubules in cells. In addition, PKR overexpression and knockdown, respectively, increase and decrease tau protein and mRNA levels in cells. This regulation occurs independent of noncoding transcriptional elements, suggesting an underlying mechanism involving intra-exonic regulation of the tau-encoding microtubule-associated protein tau (MAPT) gene. Finally, acute encephalopathy in wild type mice, induced by intracranial Langat virus infection, results in robust inflammation and PKR upregulation accompanied by abnormally phosphorylated full-length- and truncated tau. These findings indicate that PKR, independent of other kinases and upon acute brain inflammation, is capable of triggering pathological modulation of tau, which, in turn, might form the initial pathologic seed in several tauopathies such as Alzheimer's disease and Chronic traumatic encephalopathy where inflammation is severe.
Topics: Alzheimer Disease; Animals; Humans; Inflammation; Mice; Mice, Inbred C57BL; Phosphorylation; eIF-2 Kinase; tau Proteins
PubMed: 32716602
DOI: 10.1111/bpa.12883 -
ELife Oct 2015The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at...
The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly.
Topics: Acetylation; Histone Acetyltransferases; Histones; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 26468616
DOI: 10.7554/eLife.09886 -
The Journal of Biological Chemistry Jan 2019Serine-arginine (SR) proteins are essential splicing factors containing a canonical RNA recognition motif (RRM), sometimes followed by a pseudo-RRM, and a C-terminal...
Serine-arginine (SR) proteins are essential splicing factors containing a canonical RNA recognition motif (RRM), sometimes followed by a pseudo-RRM, and a C-terminal arginine/serine-rich (RS) domain that undergoes multisite phosphorylation. Phosphorylation regulates the localization and activity of SR proteins, and thus may provide insight into their differential biological roles. The phosphorylation mechanism of the prototypic SRSF1 by serine-arginine protein kinase 1 (SRPK1) has been well-studied, but little is known about the phosphorylation of other SR protein members. In the present study, interaction and kinetic assays unveiled how SRSF1 and the single RRM-containing SRSF3 are phosphorylated by SRPK2, another member of the SRPK family. We showed that a conserved SRPK-specific substrate-docking groove in SRPK2 impacts the binding and phosphorylation of both SR proteins, and the localization of SRSF3. We identified a nonconserved residue within the groove that affects the kinase processivity. We demonstrated that, in contrast to SRSF1, for which SRPK-mediated phosphorylation is confined to the N-terminal region of the RS domain, SRSF3 phosphorylation sites are spread throughout its entire RS domain Despite this, SRSF3 appears to be hypophosphorylated in cells at steady state. Our results suggest that the absence of a pseudo-RRM renders the single RRM-containing SRSF3 more susceptible to dephosphorylation by phosphatase. These findings suggest that the single RRM- and two RRM-containing SR proteins represent two subclasses of phosphoproteins in which phosphorylation statuses are maintained by unique mechanisms, and pose new directions to explore the distinct roles of SR proteins .
Topics: Amino Acid Sequence; HEK293 Cells; Humans; Models, Molecular; Phosphorylation; Protein Serine-Threonine Kinases; Sequence Alignment; Serine-Arginine Splicing Factors
PubMed: 30478176
DOI: 10.1074/jbc.RA118.003392 -
STAR Protocols Sep 2023Quantification of intracellular proteins is essential to understand signaling. Here, we describe quantification of the expression and phosphorylation of the...
Quantification of intracellular proteins is essential to understand signaling. Here, we describe quantification of the expression and phosphorylation of the transcription factor STAT3. We present isolation of total and phosphorylated STAT3 from cell lysates by immunoprecipitation, followed by SDS-PAGE and western blot together with known amounts of a calibrator protein that shares an epitope with the precipitated proteins. Finally, we explain how to relate the amount of precipitated protein to the amount of calibrator protein considering the efficiency of immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Dittrich et al. (2012) and Reeh et al. (2019)..
Topics: Blotting, Western; Signal Transduction; Proteins; Phosphorylation
PubMed: 37669163
DOI: 10.1016/j.xpro.2023.102508 -
Biochimica Et Biophysica Acta.... Jan 2024AP2 forms AP2 CCV with clathrin and over 60 additional coat proteins. Due to this complexity, we have a limited understanding of CCV life cycle regulation. Synapses...
AP2 forms AP2 CCV with clathrin and over 60 additional coat proteins. Due to this complexity, we have a limited understanding of CCV life cycle regulation. Synapses contain canonical AP2 CCV, canCCV, and more stable, thereby longer lived, AP2 CCV. The more stable AP2 CCV can be distinguished from canCCV due to the stable binding of Hsc70 to clathrin. The AP1/σ1B complex knockout leads to impaired synaptic vesicle recycling and altered endosomal protein sorting. This causes as a secondary phenotype the twofold upregulation of endocytosis by canCCV and by more stable AP2 CCV. These stable CCV are more stabilized than their wt counterpart, hence stCCV. They have less of the uncoating proteins synaptojanin1 and Hsc70, and more of the coat stabilizing AAK1. Hsc70 clathrin dissociation activity is regulated by complex phosphorylation patterns. Two major groups of hyper- and of hypo-phosphorylated Hsc70 proteins are formed. The latter are enriched in wt stable CCV and stabilized stCCV. Hsc70 T265 phosphorylation regulates binding of CaM/Ca. CaM/Ca binding to the T265 domain blocks Hsc70 homodimerization and its concentration in stCCV required for clathrin disassembly. Kinases DYRK1A and CaMK-IIδ can phosphorylate T265 preventing CaM/Ca binding. Their and the levels of STK38L and STK39/Cab39, which are able to phosphorylate additional Hsc70 residues are reduced in stCCV. The stCCV pathway sorts specifically the cell adhesion proteins CHL1 and Neurocan, supporting our model of that the stCCV pathway fulfills specific functions in synaptic plasticity.
Topics: Phosphorylation; Calmodulin; Longevity; Cell Adhesion; Clathrin; Cell Adhesion Molecules; Protein Transport
PubMed: 37926156
DOI: 10.1016/j.bbamcr.2023.119611 -
Autophagy Feb 2022ULK1 kinase is the gatekeeper of canonical macroautophagy (hereafter referred to as autophagy) phosphorylating an array of substrates critical for autophagosome...
ULK1 kinase is the gatekeeper of canonical macroautophagy (hereafter referred to as autophagy) phosphorylating an array of substrates critical for autophagosome biogenesis. To uncover if ULK1 has broader functions also regulating subsequent steps of autophagosome turnover, i.e., maturation, lysosomal fusion, and degradation, we performed a set of unbiased phosphoproteomic experiments employing mouse and human cells in combination with genetic and environmental perturbations. We characterized more than 1,000 potential ULK1 target sites of which many affect proteins known to be involved in all phases of the autophagosome life cycle. To better understand which of these 1,000 phosphosites were directly phosphorylated by ULK1, in contrast to downstream kinases being activated or phosphatases being inhibited by ULK1, we developed a proteome-scale kinase assay and characterized 187 phosphosites on 157 proteins as ULK1 target sites. Interestingly, our results highlight an intricate crosstalk between ULK1 and protein phosphatases. Focusing on STRN (striatin), a regulatory subunit of PPP2/PP2A (protein phosphatase 2), we identified a positive feedback loop linked to ULK1 and promoting autophagy.
Topics: Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Intracellular Signaling Peptides and Proteins; Mice; Phosphorylation; Protein Phosphatase 2
PubMed: 34839766
DOI: 10.1080/15548627.2021.2002546 -
Biochimica Et Biophysica Acta. General... Apr 2019Ca/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca-dependent...
BACKGROUND
Ca/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear.
METHODS
Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ.
RESULTS
Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca/CaM-dependent enzyme.
CONCLUSION
cAMP/PKA signaling may confer Ca-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells.
GENERAL SIGNIFICANCE
Our results suggest a novel cross-talk between cAMP/PKA and Ca/CaM/CaMKKβ signaling through regulatory phosphorylation.
Topics: Animals; COS Cells; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Chlorocebus aethiops; Cyclic AMP; HeLa Cells; Humans; Phosphorylation; Rats; Recombinant Proteins; Signal Transduction
PubMed: 30660766
DOI: 10.1016/j.bbagen.2018.12.012 -
The EMBO Journal Jun 2022PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3...
PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observed that phospho-ubiquitin can bind to two distinct sites on parkin, a high-affinity site on RING1 that controls parkin localization and a low-affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho-ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.
Topics: Phosphorylation; Protein Domains; Protein Kinases; Ubiquitin; Ubiquitin-Protein Ligases
PubMed: 35491809
DOI: 10.15252/embj.2021109460 -
Scientific Reports Jan 2017Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences...
Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO's substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis.
Topics: Actins; Adenosine Diphosphate; Bacterial Proteins; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Substrate Specificity; Yersinia enterocolitica
PubMed: 28051168
DOI: 10.1038/srep39998 -
The Journal of Biological Chemistry Apr 2016P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes...
P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeastSaccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg(2+), and EGTA. Low concentrations of Ca(2+)in the phosphorylation medium decreased the rate of phosphorylation and the maximal level of phosphoenzyme. Neither Mn(2+)nor Mg(2+)had an inhibitory effect on the formation of the phosphoenzyme similar to that of Ca(2+) TheKmfor ATP in the phosphorylation reaction was ∼1 μmand did not significantly change in the presence of Ca(2+) Half-maximal phosphorylation was attained at 8 μmMg(2+), but higher concentrations partially protected from Ca(2+)inhibition. In conditions similar to those used for phosphorylation, Ca(2+)had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.
Topics: ATP-Binding Cassette Transporters; Calcium; Endoplasmic Reticulum Stress; Phosphorylation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 26858246
DOI: 10.1074/jbc.M115.695122