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Biochemical and Biophysical Research... Mar 2020Protein-protein interactions can be modulated by phosphorylation of either binding partner, thereby altering subcellular localization and/or physiological function....
Protein-protein interactions can be modulated by phosphorylation of either binding partner, thereby altering subcellular localization and/or physiological function. Shank3, a master postsynaptic scaffolding protein that controls the developmental maturation of excitatory synapses, was recently shown to be phosphorylated by Protein Kinase A (PKA) at Ser685 in vivo. Mutation of Shank3 Ser685 was shown to modulate the binding of Abelson interactor 1 (ABI1), a component of the WAVE regulatory complex for actin remodeling, but a direct effect of Ser685 phosphorylation on ABI1 binding was not investigated. Here, we demonstrate that Ca/calmodulin-dependent protein kinase II alpha (CaMKIIα) also phosphorylates Shank3 at Ser685. Mutation of Ser685 to phospho-null alanine (S685A) prevented both CaMKIIα and PKA phosphorylation of a GST-Shank3 fusion protein. The co-immunoprecipitation of ABI1 with Shank3 from HEK293 cell extracts is reduced by mutation of Ser685 to either Ala or Asp. However, pre-phosphorylation of GST-Shank3 by purified CaMKIIα significantly increased binding of ABI1, and this effect was abrogated by Ser685 to Ala mutation in GST-Shank3. Taken together, our data suggest that neuronal ABI1-Shank3 interactions may be convergently regulated by Shank3 Ser685 phosphorylation in response to both Ca and cAMP signaling, potentially modulating dendritic spine morphology.
Topics: Adaptor Proteins, Signal Transducing; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cattle; Cyclic AMP-Dependent Protein Kinases; HEK293 Cells; Humans; Mice; Mutation; Nerve Tissue Proteins; Phosphorylation; Phosphoserine; Protein Binding
PubMed: 31983435
DOI: 10.1016/j.bbrc.2020.01.089 -
Acta Biomaterialia Mar 2020Calcium phosphate-based bone cements have been widely adopted in both orthopedic and dental applications. Phosphoserine (pSer), which has a natural role in...
Calcium phosphate-based bone cements have been widely adopted in both orthopedic and dental applications. Phosphoserine (pSer), which has a natural role in biomineralization, has been identified to possess the functionality to react with calcium phosphate phases, such as tetracalcium phosphate (TTCP) and α-tricalcium phosphate (α-TCP), and form a uniquely adhesive cement. This study investigated the chemical composition and phase evolution of a heterogeneous calcium phosphate (56% TTCP and 15% α-TCP) and pSer cement system with respect to pH. The coordination network of calcium phosphoserine monohydrate was discovered as the predominant crystalline phase of this adhesive apatitic cement system. Furthermore, it was determined that pH has a significant effect on the reaction kinetics of the system, whereby a lower pH tends to accelerate the reaction rate and favor products with lower Ca/P ratios. These findings provide a better understanding of the reaction and products of this adhesive organo-ceramic cement, which can be compositionally tuned for broad applications in the orthopedic and dental spaces. STATEMENT OF SIGNIFICANCE: The application of self-setting calcium phosphate cements (CPCs) in hard tissue regeneration has been a topic of significant research since their introduction to the field 30 years ago. Traditional CPCs, however, are limited by their suboptimal mechanical properties due to their solely inorganic composition. Recently, it was discovered that monomeric phosphoserine (pSer) is capable of serving as a setting reagent for a subset of CPC systems, resulting in an adhesive organo-ceramic composite. Despite its adhesive functionality and biomedical potential, its reaction chemistry and product composition were not well characterized. The present study identifies a calcium phosphoserine coordination network as the primary crystalline phase of this apatitic cement system and further characterizes compositional tunability of the products with respect to pH.
Topics: Apatites; Bone Cements; Calcium; Calcium Phosphates; Hydrogen-Ion Concentration; Models, Molecular; Phosphoserine; Resin Cements; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction
PubMed: 31945507
DOI: 10.1016/j.actbio.2020.01.007 -
The Journal of Biological Chemistry Jun 2020Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via...
Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 "known" substrates and two independent and larger oriented peptide array libraries (OPALs) of ∼10 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.
Topics: Amino Acid Motifs; Amino Acid Sequence; Humans; Peptide Library; Peptides; Phosphorylation; Phosphoserine; Proto-Oncogene Proteins c-akt; ROC Curve; Substrate Specificity
PubMed: 32350110
DOI: 10.1074/jbc.RA119.012425 -
Archives of Pharmacal Research Feb 2021The high incidence of obesity has increased the need to discover new therapeutic targets to combat obesity and obesity-related metabolic diseases. Obesity is defined as... (Review)
Review
The high incidence of obesity has increased the need to discover new therapeutic targets to combat obesity and obesity-related metabolic diseases. Obesity is defined as an abnormal accumulation of adipose tissue, which is one of the major metabolic organs that regulate energy homeostasis. However, there are currently no approved anti-obesity therapeutics that directly target adipose tissue metabolism. With recent advances in the understanding of adipose tissue biology, molecular mechanisms involved in brown adipose tissue expansion and metabolic activation have been investigated as potential therapeutic targets to increase energy expenditure. This review focuses on G-protein coupled receptors (GPCRs) as they are the most successful class of druggable targets in human diseases and have an important role in regulating adipose tissue metabolism. We summarize recent findings on the major GPCR classes that regulate thermogenesis and mitochondrial metabolism in adipose tissue. Improved understanding of GPCR signaling pathways that regulate these processes could facilitate the development of novel pharmacological approaches to treat obesity and related metabolic disorders.
Topics: Adipose Tissue; Adipose Tissue, Brown; Animals; Energy Metabolism; Humans; Metabolic Diseases; Obesity; Phosphoserine; Pyrazoles; Pyridines; Receptors, G-Protein-Coupled; Sphingosine 1 Phosphate Receptor Modulators; Thermogenesis
PubMed: 33550564
DOI: 10.1007/s12272-021-01314-w -
Cell Death and Differentiation Oct 2016Caspases are a family of proteases found in all metazoans, including a dozen in humans, that drive the terminal stages of apoptosis as well as other cellular remodeling...
Caspases are a family of proteases found in all metazoans, including a dozen in humans, that drive the terminal stages of apoptosis as well as other cellular remodeling and inflammatory events. Caspases are named because they are cysteine class enzymes shown to cleave after aspartate residues. In the past decade, we and others have developed unbiased proteomic methods that collectively identified ~2000 native proteins cleaved during apoptosis after the signature aspartate residues. Here, we explore non-aspartate cleavage events and identify 100s of substrates cleaved after glutamate in both human and murine apoptotic samples. The extended consensus sequence patterns are virtually identical for the aspartate and glutamate cleavage sites suggesting they are cleaved by the same caspases. Detailed kinetic analyses of the dominant apoptotic executioner caspases-3 and -7 show that synthetic substrates containing DEVD↓ are cleaved only twofold faster than DEVE↓, which is well within the 500-fold range of rates that natural proteins are cut. X-ray crystallography studies confirm that the two acidic substrates bind in virtually the same way to either caspases-3 or -7 with minimal adjustments to accommodate the larger glutamate. Lastly, during apoptosis we found 121 proteins cleaved after serine residues that have been previously annotated to be phosphorylation sites. We found that caspase-3, but not caspase-7, can cleave peptides containing DEVpS↓ at only threefold slower rate than DEVD↓, but does not cleave the unphosphorylated serine peptide. There are only a handful of previously reported examples of proteins cleaved after glutamate and none after phosphorserine. Our studies reveal a much greater promiscuity for cleaving after acidic residues and the name 'cacidase' could aptly reflect this broader specificity.
Topics: Amino Acid Sequence; Animals; Apoptosis; Aspartic Acid; Caspases; Conserved Sequence; Crystallography, X-Ray; Glutamic Acid; HEK293 Cells; Humans; Kinetics; Mice; Peptides; Phosphorylation; Phosphoserine; Proteolysis; Substrate Specificity
PubMed: 27367566
DOI: 10.1038/cdd.2016.62 -
International Journal of Molecular... Dec 2021YB-1 is a multifunctional DNA- and RNA-binding protein involved in cell proliferation, differentiation, and migration. YB-1 is a predominantly cytoplasmic protein that...
YB-1 is a multifunctional DNA- and RNA-binding protein involved in cell proliferation, differentiation, and migration. YB-1 is a predominantly cytoplasmic protein that is transported to the nucleus in certain conditions, including DNA-damaging stress, transcription inhibition, and viral infection. In tumors, YB-1 nuclear localization correlates with high aggressiveness, multidrug resistance, and a poor prognosis. It is known that posttranslational modifications can regulate the nuclear translocation of YB-1. In particular, well-studied phosphorylation at serine 102 (S102) activates YB-1 nuclear import. Here, we report that Akt kinase phosphorylates YB-1 in vitro at serine 209 (S209), which is located in the vicinity of the YB-1 nuclear localization signal. Using phosphomimetic substitutions, we showed that S209 phosphorylation inhibits YB-1 nuclear translocation and prevents p-S102-mediated YB-1 nuclear import.
Topics: Amino Acid Sequence; Animals; Cell Nucleus; HeLa Cells; Humans; Mice; NIH 3T3 Cells; Phosphorylation; Phosphoserine; Protein Binding; Protein Transport; Proto-Oncogene Proteins c-akt; RNA; Serum; Y-Box-Binding Protein 1
PubMed: 35008856
DOI: 10.3390/ijms23010428 -
The Journal of Biological Chemistry Aug 2008Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA...
Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.
Topics: Adenosine Triphosphate; Amino Acyl-tRNA Synthetases; Aminoacylation; Kinetics; Methanosarcina; Models, Molecular; Phosphoserine; Protein Binding; Protein Structure, Tertiary; Time Factors; Transfer RNA Aminoacylation
PubMed: 18559342
DOI: 10.1074/jbc.M801838200 -
Cell Reports May 2016PTPN12 is an important tumor suppressor that plays critical roles in various physiological processes. However, the molecular basis underlying the substrate specificity...
PTPN12 is an important tumor suppressor that plays critical roles in various physiological processes. However, the molecular basis underlying the substrate specificity of PTPN12 remains uncertain. Here, enzymological and crystallographic studies have enabled us to identify two distinct structural features that are crucial determinants of PTPN12 substrate specificity: the pY+1 site binding pocket and specific basic charged residues along its surface loops. Key structurally plastic regions and specific residues in PTPN12 enabled recognition of different HER2 phosphorylation sites and regulated specific PTPN12 functions. In addition, the structure of PTPN12 revealed a CDK2 phosphorylation site in a specific PTPN12 loop. Taken together, our results not only provide the working mechanisms of PTPN12 for desphosphorylation of its substrates but will also help in designing specific inhibitors of PTPN12.
Topics: Amino Acid Sequence; Catalytic Domain; Crystallography, X-Ray; Cyclin-Dependent Kinase 2; Humans; Kinetics; Models, Molecular; Peptides; Phosphorylation; Phosphoserine; Protein Structure, Secondary; Protein Tyrosine Phosphatase, Non-Receptor Type 12; Substrate Specificity
PubMed: 27134172
DOI: 10.1016/j.celrep.2016.04.016 -
Cell Communication and Signaling : CCS Jul 2020Estrogen receptor α (ERα) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ERα conserves...
BACKGROUND
Estrogen receptor α (ERα) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ERα conserves the phosphorylation motif at Ser216 within the DNA binding domain. Previously, Ser216 was found to be phosphorylated in neutrophils infiltrating into the mouse uterus and to enable ERα to regulate migration. Given the implication of this phosphorylation in immune regulation, ERα was examined in mouse microglia to determine if Ser216 is phosphorylated and regulates microglia's inflammation. It was found that Ser216 was constitutively phosphorylated in microglia and demonstrated that in the absence of phosphorylated ERα in ERα KI brains microglia inflamed, confirming that phosphorylation confers ERα with anti-inflammatory capability. ERα KI mice were obese and weakened motor ability.
METHODS
Mixed glia cells were prepared from brains of 2-days-old neonates and cultured to mature and isolate microglia. An antibody against an anti-phospho-S216 peptide of ERα (αP-S216) was used to detect phosphorylated ERα in double immunofluorescence staining with ERα antibodies and a microglia maker Iba-1 antibody. A knock-in (KI) mouse line bearing the phosphorylation-blocked ERα S216A mutation (ERα KI) was generated to examine inflammation-regulating functions of phosphorylated ERα in microglia. RT-PCR, antibody array, ELISA and FACS assays were employed to measure expressions of pro- or anti-inflammatory cytokines at their mRNA and protein levels. Rotarod tests were performed to examine motor connection ability.
RESULTS
Double immune staining of mixed glia cells showed that ERα is phosphorylated at Ser216 in microglia, but not astrocytes. Immunohistochemistry with an anti-Iba-1 antibody showed that microglia cells were swollen and shortened branches in the substantial nigra (SN) of ERα KI brains, indicating the spontaneous activation of microglia as observed with those of lipopolysaccharide (LPS)-treated ERα WT brains. Pro-inflammatory cytokines were up-regulated in the brain of ERα KI brains as well as cultured microglia, whereas anti-inflammatory cytokines were down-regulated. FACS analysis showed that the number of IL-6 producing and apoptotic microglia increased in those prepared from ERα KI brains. Times of ERα KI mice on rod were shortened in Rotarod tests.
CONCLUSIONS
Blocking of Ser216 phosphorylation aggravated microglia activation and inflammation of mouse brain, thus confirming that phosphorylated ERα exerts anti-inflammatory functions. ERα KI mice enable us to further investigate the mechanism by which phosphorylated ERα regulates brain immunity and inflammation and brain diseases. Video abstract.
Topics: Animals; Brain; Cells, Cultured; Estrogen Receptor alpha; Gene Knock-In Techniques; Inflammation; Mice; Microglia; Motor Activity; Phosphorylation; Phosphoserine; Reaction Time
PubMed: 32727504
DOI: 10.1186/s12964-020-00578-x -
The Journal of Biological Chemistry Dec 2022Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure...
Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of N- (and C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic ΔserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) ΔserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain ≥10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses.
Topics: Humans; Phosphoserine; COVID-19; SARS-CoV-2; Magnetic Resonance Spectroscopy; Recombinant Proteins; Serine; Escherichia coli
PubMed: 36265582
DOI: 10.1016/j.jbc.2022.102613