The Experts below are selected from a list of 23830422 Experts worldwide ranked by ideXlab platform
Hiroyasu Nakano - One of the best experts on this subject based on the ideXlab platform.
-
nf κb rela phosphorylation regulates rela acetylation
Molecular and Cellular Biology, 2005Co-Authors: Lin Feng Che, Samuel A Williams, Hiroyasu Nakano, James M Due, Leonard Uckbinde, Warne C GreeneAbstract:The nuclear functions of NF-kappaB p50/RelA heterodimers are regulated in part by posttranslational modifications of its RelA subunit, including phosphorylation and acetylation. Acetylation at lysines 218, 221, and 310 differentially regulates RelA's DNA binding activity, assembly with IkappaBalpha, and transcriptional activity. However, it remains unclear whether the acetylation is regulated or simply due to stimulus-coupled nuclear translocation of NF-kappaB. Using anti-acetylated lysine 310 RelA antibodies, we detected p300-mediated acetylation of RelA in vitro and in vivo after stimulation of cells with tumor necrosis factor alpha (TNF-alpha). Coexpression of catalytically inactive mutants of the catalytic subunit of protein kinase A/mitogen- and stress-activated kinase 1 or IKK1/IKK2, which phosphorylate RelA on serine 276 or serine 536, respectively, sharply inhibited RelA acetylation on lysine 310. Furthermore, phosphorylation of RelA on serine 276 or serine 536 increased assembly of phospho-RelA with p300, which enhanced acetylation on lysine 310. Reconstitution of RelA-deficient murine embryonic fibroblasts with RelA S276A or RelA S536A decreased TNF-alpha-induced acetylation of lysine 310 and expression of the endogenous NF-kappaB-responsive E-selectin gene. These findings indicate that the acetylation of RelA at lysine 310 is importantly regulated by prior phosphorylation of serines 276 and 536. Such phosphorylated and acetylated forms of RelA display enhanced transcriptional activity.
-
transient and selective nf κb p65 serine 536 phosphorylation induced by t cell costimulation is mediated by iκb kinase β and controls the kinetics of p65 nuclear import
Journal of Immunology, 2004Co-Authors: Ivan Mattioli, Andrea Sebald, Cyril Bucher, Rochphilippe Charles, Michael Kracht, Hiroyasu Nakano, Lienhard M SchmitzAbstract:Full transcriptional activity of the nuclear, DNA-bound form of NF-κB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans -activating NF-κB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IκBα and IκB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Cθ, and NF-κB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKβ, but not by IKKα. Phosphorylation occurs within the cytoplasmic and intact NF-κB/IκBα complex and requires prior phosphorylation of IκBα at serines 32 and 36. Reconstitution of p65 −/− cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IκBα localization and the kinetics of p65 nuclear import.
-
transient and selective nf κb p65 serine 536 phosphorylation induced by t cell costimulation is mediated by iκb kinase β and controls the kinetics of p65 nuclear import
Journal of Immunology, 2004Co-Authors: Ivan Mattioli, Andrea Sebald, Cyril Bucher, Rochphilippe Charles, Michael Kracht, Hiroyasu Nakano, Takahiro Doi, Lienhard M SchmitzAbstract:Full transcriptional activity of the nuclear, DNA-bound form of NF-kappaB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans-activating NF-kappaB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IkappaBalpha and IkappaB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Ctheta, and NF-kappaB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKbeta, but not by IKKalpha. Phosphorylation occurs within the cytoplasmic and intact NF-kappaB/IkappaBalpha complex and requires prior phosphorylation of IkappaBalpha at serines 32 and 36. Reconstitution of p65(-/-) cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IkappaBalpha localization and the kinetics of p65 nuclear import.
Herman Wolosker - One of the best experts on this subject based on the ideXlab platform.
-
Purification of serine racemase: ) neuromodulator D-Serine
2016Co-Authors: Herman Wolosker, Masaaki Takaha, Christopher D. Ferris, Solomon H. Snyder, Kevin N Sheth, Jean-pierre MothetAbstract:High levels of D-Serine occur in mammalian brain, where it appears to be an endogenous ligand of the glycine site of N-methyl-D-aspartate receptors. In glial cul- tures of rat cerebral cortex, D-Serine is enriched in type II astrocytes and is released upon stimulation with agonists of non-N-methyl-D-aspartate glutamate receptors. The high lev- els of D-Serine in discrete areas of rat brain imply the existence of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of L-serine to D-Serine. Purified serine racemase has a molecular mass of 37 kDa and requires pyridoxal 5'-phosphate for its activity. The enzyme is highly selective toward L-serine, failing to racemize any other amino acid tested. Properties such as pH optimum, Km values, and the requirement for pyridoxal phos- phate resemble those of bacterial racemases, suggesting that the biosynthetic pathway for D-amino acids is conserved from bacteria to mammalian brain.
-
Nuclear Compartmentalization of Serine Racemase Regulates D-Serine Production
The Journal of biological chemistry, 2015Co-Authors: Goren Kolodney, Elena Dumin, Hazem Safory, Dina Rosenberg, Hisashi Mori, Inna Radzishevisky, Herman WoloskerAbstract:d- Serine is a physiological co-agonist that activates N-methyl d- aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity, and behavior. d- Serine may also trigger NMDAR-mediated neurotoxicity, and its dysregulation may play a role in neurodegeneration. d- Serine is synthesized by the enzyme serine racemase (SR), which directly converts l- serine to d- serine. However, many aspects concerning the regulation of d- serine production under physiological and pathological conditions remain to be elucidated. Here, we investigate possible mechanisms regulating the synthesis of d- serine by SR in paradigms relevant to neurotoxicity. We report that SR undergoes nucleocytoplasmic shuttling and that this process is dysregulated by several insults leading to neuronal death, typically by apoptotic stimuli. Cell death induction promotes nuclear accumulation of SR, in parallel with the nuclear translocation of GAPDH and Siah proteins at an early stage of the cell death process. Mutations in putative SR nuclear export signals (NESs) elicit SR nuclear accumulation and its depletion from the cytosol. Following apoptotic insult, SR associates with nuclear GAPDH along with other nuclear components, and this is accompanied by complete inactivation of the enzyme. As a result, extracellular d- serine concentration is reduced, even though extracellular glutamate concentration increases severalfold. Our observations imply that nuclear translocation of SR provides a fail-safe mechanism to prevent or limit secondary NMDAR-mediated toxicity in nearby synapses.
-
Phosphorylation of mouse serine racemase regulates D-Serine synthesis
FEBS Letters, 2010Co-Authors: Veronika N. Foltyn, Martin Zehl, Elena Dikopoltsev, Ole Nørregaard Jensen, Herman WoloskerAbstract:Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-Serine, which plays a major role in synaptic plasticity and N-methyl d-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in vivo phosphorylation assays. Thr71 phosphorylation was observed in the cytosolic and membrane-bound SR while Thr227 phosphorylation was restricted to the membrane fraction. The Thr71 site has a motif for proline-directed kinases and is the main phosphorylation site of SR. Experiments with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating D-Serine production.
-
a new strategy to decrease n methyl d aspartate nmda receptor coactivation inhibition of d serine synthesis by converting serine racemase into an eliminase
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Rogerio Panizzutti, Joari De Miranda, Catia S Ribeiro, Simone Engelender, Herman WoloskerAbstract:Serine racemase is a brain-enriched enzyme that synthesizes Dserine, an endogenous modulator of the glycine site of N-methylD-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of L-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of L-serine is inhibited. Likewise, L-serine O-sulfate inhibits the synthesis of D-Serine in primary astrocyte cultures. We conclude that the synthetic compound L-serine O-sulfate is a better substrate than L-serine as well as an inhibitor of D-Serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.
-
Purification of serine racemase: Biosynthesis of the neuromodulator D-Serine
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Herman Wolosker, Christopher D. Ferris, Jean-pierre Mothet, Kevin N Sheth, Masaaki Takahashi, Roscoe O. Brady, Solomon H. SnyderAbstract:High levels of D-Serine occur in mammalian brain, where it appears to be an endogenous ligand of the glycine site of N-methyl-d-aspartate receptors. In glial cultures of rat cerebral cortex, D-Serine is enriched in type II astrocytes and is released upon stimulation with agonists of non-N-methyl-d-aspartate glutamate receptors. The high levels of D-Serine in discrete areas of rat brain imply the existence of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of l-serine to D-Serine. Purified serine racemase has a molecular mass of 37 kDa and requires pyridoxal 5′-phosphate for its activity. The enzyme is highly selective toward l-serine, failing to racemize any other amino acid tested. Properties such as pH optimum, Km values, and the requirement for pyridoxal phosphate resemble those of bacterial racemases, suggesting that the biosynthetic pathway for d-amino acids is conserved from bacteria to mammalian brain.
Lienhard M Schmitz - One of the best experts on this subject based on the ideXlab platform.
-
transient and selective nf κb p65 serine 536 phosphorylation induced by t cell costimulation is mediated by iκb kinase β and controls the kinetics of p65 nuclear import
Journal of Immunology, 2004Co-Authors: Ivan Mattioli, Andrea Sebald, Cyril Bucher, Rochphilippe Charles, Michael Kracht, Hiroyasu Nakano, Lienhard M SchmitzAbstract:Full transcriptional activity of the nuclear, DNA-bound form of NF-κB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans -activating NF-κB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IκBα and IκB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Cθ, and NF-κB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKβ, but not by IKKα. Phosphorylation occurs within the cytoplasmic and intact NF-κB/IκBα complex and requires prior phosphorylation of IκBα at serines 32 and 36. Reconstitution of p65 −/− cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IκBα localization and the kinetics of p65 nuclear import.
-
transient and selective nf κb p65 serine 536 phosphorylation induced by t cell costimulation is mediated by iκb kinase β and controls the kinetics of p65 nuclear import
Journal of Immunology, 2004Co-Authors: Ivan Mattioli, Andrea Sebald, Cyril Bucher, Rochphilippe Charles, Michael Kracht, Hiroyasu Nakano, Takahiro Doi, Lienhard M SchmitzAbstract:Full transcriptional activity of the nuclear, DNA-bound form of NF-kappaB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans-activating NF-kappaB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IkappaBalpha and IkappaB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Ctheta, and NF-kappaB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKbeta, but not by IKKalpha. Phosphorylation occurs within the cytoplasmic and intact NF-kappaB/IkappaBalpha complex and requires prior phosphorylation of IkappaBalpha at serines 32 and 36. Reconstitution of p65(-/-) cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IkappaBalpha localization and the kinetics of p65 nuclear import.
Ivan Mattioli - One of the best experts on this subject based on the ideXlab platform.
-
transient and selective nf κb p65 serine 536 phosphorylation induced by t cell costimulation is mediated by iκb kinase β and controls the kinetics of p65 nuclear import
Journal of Immunology, 2004Co-Authors: Ivan Mattioli, Andrea Sebald, Cyril Bucher, Rochphilippe Charles, Michael Kracht, Hiroyasu Nakano, Lienhard M SchmitzAbstract:Full transcriptional activity of the nuclear, DNA-bound form of NF-κB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans -activating NF-κB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IκBα and IκB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Cθ, and NF-κB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKβ, but not by IKKα. Phosphorylation occurs within the cytoplasmic and intact NF-κB/IκBα complex and requires prior phosphorylation of IκBα at serines 32 and 36. Reconstitution of p65 −/− cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IκBα localization and the kinetics of p65 nuclear import.
-
transient and selective nf κb p65 serine 536 phosphorylation induced by t cell costimulation is mediated by iκb kinase β and controls the kinetics of p65 nuclear import
Journal of Immunology, 2004Co-Authors: Ivan Mattioli, Andrea Sebald, Cyril Bucher, Rochphilippe Charles, Michael Kracht, Hiroyasu Nakano, Takahiro Doi, Lienhard M SchmitzAbstract:Full transcriptional activity of the nuclear, DNA-bound form of NF-kappaB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans-activating NF-kappaB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IkappaBalpha and IkappaB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Ctheta, and NF-kappaB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKbeta, but not by IKKalpha. Phosphorylation occurs within the cytoplasmic and intact NF-kappaB/IkappaBalpha complex and requires prior phosphorylation of IkappaBalpha at serines 32 and 36. Reconstitution of p65(-/-) cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IkappaBalpha localization and the kinetics of p65 nuclear import.
Joseph A. Adams - One of the best experts on this subject based on the ideXlab platform.
-
directional phosphorylation and nuclear transport of the splicing factor srsf1 is regulated by an rna recognition motif
Journal of Molecular Biology, 2016Co-Authors: P Serrano, Brandon E. Aubol, Malik M. Keshwani, Stefano Forli, S K Dutta, Michael Geralt, Kurt Wuthrich, Joseph A. AdamsAbstract:Multisite phosphorylation is required for the biological function of serine-arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine-arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine-serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism. Using NMR spectroscopy with two separately expressed domains of SRSF1, we showed that several residues in the RNA-binding motif 2 interact with the N-terminal region of the RS domain (RS1). These contacts provide a structural framework that balances the activities of SRPK1 and the protein phosphatase PP1, thereby regulating the phosphoryl content of the RS domain. Disruption of the implicated intramolecular RNA-binding motif 2-RS domain interaction impairs both the directional phosphorylation mechanism and the nuclear translocation of SRSF1 demonstrating that the intrinsic phosphorylation bias is obligatory for SR protein biological function.
-
Conserved proline-directed phosphorylation regulates SR protein conformation and splicing function.
The Biochemical journal, 2015Co-Authors: Malik M. Keshwani, Brandon E. Aubol, Laurent Fattet, Patricia A. Jennings, Jinsong Qiu, Joseph A. AdamsAbstract:The alternative splicing of human genes is dependent on SR proteins, a family of essential splicing factors whose name derives from a signature C-terminal domain rich in arginine–serine dipeptide repeats (RS domains). Although the SRPKs (SR-specific protein kinases) phosphorylate these repeats, RS domains also contain prolines with flanking serines that are phosphorylated by a second family of protein kinases known as the CLKs (Cdc2-like kinases). The role of specific serine–proline phosphorylation within the RS domain has been difficult to assign since CLKs also phosphorylate arginine–serine dipeptides and, thus, display overlapping residue specificities with the SRPKs. In the present study, we address the effects of discrete serine–proline phosphorylation on the conformation and cellular function of the SR protein SRSF1 (SR protein splicing factor 1). Using chemical tagging and dephosphorylation experiments, we show that modification of serine–proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over 100 human genes based on a global splicing assay. Overall, these studies draw a direct causal relationship between a specific type of chemical modification in an SR protein and the regulation of alternative gene splicing programmes. Abbreviations: CLK1, Cdc2-like kinase 1; ESE, exonic splicing enhancer; PP1, protein phosphatase-1; RRM, RNA recognition motif; RS, domain, domain rich in arginine–serine repeats; SR, protein, splicing factor containing a C-terminal RS domain; SRPK1, SR-specific protein kinase 1; SRSF1, SR protein splicing factor 1 (aka ASF/SF2)
-
Splicing kinase SRPK1 conforms to the landscape of its SR protein substrate.
Biochemistry, 2013Co-Authors: Brandon E. Aubol, Maria L. Mcglone, Michael A. Jamros, Joseph A. AdamsAbstract:The splicing function of SR proteins is regulated by multisite phosphorylation of their C-terminal RS (arginine–serine rich) domains. SRPK1 has been shown to phosphorylate the prototype SR protein SRSF1 using a directional mechanism in which 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site. Although this process is expected to operate on lengthy arginine–serine repeats (≥8), many SR proteins contain smaller repeats of only 1–4 dipeptides, raising the question of how alternate RS domain configurations are phosphorylated. To address this, we studied a splice variant of Tra2β that contains a C-terminal RS domain with short arginine–serine repeats [Tra2β(ΔN)]. We showed that SRPK1 selectively phosphorylates several serines near the C-terminus of the RS domain. SRPK1 uses a distributive mechanism for Tra2β(ΔN) where the rate-limiting step is the dissociation of the protein substrate rather than nucleotide exchange as in the cas...
-
regiospecific phosphorylation control of the sr protein asf sf2 by srpk1
Journal of Molecular Biology, 2009Co-Authors: Jonathan C. Hagopian, Gourisankar Ghosh, Joseph A. AdamsAbstract:SR proteins (splicing factors containing arginine-serine repeats) are essential factors that control the splicing of precursor mRNA by regulating multiple steps in spliceosome development. The prototypical SR protein ASF/SF2 (human alternative splicing factor) contains two N-terminal RNA recognition motifs (RRMs) (RRM1 and RRM2) and a 50-residue C-terminal RS (arginine-serine-rich) domain that can be phosphorylated at numerous serines by the protein kinase SR-specific protein kinase (SRPK) 1. The RS domain [C-terminal domain that is rich in arginine-serine repeats (residues 198-248)] is further divided into N-terminal [RS1: N-terminal portion of the RS domain (residues 198-227)] and C-terminal [RS2: C-terminal portion of the RS domain (residues 228-248)] segments whose modification guides the nuclear localization of ASF/SF2. While previous studies revealed that SRPK1 phosphorylates RS1, regiospecific and temporal-specific control within the largely redundant RS domain is not well understood. To address this issue, we performed engineered footprinting and single-turnover experiments to determine where and how SRPK1 initiates phosphorylation within the RS domain. The data show that local sequence elements in the RS domain control the strong kinetic preference for RS1 phosphorylation. SRPK1 initiates phosphorylation in a small region of serines (initiation box) in the middle of the RS domain at the C-terminal end of RS1 and then proceeds in an N-terminal direction. This initiation process requires both a viable docking groove in the large lobe of SRPK1 and one RRM (RRM2) on the N-terminal flank of the RS domain. Thus, while local RS/SR content steers regional preferences in the RS domain, distal contacts with SRPK1 guide initiation and directional phosphorylation within these regions.
-
adaptable molecular interactions guide phosphorylation of the sr protein asf sf2 by srpk1
Journal of Molecular Biology, 2008Co-Authors: Jonathan C. Hagopian, Patricia A. Jennings, Gourisankar Ghosh, Jacky Chi Ki Ngo, Bryan R Meade, Claudio Ponte De Albuquerque, Joseph A. AdamsAbstract:Abstract The SR (arginine-serine rich) protein ASF/SF2 (also called human alternative splicing factor), an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRMs; RRM1–RRM2) and a C-terminal arginine-serine repeat region (RS domain, a domain rich in arginine-serine repeats). The SR-specific protein kinase (SRPK) 1 phosphorylates the RS domain at multiple serines using a directional (C-terminal-to-N-terminal) and processive mechanism—a process that directs the SR protein to the nucleus and influences protein–protein interactions associated with splicing function. To investigate how SRPK1 accomplishes this feat, the enzyme–substrate complex was analyzed using single-turnover and multiturnover kinetic methods. Deletion studies revealed that while recognition of the RS domain by a docking groove on SRPK1 is sufficient to initiate the processive and directional mechanism, continued processive phosphorylation in the presence of building repulsive charge relies on the fine-tuning of contacts with the RRM1–RRM2 module. An electropositive pocket in SRPK1 that stabilizes newly phosphorylated serines enhanced processive phosphorylation of later serines. These data indicate that SRPK1 uses stable, yet highly flexible protein–protein interactions to facilitate both early and late phases of the processive phosphorylation of SR proteins.
