Functional coexpression of serine protein kinase SRPK1 and its substrate ASF/SF2 in Escherichia coli

Mammalian proteins expressed in Escherichia coli are used in a variety of applications. A major drawback in producing eukaryotic proteins in E.coli is that the bacteria lack most eukaryotic post-translational modification systems, including serine/threonine protein kinase(s). Here we show that a eukaryotic protein can be phosphorylated in E.coli by simultaneous expression of a mammalian protein kinase and its substrate. We show that in bacteria expressing SRPK1, ASF/SF2 becomes phosphorylated to a degree resembling native ASF/SF2 present in interphase HeLa cell nuclei. The E.coli phosphorylated ASF/SF2 is functional in splicing and, contrary to the unphosphorylated protein, soluble under native conditions.     

A peptide substrate for Escherichia coli protein kinase activity in vitro

The phosphorylation in vitro of a series of exogenous peptides by E. coli protein kinases was studied. One of the substrates assayed, the hexapeptide Arg-Gly-Tyr-Ser-Leu-Gly, was found to be significantly phosphorylated at its serine residue. This finding provides the first example of an exogenous substrate utilizable by bacterial protein kinases. The kinetic parameters of the corresponding reaction were determined and the effect of various cations were analyzed. Magnesium, cobalt, manganese and zinc ions were all found to be activators, although to a varying extent. The results were discussed in terms of substrate specificity of bacterial protein kinases.     

Adaptable molecular interactions guide phosphorylation of the SR protein ASF/SF2 by SRPK1

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.     

Ordered multi-site phosphorylation of the splicing factor ASF/SF2 by SRPK1

The human alternative splicing factor ASF/SF2, an SR (serine-arginine-rich) protein involved in mRNA splicing control, is activated by the multisite phosphorylation of its C-terminal RS domain, a segment containing numerous arginine-serine dipeptide repeats. The protein kinase responsible for this modification, SR-specific protein kinase 1 (SRPK1), catalyzes the selective phosphorylation of approximately a dozen serines in only the N-terminal portion of the RS domain (RS1). To gain insights into the nature of selective phosphate incorporation in ASF/SF2, region-specific phosphorylation in the RS domain was monitored as a function of reaction progress. Arg-to-Lys mutations were made at several positions to produce unique protease cleavage sites that separate the RS domain into identifiable N- and C-terminal phosphopeptides upon treatment with lysyl endoproteinase. These studies reveal that SRPK1 docks near the C-terminus of the RS1 segment and then moves in an N-terminal direction along the RS domain. Multiple quadruple Ser-to-Ala and deletion mutations did not disrupt the phosphorylation of other sites regardless of position, suggesting that the active site of SRPK1 docks in a flexible manner at the center of the RS domain. Taken together, these data suggest that SRPK1 uses a unique ‘grab-and-pull’ mechanism to control the regiospecific phosphorylation of its protein substrate.     

A protein kinase C-like activity in Escherichia coli

The protein kinase C (PKC) family comprises calcium- and phospholipid-dependent kinases whose activity is stimulated by diacylglycerol and tumour-promoting phorbol esters such as 12-tetradecanoyl phorbol-13-acetate (TPA). In the Gram-negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid-dependent, TPA-stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross-reactivity with several monoclonal antibodies. This revealed isozyme-specific homology between a protein(s) of relative molecular mass 80-85,000 in E. coli and the alpha- and gamma-isozymes, but probably not the beta-isozyme, of eukaryotic PKC.     

Minimal methylated substrate and extended substrate range of Escherichia coli AlkB protein,a 1-methyladenine-DNA dioxygenase

The Escherichia coli AlkB protein, and two human homologs ABH2 and ABH3, directly demethylate 1-methyladenine and 3-methylcytosine in DNA. They couple Fe(II)-dependent oxidative demethylation of these damaged bases to decarboxylation of alpha-ketoglutarate. Here, we have determined the kinetic parameters for AlkB oxidation of 1-methyladenine in poly(dA), short oligodeoxyribonucleotides, nucleotides, and nucleoside triphosphates. Methylated poly(dA) was the preferred AlkB substrate of those tested. The oligonucleotide trimer d(Tp1meApT) and even 5'-phosphorylated 1-me-dAMP were relatively efficiently demethylated, and competed with methylated poly(dA) for AlkB activity. A polynucleotide structure was clearly not essential for AlkB to repair 1-methyladenine effectively, but a nucleotide 5' phosphate group was required. Consequently, 1-me-dAMP(5') was identified as the minimal effective AlkB substrate. The nucleoside triphosphate, 1-me-dATP, was inefficiently but actively demethylated by AlkB; a reaction with 1-me-ATP was even slower. E. coli DNA polymerase I Klenow fragment could employ 1-me-dATP as a precursor for DNA synthesis in vitro, suggesting that demethylation of alkylated deoxynucleoside triphosphates by AlkB could have biological significance. Although the human enzymes, ABH2 and ABH3, demethylated 1-methyladenine residues in poly(dA), they were inefficient with shorter substrates. Thus, ABH3 had very low activity on the trimer, d(Tp1meApT), whereas no activity was detected with ABH2. AlkB is known to repair methyl and ethyl adducts in DNA; to extend this substrate range, AlkB was shown to reduce the toxic effects of DNA damaging agents that generate hydroxyethyl, propyl, and hydroxypropyl adducts.     

Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.     

Enhanced expression of cysteine‐rich antimicrobial peptide snakin‐1 in Escherichia coli using an aggregation‐prone protein coexpression system

Snakin‐1 (SN‐1) is a cysteine‐rich plant antimicrobial peptide and the first purified member of the snakin family. SN‐1 shows potent activity against a wide range of microorganisms, and thus has great biotechnological potential as an antimicrobial agent. Here, we produced recombinant SN‐1 in Escherichia coli by a previously developed coexpression method using an aggregation‐prone partner protein. Our goal was to increase the productivity of SN‐1 via the enhanced formation of insoluble inclusion bodies in E. coli cells. The yield of SN‐1 by the coexpression method was better than that by direct expression in E. coli cells. After refolding and purification, we obtained several milligrams of functionally active SN‐1, the identity of which was verified by MALDI‐TOF MS and NMR studies. The purified recombinant SN‐1 showed effective antimicrobial activity against test organisms. Our studies indicate that the coexpression method using an aggregation‐prone partner protein can serve as a suitable expression system for the efficient production of functionally active SN‐1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1520–1528, 2017     

The E1circumflexE4 protein of human papillomavirus interacts with the serine-arginine-specific protein kinase SRPK1

Human papillomavirus (HPV) infections of the squamous epithelium are associated with high-level expression of the E1circumflexE4 protein during the productive phase of infection. However, the precise mechanisms of how E1circumflexE4 contributes to the replication cycle of the virus are poorly understood. Here, we show that the serine-arginine (SR)-specific protein kinase SRPK1 is a novel binding partner of HPV type 1 (HPV1) E1circumflexE4. We map critical residues within an arginine-rich domain of HPV1 E1circumflexE4, and in a region known to facilitate E1circumflexE4 oligomerization, that are requisite for SRPK1 binding. In vitro kinase assays show that SRPK1 binding is associated with phosphorylation of an HPV1 E1circumflexE4 polypeptide and modulates autophosphorylation of the kinase. We show that SRPK1 is sequestered into E4 inclusion bodies in terminally differentiated cells within HPV1 warts and that colocalization between E1circumflexE4 and SRPK1 is not dependent on additional HPV1 factors. Moreover, we also identify SRPK1 binding of E1circumflexE4 proteins of HPV16 and HPV18. Our findings indicate that SRPK1 binding is a conserved function of E1circumflexE4 proteins of diverse virus types. SRPK1 influences important biochemical processes within the cell, including nuclear organization and RNA metabolism. While phosphorylation of HPV1 E4 by SRPK1 may directly influence HPV1 E4 function during the infectious cycle, the modulation and sequestration of SRPK1 by E1circumflexE4 may affect the ability of SRPK1 to phosphorylate its cellular targets, thereby facilitating the productive phase of the HPV replication cycle.     

Regulating SR protein phosphorylation through regions outside the kinase domain of SRPK1

SR proteins (splicing factors containing arginine-serine repeats) are essential splicing factors whose phosphorylation by the SR-specific protein kinase (SRPK) family regulates nuclear localization and mRNA processing activity. In addition to an N-terminal extension with unknown function, SRPKs contain a large, nonhomologous spacer insert domain (SID) that bifurcates the kinase domain and anchors the kinase in the cytoplasm through interactions with chaperones. While structures for the kinase domain are now available, constructs that include regions outside this domain have been resistant to crystallographic elucidation. To investigate the conformation of the full-length kinase and the functional role of noncatalytic regions, we performed hydrogen-deuterium exchange and steady-state kinetic experiments on SRPK1. Unlike the kinase core, the large SID lacks stable, hydrogen-bonded structure and may provide an intrinsically disordered region for chaperone interactions. Conversely, the N-terminus, which positively regulates SR protein binding, adopts a stable structure when the insert domain is present and stabilizes a docking groove in the large lobe of the kinase domain. The N-terminus and SID equally enhance SR protein turnover by altering the stability of several catalytic loop segments. These studies reveal that SRPK1 uses an N-terminal extension and a large, intrinsically disordered region juxtaposed to a stable structure to facilitate high-affinity SR protein interactions and phosphorylation rates.     

Target of serine inhibition in Escherichia coli

L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I.     

Protein phosphorylation in Escherichia coli and purification of a protein kinase

More than 40 protein species including RNA polymerase were found to be phosphorylated in Escherichia coli on analyses of 32P-labeled cell lysates by single and two-dimensional gel electrophoresis and autoradiography. The protein species and the level of phosphorylation varied depending on the cell growth phase. With [gamma-32P]ATP as a substrate, cell lysates phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. Both serine and threonine were the major phosphate acceptors in whole cell lysates. Starting from a partially purified RNA polymerase preparation with the protein phosphorylation activity and using an E. coli protein with an apparent Mr = 90K (K represents X 1000) as the substrate, we purified a protein kinase with a native Mr approximately 120K to apparent homogeneity. The protein kinase is either a heterodimer of 61K and 66K polypeptides or a homodimer of one of these polypeptides. We also isolated a 100K protein with self-phosphorylation activity.     

Expression of a lipid transfer protein in Escherichia coli and its phosphorylation by a membrane-bound calcium-dependent protein kinase

Ha-AP10 is a basic antifungal peptide from sunflower seeds (Helianthus annuus antifungal peptide of 10 kDa) belonging to the family of plant lipid transfer proteins. We report here its expression in E. coli [Glutathione S-transferase (GST) system] and its phosphorylation by endogenous membrane-bound calcium-dependent protein kinases.     

Localization of serine kinases, SRPK1 (SFRSK1) and SRPK2 (SFRSK2), specific for the SR family of splicing factors in mouse and human chromosomes

The serine- and arginine-rich (SR) splicing factors play an important role in both constitutive and alternative pre-mRNA splicing, and the functions of these splicing factors are regulated by phosphorylation. We have previously characterized SRPK1 (SFRSK1) and SRPK2 (SFRSK2), which are highly specific protein kinases for the SR family of splicing factors. Here we report the chromosomal localization of the mouse and human genes for both kinases. SRPK1 probes detected two loci that were mapped to mouse Chromosomes 17 and X using The Jackson Laboratory interspecific backcross DNA panel, and SRPK2 probes identified a single locus on mouse Chromosome 5. Using a somatic cell hybrid mapping panel and by fluorescence in situ hybridization, SRPK1 and SRPK2 were respectively mapped to human chromosomes 6p21.2-p21.3 (a region of conserved synteny to mouse Chromosome 17) and 7q22-q31.1 (a region of conserved synteny to mouse Chromosome 5). In addition, we also found multiple SRPK-related sequences on other human chromosomes, one of which appears to correspond to a SRPK2 pseudogene on human chromosome 8.     

Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies

Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) was investigated by using both kinetics and binding methods. Initial-velocity studies in both reaction directions show a sequential kinetic mechanism with apparent substrate activation by ATP and substrate inhibition by ADP. In addition, the Michaelis constants differ greatly from the substrate dissociation constants. Results of product inhibition studies and dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme has a random kinetic mechanism, which is consistent with the observed formation of binary complexes with all the substrates and the glycerol-independent MgATPase activity of the enzyme. Dissociation constants for substrate binding determined by using ligand protection from inactivation by N-ethylmaleimide agree with those estimated from the initial-velocity studies. Determinations of substrate binding stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP, ADP, and glycerol. Thus, the regulation by nucleotides does not appear to reflect binding at a separate regulatory site. The random kinetic mechanism obviates the need to postulate such a site to explain the formation of binary complexes with the nucleotides. The observed stoichiometry is consistent with a model for the nucleotide regulatory behavior in which the dimer is the enzyme form present in the assay and its subunits display different substrate binding affinities. Several properties of the enzyme are consistent with negative cooperativity as the basis for the difference in affinities. The possible physiological importance of the regulatory behavior with respect to ATP is considered.     

Two amino acid replacements change the substrate preference of DNA mismatch glycosylase Mig.MthI from T/G to A/G

Mig.MthI from Methanobacterium thermoautotrophicum and MutY of Escherichia coli are both DNA mismatch glycosylases of the ‘helix-hairpin-helix’ (HhH) superfamily of DNA repair glycosylases; the former excises thymine from T/G, the latter adenine from A/G mismatches. The structure of MutY, in complex with its low molecular weight product, adenine, has previously been determined by X-ray crystallography. Surprisingly, the set of amino acid residues of MutY that are crucial for adenine recognition is largely conserved in Mig.MthI. Here we show that replacing two amino acid residues in the (modeled) thymine binding site of Mig.MthI (Leu187 to Gln and Ala50 to Val) changes substrate discrimination between T/G and A/G by a factor of 117 in favor of the latter (from 56-fold slower to 2.1-fold faster). The Ala to Val exchange also affects T/G versus U/G selectivity. The data allow a plausible model of thymine binding and of catalytic mechanism of Mig.MthI to be constructed, the key feature of which is a bidentate hydrogen bridge of a protonated glutamate end group (number 42) with thymine centers NH-3 and O-4, with proton transfer to the exocyclic oxygen atom neutralizing the negative charge that builds up in the pyrimidine ring system as the glycosidic bond is broken in a heterolytic fashion. The results also offer an explanation for why so many different substrate specificities are realized within the HhH superfamily of DNA repair glycosylases, and they widen the scope of these enzymes as practical tools.     

Mass spectrometric and kinetic analysis of ASF/SF2 phosphorylation by SRPK1 and Clk/Sty

Assembly of the spliceosome requires the participation of SR proteins, a family of splicing factors rich in arginine-serine dipeptide repeats. The repeat regions (RS domains) are polyphosphorylated by the SRPK and Clk/Sty families of kinases. The two families of kinases have distinct enzymatic properties, raising the question of how they may work to regulate the function of SR proteins in RNA metabolism in mammalian cells. Here we report the first mass spectral analysis of the RS domain of ASF/SF2, a prototypical SR protein. We found that SRPK1 was responsible for efficient phosphorylation of a short stretch of amino acids in the N-terminal portion of the RS domain of ASF/SF2 while Clk/Sty was able to transfer phosphate to all available serine residues in the RS domain, indicating that SR proteins may be phosphorylated by different kinases in a stepwise manner. Both kinases bind with high affinity and use fully processive catalytic mechanisms to achieve either restrictive or complete RS domain phosphorylation. These findings have important implications on the regulation of SR proteins in vivo by the SRPK and Clk/Sty families of kinases.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.