Cluster Of Serine Residues Research Articles

Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons.

A synaptic pool of extracellular signal-regulated kinases (ERK) controls synaptic transmission, although little is known about its underlying signaling mechanisms. Here, we found that synaptic ERK2 directly binds to postsynaptic metabotropic glutamate receptor 1a (mGluR1a). This binding is direct and the ERK-binding site is located in the intracellular C-terminus (CT) of mGluR1a. Parallel with this binding, ERK2 phosphorylates mGluR1a at a cluster of serine residues in the distal part of mGluR1a-CT. In rat cerebellar neurons, ERK2 interacts with mGluR1a at synaptic sites, and active ERK constitutively phosphorylates mGluR1a under normal conditions. This basal phosphorylation is critical for maintaining adequate surface expression of mGluR1a. ERK is also essential for controlling mGluR1a signaling in triggering distinct postreceptor signaling transduction pathways. In summary, we have demonstrated that mGluR1a is a sufficient substrate of ERK2. ERK that interacts with and phosphorylates mGluR1a is involved in the regulation of the trafficking and signaling of mGluR1.

Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner

The activation of Smoothened (Smo) requires phosphorylation at three clusters of Serine residues in Drosophila Hedgehog (Hh) signaling. However, the mechanism by which phosphorylation promotes Smo conformational change and subsequently activates Smo in response to Hh gradient remains unclear. Here, we show that the conformational states of Smo are determined by not only the amount but also the position of the negative charges provided by phosphorylation. By using a Smo phospho-specific antibody, we demonstrate that Smo is differentially phosphorylated at three clusters of serine residues in response to levels of Hh activity. Mutating the first cluster, compared to mutating the other clusters, impairs Smo activity more severely, whereas mutating the last cluster prohibits C-terminus dimerization. In addition, phosphorylation of the membrane proximal cluster promotes phosphorylation of the distal cluster. We propose a zipper-lock model in which the gradual phosphorylation at these clusters induces a gradual conformational change in the Smo cytoplasmic tail, which promotes the interaction between Smo and Costal2 (Cos2). Moreover, we show that Hh regulates both PKA and CK1 phosphorylation of Smo. Thus, the differential phosphorylation of Smo mediates the thresholds of Hh activity.

Purification and characterization of the ternary complex between phosducin‐like protein, the G protein β subunit and the cytosolic chaperonin ‐ an important intermediate in the assembly of the G protein βγ dimer

Phosducin‐like protein 1 (PhLP1) has been recently shown to be an essential co‐chaperone, along with the cytosolic chaperonin containing the tailless‐complex polypeptide 1 (CCT), in the folding and assembly of the nascent G protein βγ subunit dimer (Gβγ). It has been proposed that a ternary PhLP1‐Gβ‐CCT complex forms during the folding process with nascent Gβ in the CCT folding cavity and PhLP above the cavity contacting Gβ and the apical domains of CCT. If PhLP1 is phosphorylated in a cluster of serine residues near its N‐terminus (S18–20) by the protein kinase CK2, it will release Gβ from CCT in a PhLP1‐Gβ complex that is then able to interact with Gγ and form the Gβγ dimer. To further test the proposed mechanism of Gβγ assembly, we have isolated the PhLP1‐Gβ‐CCT ternary complex in both states of PhLP1 phosphorylation from insect cells over‐expressing both PhLP and Gβ using a tandem affinity purification method. In addition, we have isolated the Gβ‐CCT complex in the absence of PhLP1. These complexes have been subjected to structural determination by cryo‐electron microscopy in an effort to determine the mechanism by which PhLP1 phosphorylation results in the release of PhLP1‐Gβ from CCT.

Protein Kinase CK1δ Phosphorylates Key Sites in the Acidic Domain of Murine Double-Minute Clone 2 Protein (MDM2) That Regulate p53 Turnover

Murine double-minute clone 2 protein (MDM2) is an E3 ubiquitin ligase that regulates the turnover of several cellular factors including the p53 tumor suppressor protein. As part of the mechanism of p53 induction in response to DNA damage, a cluster of serine residues within the central acidic domain of MDM2 become hypophosphorylated, leading to attenuation of MDM2-mediated p53 destruction. In the present study, we identify the protein kinase CK1delta as a major cellular activity that phosphorylates MDM2. Amino acid substitution, coupled with phosphopeptide analyses, indicates that several serine residues in the acidic domain, including Ser-240, Ser-242, and Ser-246, as well as Ser-383 in the C-terminal region, are phosphorylated by CK1delta in vitro. We also show, through expression of a dominant negative mutant of CK1delta or treatment of cells with IC261, a CK1delta-selective inhibitor, that MDM2 is phosphorylated by CK1delta in cultured cells. These data establish the identity of a key signaling molecule that promotes the phosphorylation of a major regulatory region in MDM2 under normal growth conditions.

Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro.

SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus. A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CL(pro) (also termed SARS-CoV 3CL(pro)). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CL(pro) as well as a truncated form containing only the catalytic domains. The recombinant proteins have been characterized enzymatically using a fluorescently labeled substrate; their structural stability in solution has been determined by differential scanning calorimetry, and novel inhibitors have been discovered. Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the alpha-helical domain. Differential scanning calorimetry indicates that the alpha-helical domain does not contribute to the structural stability of the catalytic domains. Analysis of the active site cavity reveals the presence of subsites that can be targeted with specific chemical functionalities. In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compounds containing boronic acid. This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development. It was found that bifunctional aryl boronic acid compounds were particularly effective at inhibiting the protease, with inhibition constants as strong as 40 nM. Isothermal titration microcalorimetric experiments indicate that these inhibitors bind reversibly to 3CL(pro) in an enthalpically favorable fashion, implying that they establish strong interactions with the protease molecule, thus defining attractive molecular scaffolds for further optimization.

The major phosphorylation sites of the respiratory syncytial virus phosphoprotein are dispensable for virus replication in vitro.

The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of (33)P(i)-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.

Molecular cloning and characterization of ryanodine receptor from unfertilized sea urchin eggs.

Unfertilized eggs of sea urchins (Hemicentrotus pulcherrimus) demonstrated cyclic ADP-ribose (cADPR)-induced Ca(2+) release and caffeine-induced Ca(2+) release, both of which were considered to be mediated through the ryanodine receptor (RyR). We cloned cDNAs for sea urchin egg RyR (suRyR), which encode a 597-kDa protein of 5,317 amino acids. suRyR shares common structural features with known RyRs: the well-conserved COOH-terminal domain, which forms a functional Ca(2+) channel, and a large hydrophilic NH2-terminal domain. suRyR shows amino acid sequence identity (43-45%) similar to the three mammalian RyR isoforms. Phylogenetic analysis indicates that suRyR branched from three isoforms of vertebrates before they diverged, suggesting that suRyR may be the only RyR isoform in the sea urchin. Four in-frame insertions were found in suRyR cDNAs, one of which was novel and unique, in that it had a cluster of serine residues. The transcripts with and without these insertions were found in the egg RNA. These results suggest that suRyR may be expressed as a functional Ca(2+)-induced Ca(2+) release channel, which might also be involved in cADPR-induced Ca(2+) release.

Contribution of serine residues to constitutive and agonist-induced signaling via the D2S dopamine receptor: evidence for multiple, agonist-specific active conformations.

Dopamine D2 receptors contain a cluster of serine residues in the fifth transmembrane domain that contribute to activation of the receptor as well as to the binding of agonists. We used rat D2S dopamine receptor mutants, each containing a serine-to-alanine substitution (S193A, S194A, S197A), to investigate the mechanism through which these residues affect activation of the receptor. Activation of the mutant receptor S194A was abolished in an agonist-dependent manner, such that dopamine no longer inhibited cAMP accumulation in C6 glioma cells or activated G protein-regulated K+ channels in Xenopus laevis oocytes, whereas the efficacy of several other agonists was unaffected. Dihydrexidine did not inhibit cAMP accumulation at either S193A or S194A. The decreased efficacy of dihydrexidine at S193A and S194A and dopamine at S194A was associated with a decreased ability to detect a GTP-sensitive high affinity binding state for these agonists. The ability of dopamine to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding via S194A also was decreased by approximately 50%. Finally, constitutive stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate binding and inhibition of adenylate cyclase by the D2S receptor was reduced by mutation of either S193 or S194. These data support the existence of multiple active receptor conformations that are differentially sensitive to mutation of serine residues in the fifth-transmembrane domain.

Ordered multisite phosphorylation of Xenopus ribosomal protein S6 by S6 kinase II.

Phosphorylated ribosomal proteins were isolated from Xenopus 40 S ribosomal subunits by reversed-phase high performance liquid chromatography (HPLC) to enable direct analysis of the phosphorylation sites in ribosomal protein S6. Xenopus S6 closely resembled mammalian S6 with respect to the following properties: (i) reversed-phase HPLC elution behavior, (ii) amino-terminal sequence (96% identity in the first 37 residues), and (iii) an identical sequence within the region of its phosphorylation sites. Whereas S6 was the only ribosomal protein phosphorylated in vitro by Xenopus S6 kinase II, ribosomes phosphorylated in vivo were found to be associated with an additional phosphoprotein having an amino-terminal sequence identical to that of the ubiquitin carboxyl-terminal extension protein CEP 80. S6 kinase II phosphorylated at least four sites (serines 1-3 and 5) in the sequence Arg-Arg-Leu-Ser(1)-Ser(2)-Leu-Arg-Ala-Ser(3)-Thr-Ser(4)-Lys-Ser(5)-, which correspond to the residues known to be phosphorylated in the carboxyl-terminal region of mammalian S6. The in vivo S6 phosphorylation sites in maturing Xenopus oocytes were shown to be located within the same cluster of serine residues, although individual sites were not identified. Kinetic analysis of S6 kinase II-catalyzed phosphorylation events indicated a simple sequential mechanism of multisite phosphorylation initiating at either serine 2 (preferred) or serine 1, with the rates of phosphorylation of individual sites occurring in the order serine 2 greater than serine 1 greater than serine 3 greater than serine 5.

Phosphorylation of maize RAB-17 protein by casein kinase 2.

The maize gene RAB-17, which is responsive to abscisic acid, encodes a basic glycine-rich protein containing, in the middle part of its sequence, a cluster of 8 serine residues followed by a putative casein kinase 2-type substrate consensus sequence. This protein was found to be highly phosphorylated in vivo. Here, we show that RAB-17 protein is a real substrate for casein kinase 2. RAB-17 protein is phosphorylated in vitro by casein kinase 2 isolated from rat liver cytosol and from maize embryos. A maximum of 4 mol of phosphate were incorporated per mol of RAB-17 protein following incubation with casein kinase 2. Phosphopeptide mapping experiments show that the peptide phosphorylated by casein kinase 2 in vitro is identical to that derived from the protein phosphorylated in vivo. Purification by high performance liquid chromatography and partial sequencing of the phosphopeptide indicate that it corresponds to the region of the protein (residues 56-89) containing the cluster of serine residues. Our results indicate that RAB-17 is phosphorylated by casein kinase 2 or a kinase with a similar specificity and that phosphorylation takes place in the serine cluster region of the protein both in vitro and in vivo.

Structural analysis of cDNA for rat peroxisomal 3-ketoacyl-CoA thiolase.

cDNA clones of rat peroxisomal 3-ketoacyl-CoA thiolase were isolated. By blotting analysis using the cDNAs as probes, the mRNA for this enzyme was estimated to be about 1.9-kilobase pairs. Elevation of mRNA levels in the liver with administration of di(2-ethylhexyl)phthalate was also evident. Sequencing analysis revealed 1,272 bases of the open reading frame which encoded 424 amino acid residues. Amino acid sequence data on six tryptic peptides and the amino terminus of the purified enzyme confirmed the cDNA sequence. The precursor of peroxisomal thiolase contains at its amino terminus a peptide extension of 26 residues. The mature enzyme is composed of 398 amino acids and the molecular weight is 41,074. The presequence has a net positive charge, lacks a long stretch of hydrophobic residues, and contains a cluster of serine residues. When the primary structure of the precursor was compared to structure of known peroxisomal proteins, there was no common homologous sequence. Peroxisomal thiolase exhibits a significant sequence homology with the mitochondrial thiolase. Possible location of the transport signal of the peroxisomal thiolase is discussed. Acyl-CoA binding sites were also located on primary structures of the two thiolases. The occurrence of interrupting sequences in several clones likely originates from intron sequences.