Phosphorylation mechanism and structure of serine-arginine protein kinases

The splicing of mRNA requires a group of essential factors known as SR proteins, which participate in the maturation of the spliceosome. These proteins contain one or two RNA recognition motifs and a C-terminal domain rich in Arg-Ser repeats (RS domain). SR proteins are phosphorylated at numerous serines in the RS domain by the SR-specific protein kinase (SRPK) family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus, and may also play important roles in alternative splicing, mRNA export, and other processing events. Although SR proteins are polyphosphorylated in vivo, the mechanism underlying this complex reaction has only been recently elucidated. Human alternative splicing factor [serine/arginine-rich splicing factor 1 (SRSF1)], a prototype for the SR protein family, is regiospecifically phosphorylated by SRPK1, a post-translational modification that controls cytoplasmic-nuclear localization. SRPK1 binds SRSF1 with unusually high affinity, and rapidly modifies about 10-12 serines in the N-terminal region of the RS domain (RS1), using a mechanism that incorporates sequential, C-terminal to N-terminal phosphorylation and several processive steps. SRPK1 employs a highly dynamic feeding mechanism for RS domain phosphorylation in which the N-terminal portion of RS1 is initially bound to a docking groove in the large lobe of the kinase domain. Upon subsequent rounds of phosphorylation, this N-terminal segment translocates into the active site, and a β-strand in RNA recognition motif 2 unfolds and occupies the docking groove. These studies indicate that efficient regiospecific phosphorylation of SRSF1 is the result of a contoured binding cavity in SRPK1, a lengthy Arg-Ser repetitive segment in the RS domain, and a highly directional processing mechanism.     

Slow posttranslational modification of a neurofilament protein

The synthesis and subsequent modification of neurofilament (NF) polypeptides has been examined in pulse-chase experiments, using cultured chick spinal cord neurons. Fluorography of the [35S]methionine-labeled cytoskeletal proteins, after separation by two-dimensional gel electrophoresis, revealed that (a) the mid-size chicken NF protein, NF-M160, is synthesized as a smaller and more basic precursor, NF-M130; (b) beginning approximately 8 h after translation, NF-M130 slowly and continuously becomes larger and more acidic, attaining the size and charge of NF-M160 16 or more h later, and undergoing no further change in mobility for many days thereafter; and (c) in contrast, the low molecular weight NF protein, NF-L, is synthesized as such, and undergoes no subsequent change in apparent size or charge. Additional experiments provided evidence that the conversion of NF-M130 to NF-M160 is due, at least in part, to phosphorylation: (a) Incubation of similar cultures in 32PO4 resulted in incorporation into NF-M160 and transitional forms, but not into NF-M130. (b) An antiserum to NF-M160 was found by immunoblot analysis to bind strongly to untreated NF-M160, but poorly to phosphatase-treated NF-M160, and not at all to NF-M130. It has already been demonstrated (Bennett, G. S., S. J. Tapscott, C. DiLullo, and H. Holtzer, 1984, Brain Res., 304:291-302) that this anti-NF-M160 fails to stain the soma of motor neurons in sections of chick spinal cord, but detects an increasing gradient of immunoreactivity in the proximal axons. These results, together with the known kinetics of axoplasmic transport of NF, suggest that the mid-size chicken NF protein is synthesized as NF-M130 and is extensively modified, at least in part by phosphorylation, to become NF-M160 during transport along proximal neurites. Once maximally modified, NF-M160 undergoes no further net change during transport along distal neurites.     

Towards posttranslational modification proteome of royal jelly

Royal jelly (RJ) is a secretory protein from the hypopharyngeal glands of nurse honeybee workers, which contains a variety of proteins of which major royal jelly proteins (MRJPs) are some of the most important. It plays important roles both for honeybee and human. Each family of MRJP 1-5 displays a string of modified protein spots in the RJ proteome profile, which may be caused by posttranslational modifications (PTMs) of MRJPs. However, information on the RJ PTMs is still limited. Therefore, the PTM status of RJ was identified by using complementary proteome strategies of two-dimensional gel electrophoresis (2-DE), shotgun analysis in combination with high performance liquid chromatography-chip/electrospray ionization quadrupole time-of-flight/tandem mass spectrometry and bioinformatics. Phosphorylation was characterized in MRJP 1, MRJP 2 and apolipophorin-III-like protein for the first time and a new site was localized in venom protein 2 precursor. Methylation and deamidation were also identified in most of the MRJPs. The results indicate that methylation is the most important PTM of MRJPs that triggers the polymorphism of MRJP 1-5 in the RJ proteome. Our data provide a comprehensive catalog of several important PTMs in RJ and add valuable information towards assessing both the biological roles of these PTMs and deciphering the mechanisms underlying the beneficial effects of RJ for human health.     

Generation of protein-derived redox cofactors by posttranslational modification

Redox enzymes which catalyze the oxidation and reduction of substrates are ubiquitous in nature. These enzymes typically possess exogenous cofactors to allow them to perform catalytic functions which cannot be accomplished using only amino acid residues. It is now evident that nature also employs an alternative strategy of generating catalytic and redox-active sites in proteins by posttranslational modification of amino acid residues. This review describes the structures and functions of several of these protein-derived cofactors and the diverse mechanisms of posttranslational modification through which they are generated.     

High-throughput profiling of posttranslational modification enzymes by phage display

Phage display has been used as a high-throughput platform for identifying proteins or peptides with desired binding or catalytic activities from a complex proteome. Recently, phage display has been applied to profile the catalytic activities of posttranslational modification (PTM) enzymes. Here, we highlight recent work elucidating the downstream targets of PTM enzymes by phage display, including the genome-wide profiling of biosynthetic enzymes subject to phosphopantetheinyl transferase (PPTase) modification.     

The posttranslational modification of thyrotropin receptor and thyroid diseases

The thyrotropin receptor (TSHR), lutropin receptor, and follitropin receptor are related members of the superfamily of leucine-rich repeats containing adenylate cyclase stimulating receptors. The unique posttranslational modification of the TSHR leads to the transformation of its monomeric form to the subunit structure where the subunits A and B are connected by disulphide bonds. This natural processing occurs with the release from the receptor of a short peptide C, and is followed by the release of the subunit A. Both monomeric and dimeric forms of the receptor are stimulated by TSH, so no clear functional significance of TSHR modifications have been found. We can speculated that the processing of TSHR with the release of its large fragments contributes to the development of autoimmune diseases and production anti-TSH receptor autoantibodies. The extrathyroidal manifestations of Graves disease may also be related to metastasis of the autoimmune reaction to extrathyroidal sites via the released A subunit. The TSHR processing may, to some extent, be connected to the hyperthyroidism since the release of the subunit A from the receptor augmented the adenylate cyclase activity in the absence of TSH. According to the recent model of receptors action the TSHR is in equilibrium between the inactive (closed) and active (opened) conformations. In opened conformation it can associate with Gs protein and trigger the intracellular signal. TSH and stimulating autoantibodies preferentially bind to opened receptors and stabilizes them.     

Probing the transglutaminase-mediated, posttranslational modification of proteins during development

Sphaerechinus granularis eggs were fertilized in seawater in the presence of 0.2 mM dansylcadaverine, and development was allowed to take place with this compound in the medium. gamma-Glutamyldansylcadaverine, indicative of the utilization of the amine tracer by intrinsic transglutaminase, was isolated from the embryonic proteins, and identity of the product with the chemically synthesized gamma-glutamyl derivative of dansylcadaverine was confirmed. Covalent labeling of proteins occurring during development was examined by means of electrophoresis in NaDodSO4, followed by immunoblotting with an antibody that specifically recognized the dansyl hapten. There was an increase in the total uptake of the tracer at an essentially constant rate with each cell division, from 2- to 8- and 64-cell stages. Moreover, multiple protein labeling was evident in all specimens. The described concept of studying posttranslational modifications in vivo by transglutaminase through detection of the haptenic or specific ligand recognizable group of an incorporated small amine substrate will undoubtedly be of general utility for probing the functions of this family of enzymes in other cell types as well.     

Regulation of the water channel aquaporin-2 by posttranslational modification

The cellular functions of many eukaryotic membrane proteins, including the vasopressin-regulated water channel aquaporin-2 (AQP2), are regulated by posttranslational modifications. In this article, we discuss the experimental discoveries that have advanced our understanding of how posttranslational modifications affect AQP2 function, especially as they relate to the role of AQP2 in the kidney. We review the most recent data demonstrating that glycosylation and, in particular, phosphorylation and ubiquitination are mechanisms that regulate AQP2 activity, subcellular sorting and distribution, degradation, and protein interactions. From a clinical perspective, posttranslational modification resulting in protein misrouting or degradation may explain certain forms of nephrogenic diabetes insipidus. In addition to providing major insight into the function and dynamics of renal AQP2 regulation, the analysis of AQP2 posttranslational modification may provide general clues as to the role of posttranslational modification for regulation of other membrane proteins.     

Tryptophan tryptophylquinone biosynthesis: a radical approach to posttranslational modification

Protein-derived cofactors are formed by irreversible covalent posttranslational modification of amino acid residues. An example is tryptophan tryptophylquinone (TTQ) found in the enzyme methylamine dehydrogenase (MADH). TTQ biosynthesis requires the cross-linking of the indole rings of two Trp residues and the insertion of two oxygen atoms onto adjacent carbons of one of the indole rings. The diheme enzyme MauG catalyzes the completion of TTQ within a precursor protein of MADH. The preMADH substrate contains a single hydroxyl group on one of the tryptophans and no crosslink. MauG catalyzes a six-electron oxidation that completes TTQ assembly and generates fully active MADH. These oxidation reactions proceed via a high valent bis-Fe(IV) state in which one heme is present as Fe(IV)=O and the other is Fe(IV) with both axial heme ligands provided by amino acid side chains. The crystal structure of MauG in complex with preMADH revealed that catalysis does not involve direct contact between the hemes of MauG and the protein substrate. Rather it is accomplished through long-range electron transfer, which presumably generates radical intermediates. Kinetic, spectrophotometric, and site-directed mutagenesis studies are beginning to elucidate how the MauG protein controls the reactivity of the hemes and mediates the long range electron/radical transfer required for catalysis. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Analysis of expression and posttranslational modification of proteins during hypoxia.

The cellular responses to hypoxia are complex and characterized by alterations in the expression of a number of genes, including stress-related genes and corresponding proteins that are necessary to maintain homeostasis. The purpose of this article is to review previous and recent studies that have examined the changes in the expression and posttranslational modification of proteins in response to chronic sustained and intermittent forms of hypoxia. A large number of studies focused on the analysis of either the single protein or a subset of related proteins using one-dimensional gel electrophoresis to separate a complex set of proteins from solubilized tissues or cell extracts, followed by immunostaining of proteins using antibodies that are specific to either native or posttranslationally modified forms. On the other hand, only a limited number of studies have examined the global perturbations on protein expression by hypoxia using proteomics approach involving two-dimensional electrophoresis coupled with mass spectrometry. Results derived from specific protein analysis of a variety of tissues and cells showed that hypoxia, depending on the duration and severity of the stimulus, affects the level and the state of posttranslational modification of a subset of proteins that are associated with energy metabolism, stress response, cell injury, development, and apoptosis. Some of these earlier findings are further corroborated by recent studies that utilize a global proteomics approach, and, more importantly, results from these proteomics investigations on the effects of hypoxia provide new protein targets for further functional analysis. The anticipated new information stems from the analysis of expression, and posttranslational modification of these novel protein targets, along with gene expression profiles, offers exciting new opportunities to further define the mechanisms of cellular responses to hypoxia and to control more effectively the clinical consequences of prolonged or periodic lack of oxygen.     

Evidence for posttranslational modification and gene duplication of Campylobacter flagellin.

A gene encoding a flagellin protein of Campylobacter coli VC167 has been cloned and sequenced. The gene was identified in a pBR322 library by hybridization to a synthetic oligonucleotide probe corresponding to amino acids 4 to 9 of the N-terminal sequence obtained by direct chemical analysis (S. M. Logan, L. A. Harris, and T. J. Trust, J. Bacteriol. 169:5072-5077, 1987). The DNA was sequenced and shown to contain an open reading frame encoding a protein with a molecular weight of 58,945 and a length of 572 amino acids. The deduced amino acid sequence was identical to the published N-terminal amino acid sequence of VC167 flagellin and to four internal regions whose partial sequences were obtained by direct chemical analysis of two tryptic and two cyanogen bromide peptides of VC167 flagellin. The C. coli flagellin protein contains posttranslationally modified serine residues, most of which occur within a region containing two 9-amino-acid repeating peptides separated by 34 unique amino acids. Comparisons with the sequences of flagellins from other bacterial species revealed conserved residues at the amino- and carboxy-terminal regions. Hybridization data suggest the presence of a second flagellin copy located adjacent to the first on the VC167 chromosome.     

Chemical structure of posttranslational modification with a farnesyl group on tryptophan

Bacillus subtilis and related bacilli produce a posttranslationally modified oligopeptide, the ComX pheromone, that stimulates natural genetic competence controlled by quorum sensing. The ComX(RO-C-2) pheromone from strain RO-C-2 must be modified with a farnesyl group on the Trp residue, but the precise structure is not known. Here we report the precise nature of posttranslational farnesylation of ComX(RO-C-2) pheromone on the Trp residue, resulting in the formation of a tricyclic structure. The ComX(168) pheromone, produced by the standard laboratory strain used in the study of B. subtilis, is also posttranslationally farnesylated according to phylogenetic resemblance.     

Hypoxia potentiates microRNA-mediated gene silencing through posttranslational modification of Argonaute2

Hypoxia contributes to the pathogenesis of various human diseases, including pulmonary artery hypertension (PAH), stroke, myocardial or cerebral infarction, and cancer. For example, acute hypoxia causes selective pulmonary artery (PA) constriction and elevation of pulmonary artery pressure. Chronic hypoxia induces structural and functional changes to the pulmonary vasculature, which resembles the phenotype of human PAH and is commonly used as an animal model of this disease. The mechanisms that lead to hypoxia-induced phenotypic changes have not been fully elucidated. Here, we show that hypoxia increases type I collagen prolyl-4-hydroxylase [C-P4H(I)], which leads to prolyl-hydroxylation and accumulation of Argonaute2 (Ago2), a critical component of the RNA-induced silencing complex (RISC). Hydroxylation of Ago2 is required for the association of Ago2 with heat shock protein 90 (Hsp90), which is necessary for the loading of microRNAs (miRNAs) into the RISC, and translocation to stress granules (SGs). We demonstrate that hydroxylation of Ago2 increases the level of miRNAs and increases the endonuclease activity of Ago2. In summary, this study identifies hypoxia as a mediator of the miRNA-dependent gene silencing pathway through posttranslational modification of Ago2, which might be responsible for cell survival or pathological responses under low oxygen stress.     

Proteasome inhibition induces neurite outgrowth through posttranslational modification of TrkA receptor

The ubiquitin-proteasome pathway regulates many biological processes, including protein degradation, receptor endocytosis, protein sorting, subnuclear trafficking and neuronal differentiation. While proteasome inhibition is known to induce neurite outgrowth, the signaling mechanisms that mediate these effects have not been defined. In this study, we investigated the underlying mechanisms that link proteasome inhibition with neurite generation. We found that the proteasome inhibitors, MG132 and lactacystin, induced neurite outgrowth and also activated extracellular signal-regulated kinase/mitogen activated protein kinase and phosphatidylinositol-3-kinase/AKT pathways. These proteasome inhibitors also induced phosphorylation and ubiquitination of TrkA receptors, indicating that proteasome inhibition activates the major pathways of TrkA signaling. However, in contrast to nerve growth factor stimulation, which induces internalization of surface TrkA receptors, proteasome inhibitor-induced neurite outgrowth did not require TrkA receptor internalization. These results indicate that the ubiquitin-proteasome system regulates neurite formation through posttranslational modification of TrkA receptors.     

A novel transglutaminase-catalyzed posttranslational modification of HIV-1 aspartyl protease.

We demonstrate that HIV-1 aspartyl protease (AP), the enzyme essential for the maturation of the AIDS virus, covalently incorporates spermidine catalyzed by guinea pig liver transglutaminase (TGase) and human coagulation factor XIIIa. Preliminary evidence indicates that there are at least three reactive glutamyl and lysyl residues in AP which act as acyl donor and acceptor respectively in a TGase reaction. SDS-PAGE and chromatographic analyses indicate that the two TGases tested catalyze the incorporation of radioactive spermidine into pure HIV-1 AP. The chemical identification and quantitation of (gamma-glutamyl) spermidine isopeptide provide conclusive evidence that the formation of this derivative is catalyzed by TGase. These results imply that TGase-catalyzed post-translational modification of HIV-1 AP may take place in a manner similar to the ones demonstrated in porcine pancreatic phospholipase A2.     

Senile plaque composition and posttranslational modification of amyloid-beta peptide and associated proteins

Amyloid deposits are primarily composed of the amyloid-beta protein, although other proteins (and metal ions) also have been colocalized to these lesions. The pattern of oxidative modifications in amyloid plaques is very different to that associated with neurofibrillary tangles and neuronal cell bodies, likely reflecting the different composition of these structures, accessibility of oxidants, the generation of oxidants in and around these structures and the intrinsic antioxidant defense systems to protect these structures. Future studies directed at understanding Abeta interactions with other amyloid components, the role of oxidative modifications in stabilizing amyloid deposits and the determination of protease cleavage sites on Abeta may provide mechanistic insights regarding both amyloid formation and removal.