Use of okadaic acid to identify relevant phosphoepitopes in pathology: a focus on neurodegeneration.

Protein phosphorylation is involved in the regulation of a wide variety of physiological processes and is the result of a balance between protein kinase and phosphatase activities. Biologically active marine derived compounds have been shown to represent an interesting source of novel compounds that could modify that balance. Among them, the marine toxin and tumor promoter, okadaic acid (OA), has been shown as an inhibitor of two of the main cytosolic, broad-specificity protein phosphatases, PP1 and PP2A, thus providing an excellent cell-permeable probe for examining the role of protein phosphorylation, and PP1 and PP2A in particular, in any physiological or pathological process. In the present work, we review the use of okadaic acid to identify specific phosphoepitopes mainly in proteins relevant for neurodegeneration. We will specifically highlight those cases of highly dynamic phosphorylation-dephosphorylation events and the ability of OA to block the high turnover phosphorylation, thus allowing the detection of modified residues that could be otherwise difficult to identify. Finally, its effect on tau hyperhosphorylation and its relevance in neurodegenerative pathologies such as Alzheimer's disease and related dementia will be discussed.

Regulation of GSK3 isoforms by phosphatases PP1 and PP2A.

Dephosphorylation of phospho GSK3 isoforms, from COS-7 cells, was determined in vitro and in cultured cells in the absence or the presence of okadaic acid and lithium. Our results indicate a preferential dephosphorylation of phospho GSK3α by PP2A phosphatase, whereas dephosphorylation of phospho GSK3ß mainly takes place by PP1 phosphatase.

GSK3 inhibitors and disease.

This review describes, briefly, the characteristics and regulation of glycogen synthase kinase 3 (GSK3) together with the role of GSK3 dysfunctions in different pathologies, and GSK3 as target for therapeutic treatment in different diseases. Several GSK3 inhibitors acting at different levels are described in this work, ranging from cations like lithium to small compounds developed by different pharmaceutical companies. Also, the use of specific interference RNA (iRNA) for the specific inhibition of the expression of the different GSK3 isoforms is discussed.

Residues in human respiratory syncytial virus P protein that are essential for its activity on RNA viral synthesis.

Human respiratory syncytial virus (HRSV) P protein, 241 amino acid long, is a structural homotetrameric phosphoprotein. Viral transcription and replication processes are dependent on functional P protein interactions inside viral ribonucleoprotein complexes (RNPs). Binding capacity to RNPs proteins and transcription and replication complementation analyses, using inactive P protein variants, have identified residues essential for functional interactions with itself, L, N and M2-1 proteins. P protein may establish some of these interactions as monomer, but efficient viral transcription and replication requires P protein oligomerization through the central region of the molecule. A structurally stable three-dimensional model has been generated in silico for this region (residues 98-158). Our analysis has indicated that P protein residues L135, D139, E140 and L142 are involved in homotetramerization. Additionally, the residues D136, S156, T160 and E179 appear to be essential for P protein activity on viral RNA synthesis and very high turnover phosphorylation at S143, T160 and T210 could regulate it. Thus, compounds targeted to those of these residues, located in the modeled three-dimensional structure, could have specific anti-HRSV effect.

Role of polyglycine repeats in the regulation of glycogen synthase kinase activity.

Glycogen synthase kinase (GSK3) activity present in one cell is the consequence of the sum of the activities of two different proteins called GSK3alpha and GSK3beta. These isoenzymes are coded by two different genes and share an almost identical sequence at their catalytic domain, but differ in the sequence of putative regulatory regions. In this review, we propose that glycine repeats present only in GSK3alpha may result in the different cleavage of both isoenzymes by the protease calpain, a cleavage that modifies GSK3 activity.

Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.

The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells.

Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating.

The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) beta and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects.

Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex.

The human respiratory syncytial virus (HRSV) P protein is phosphorylated, with different turnover rates, at several serine (S) and threonine (T) residues. The role of phosphothreonines in viral RNA synthesis was studied by using P protein substitution variants and the HRSV-based minigenome pM/SH. By using liquid chromatography coupled to ion-trap mass spectrometry, it was found that P protein T108 was phosphorylated by addition of a high-turnover phosphate group. This phosphorylation occurs in P protein expressed transiently and during HRSV infection. The results suggest that phosphorylation at P protein T108 affects M2-1 transcriptional activities, because this modification prevents interaction between the P and M2-1 proteins. Therefore, P protein phosphorylation-dephosphorylation at T108 could distinguish the role of the P protein in viral transcription and replication.

Determination of phosphorylated residues from human respiratory syncytial virus P protein that are dynamically dephosphorylated by cellular phosphatases: a possible role for serine 54.

The 241 aa human respiratory synctyial virus (HRSV) Long strain P protein is phosphorylated at serines 116, 117 and/or 119, and 232. Phosphates added to these residues have slow turnover and can be detected in the absence of protein phosphatase inhibition. Inhibition of phosphatases PP1 and PP2A increases the level of phosphorylation at serines 116, 117 and/or 119, suggesting a more rapid turnover for phosphates added to these residues compared to that of S232. High-turnover phosphorylation is detected in the P-protein NH2-terminal region, mainly at S54 and, to a lesser extent, at S39, in the Long strain. When the P protein bears the T46I substitution (in the remaining HRSV strains), phosphates are added to S30, S39, S45 and S54. Phosphatase PP1 removes phosphate at residues in the central part of the P-protein molecule, whereas those in the NH2-terminal region are removed by phosphatase PP2A. The significance of the phosphorylation of the NH2-terminal region residues for some P-protein functions was studied. The results indicated that this modification is not essential for P-protein oligomerization or for its role in viral RNA synthesis. Nonetheless, dephosphorylation at S54 could facilitate P-M protein interactions that probably occur during the egress of viral particles.

Human respiratory syncytial virus matrix protein is an RNA-binding protein: binding properties, location and identity of the RNA contact residues.

The human respiratory syncytial virus (HRSV) matrix (M) protein is a structural internal membrane protein. Here we have shown that, like its orthomyxovirus and rhabdovirus counterparts, it has RNA-binding capacity, as determined by retardation of (32)P-labelled riboprobes in gel electrophoresis, cross-linking with UV light and Northern-Western assays. Its binding to RNA was neither sequence-specific nor showed a length requirement, although it had cooperative kinetics with a K(d) of 25 nM and probably two different types of RNA-binding sites. After preparative cross-linking of (32)P-labelled riboprobes with purified, renatured HRSV Long strain M protein (256 residues), the residues in contact with RNA were located between amino acids 120 and 170, in the central part of the molecule. Lysine (positions 121, 130, 156 and 157) and arginine (position 170) residues located within this region and conserved among pneumovirus M proteins of different origins were found to be essential for RNA contact. M protein expression did not affect the replication and transcription of HRSV RNA analogues in vivo (except when expressed in large amounts), in contrast to the in vitro transcription inhibition described previously. In addition, M protein was found to aggregate into high-molecular-mass oligomers, both in the presence and absence of its RNA-binding activity. The formation of these structures has been related in other viruses to either viral or host-cell RNA metabolism.