Aspects Of Phosphorylation Research Articles

Protein Tyrosine Phosphorylation and Reversible Oxidation: Two Cross-Talking Posttranslation Modifications

In addition to protein phosphorylation, redox-dependent posttranslational modification of proteins is emerging as a key signaling system, conserved throughout evolution, and influencing many aspects of cellular homeostasis. Recent data have provided new insight about the interplay between phosphorylation- and redox-dependent signaling, and reactive oxygen species have been included among intracellular signal transducers of growth factor and extracellular matrix receptors. Both tyrosine phosphorylation and thiol oxidation are reversible and dynamic, and this review will particularly focus on the cross-talk between these posttranslational protein regulatory means. Although these modifications share their reversibility, their effects on enzymatic activity of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) may be even opposite. Indeed, while tyrosine phosphorylation is frequently correlated to enzyme activation, thiol oxidation leads to inactivation of PTPs and to superactivation of PTKs. Several papers describe that both these modifications occur during the same input, (i.e., cell proliferation and motility induced by numerous growth factors and cytokines). The review will discuss several aspects of phosphorylation\oxidation interplay, describing both convergent and divergent features of the integrated and coordinated function of PTPs and PTKs during signaling.

Synthesis of homoserine phospho-, H-phosphono- and methylphosphonopeptides

Phosphopeptides and mimics thereof are useful tools for the investigation of phosphorylation, an important posttranslational modification of peptides and proteins. In order to investigate different aspects of phosphorylation and dephosphorylation processes, homoserine phospho-, H-phosphono- and methylphosphonopeptides were synthesized. The tetrapeptide H-Gly-Gly-Hse-Ala-OH was used as a model sequence; further, the heptapeptide H-Leu-Arg-Arg-Ala-Hse-Leu-Gly-OH and the octapeptide H-Glu-Ser-Leu-Hse-Ser-Ser-Glu-Glu-OH were synthesized and modified. After selective deprotection of the trityl-protected homoserine residue, phosphorylation or phosphonylation was performed on resin by the global phosphorylation approach using different phosphoamidites. Peptides were analysed by analytical RP-HPLC and electrospray mass spectrometry. All compounds were obtained in yields over 75%. The byproducts observed were both the unmodified peptide and the H-phosphonopeptide in the case of the phosphopeptides, the phosphorylated and the unmodified peptide in the case of the H-phosphonopeptides, and the unmodified peptide in the case of the methylphosphonopeptides. Due to simple purification by RP-HPLC, the method presented gives access to a new class of phosphopeptides and mimics.

On the use of seven-membered phosphorous heterocycles based on 2,2′-dihydroxy-1,1′-binaphthalene and 1,4∶3,6-dianhydro-d-mannitol in the31P NMR analysis of the enantiomeric composition of chiral alcohols

Some aspects of phosphorylation of 2,2′-dihydroxy-1,1′-binaphthalene (BINOL) and 1,4∶3,6-dianhydro-d-mannitol (isomannide) were investigated. The possibility of using the corresponding cyclic chloro- and amidophosphites for the analysis of enantiomeric compositions of chiral primary and secondary alcohols by31P NMR was examined.

Protein Kinase and Phosphatase Activity in the Lungs of Normoxic Versus Hyperoxic Rats

Studies were designed to examine aspects of phosphorylation and dephosphorylation in rat lung cells in response to hyperoxic exposure. Protein kinase and phosphatase activities were measured in preparations of lungs from normoxic rats, hyperoxia-exposed rats (95 % O2 for 60 h), and rats recovering in room air for 1 and 3 days. Protein kinase C (PKC) activity immediately postexposure was significantly lower than in normoxic controls (normoxia 127.1 ± 13 vs. hyperoxia 101.5 ± 6 pmol/min mg−1) and continued to decline during the recovery period (85.3 ± 4 and 78.2 ± 6 pmol/min mg−1 at 1 and 3 days recovery, respectively). The PKC activity did not translocate from cytoplasm to the membranes. In contrast, PKA activity did not change in response to hyperoxia exposure or recovery. Protein phosphatase activity was decreased significantly by hyperoxia exposure (normoxia 30.7 ± 3 vs. hyperoxia 21.9 ± 1 pmol/min µg−1) but returned to normoxic control levels by 1 and 3 days (24.1 ± 4 and 31.5 ± 1 pmol/min µg−1, respectively). Protein phosphatase activity was inhibited by okadaic acid (Ki = 1 nM) and calyculin A (Ki = 0.61 pM), indicating a type 2A protein phosphatase. Enzyme activities in cultured type II alveolar cells paralleled those observed in whole lung preparations. Decreased enzyme activities in the lung may be related to the development of acute lung injury during hyperoxic exposure.

Phosphorylation and Steroid Hormone Action

Publisher Summary This chapter describes the different aspects of phosphorylation and steroid hormone action. Group A receptors form complexes with heat-shock proteins in the absence of hormone. After hormone binding, the receptors dissociate from some of the heat-shock proteins and are transformed into complexes in which the receptors dimerize, bind DNA, and activate or repress the transcription of target genes. In the absence of hormone, group B receptors usually bind to DNA. Most steroid receptors are phosphorylated in vivo at multiple sites, suggesting that phosphorylation may play multiple roles in receptor function. Several features of chicken progesterone receptor (cPR) phosphorylation are common to the members of the steroid hormone receptor superfamily, especially the group A receptors. First, the phosphorylation sites can be basal, ligand-dependent, or DNA-dependent. Both cPR and human progesterone receptor (hPR) are phosphorylated in a DNA-dependent manner during cell-free transcription in HeLa cell nuclear extracts. The phosphorylation is increased in vivo after the activation of either protein kinase A (PKA) or protein kinase C (PKC) pathways, and treatment with H7, which can inhibit several kinases, including PKA and PKC, resulted in both a dose-dependent inhibition of the phosphorylation and the induction of terminal differentiation of the erythroblasts.

Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro.

Two species of polyoma virus middle T antigen were detected in both lytically infected and transformed cells by in vitro kinase assay of immunoprecipitates. A minor species with an apparent molecular weight of 58,000 (58K) represented less than 10% of the total middle T protein. This species was roughly 10 times more active as a phosphate acceptor than was the predominant 56K form. Partial proteolytic mapping experiments showed that the same site was phosphorylated in both species. Mapping of the middle T antigens from a series of deletion mutants suggested that the major site of phosphorylation is tyrosine residue 315. Phosphorylation occurred on both middle T species in vivo, involving sites predominantly other than the tyrosine labeled in vitro. The 56K and 58K middle T forms differed from each other in their in vivo phosphorylation patterns. Some phosphate was incorporated into the 58K species in a region of the molecule to which at least part of the apparent molecular weight different could be mapped. hr-t mutant NG-59, which codes for a slightly altered middle T, produced only a single species (56K) which was inactive in the in vitro kinase reaction. Moreover, no 58K species appeared in vivo with this mutant. hr-t mutants are therefore defective in both aspects of phosphorylation. Phenotypically normal revertant cells of a polyoma transformed line failed to express any middle T antigens or associated kinase activity.