Effect of the phosphonic analogue of tyrosine on tyrosine iodination

Summary Depositing ofdl-1-amino-2-(p-hydroxyphenyl)-ethylphosphonic acid (Tyr-P) on the chicken embryo induced a dose dependent decrease of the iodine uptake by the embryonic thyroid. Tyr-P interfered on iodination of tyrosine when tested with hog thyroid peroxidase (TPO) and with bovine lactoperoxidase (LPO); the analogue was recognized by the two enzymes but its affinity for TPO and LPO was respectively 3 and 7 fold higher compared with that of the natural substrate, suggesting that Tyr-P may act as an iodine trap.     

Action of the phosphonic analogue of tyrosine and of other tyrosine derivatives on rat liver tyrosine aminotransferase

Summary Tyrosine transamination has been investigatedin vitro with a preparation of rat liver tyrosine aminotransferase in the presence of several structural derivatives of the substrate, including the phosphonic analogue. The transamination by tyrosine aminotransferase (TAT) needs the presence in the substrate molecule of free amino and carboxylic groups, a three-carbon aliphatic chain, a para-phenolic hydroxylic function and al-configuration. Some tyrosine analogues can markedly disturb the Tyr-TAT association: the chief structural modifications are (i) the removal of the free amine function in a compound still possessing a para-hydroxylic and a carboxylic group, (ii) the change of the carboxylic function by another acidic group, especially a phosphonic one, (iii) a disubstitution in positions 3 and 5. In every situation, the presence of a parahydroxylic group is compulsory to observe an inhibitory effect.     

Meal-induced peptide tyrosine tyrosine inhibition of pancreatic secretion in the rat

In the present studies we examined the distribution, release, and biological actions of peptide tyrosine tyrosine (PYY) in the rat. The concentration and distribution of PYY was highest in the ileum and colon as determined by both radioimmunoassay of rat tissue extracts and immunocytochemistry. An ultrastructural comparison of rat and dog colonic PYY cells revealed a bipolar distribution of peptide-containing secretory granules in both species. Serum PYY and pancreatic exocrine secretory responses were monitored after presentation of a meal to meal-trained rats (n = 12). A significant increase in PYY concentrations was not observed until 120 min after meal presentation, a delayed response similar to that previously observed in the dog. PYY responses were also observed in rats after perfusion of the intestine at the level of the duodenum and ileum with an 80 mOsm micellar solution of sodium oleate. Duodenal instillations of the fatty acids resulted in a maximum PYY response after 120 min, whereas rats subject to ileal perfusion of fat exhibited maximum PYY release within the first hour. In other experiments, infusion of exogenous PYY at 100 pmol.kg-1.h-1, which reproduced plasma PYY levels observed after a meal and perfusion of the gut with fat, significantly inhibited CCK-stimulated bile pancreatic volume (P less than 0.02), protein (P less than 0.01), and amylase (P less than 0.01) output. These studies demonstrate a bipolar distribution of PYY-containing secretory granules in cells of the jejunal, ileal, and colonic mucosa, and show that PYY is released in response to a meal in amounts sufficient to inhibit cholecystokinin-stimulated pancreatic secretion. Evidence is presented that PYY may mediate the delayed inhibition of pancreatic secretion that is observed in the rat after ingestion of a meal.     

Modification of the tyrosine hydroxylase assay

A modification of the tyrosine hydroxylase assay is described in which ascorbate, rather than 2-mercaptoethanol or dihydropteridine reductase with NADPH, is used as the reductant. Enzyme activity is 3–4 times higher with ascorbate than with the other reducing agents. Low blanks are obtained with the ascorbate system provided that catalase is also included. The tissue distribution and kinetic activation of the enzyme have been studied with the ascorbate assay. The results obtained are consistent with the biological and regulatory properties of the enzyme which have been determined with the other reducing systems.     

Developmental expression and distinctive tyrosine phosphorylation of the Eph-related receptor tyrosine kinase Cek9

Cek9 is a receptor tyrosine kinase of the Eph subfamily for which only a partial cDNA sequence was known (Sajjadi, F.G., and E.B. Pasquale. 1993. Oncogene. 8:1807-1813). We have obtained the entire cDNA sequence and identified a variant form of Cek9 that lacks a signal peptide. We subsequently examined the spatio-temporal expression and tyrosine phosphorylation of Cek9 in the chicken embryo by using specific antibodies. At embryonic day 2, Cek9 immunoreactivity is concentrated in the eye, the brain, the posterior region of the neural tube, and the most recently formed somites. Later in development, Cek9 expression is widespread but particularly prominent in neural tissues. In the developing visual system, Cek9 is highly concentrated in areas containing retinal ganglion cell axons, suggesting a role in regulating their outgrowth to the optic tectum. Unlike other Eph-related receptors, Cek9 is substantially phosphorylated on tyrosine in many tissues at various developmental stages. Since autophosphorylation of receptor protein-tyrosine kinases typically correlates with increased enzymatic activity, this suggests that Cek9 plays an active role in embryonic signal transduction pathways.     

On the cross-regulation of protein tyrosine phosphatases and receptor tyrosine kinases in intracellular signaling

Intracellular signaling proteins are very often regulated by site-specific phosphorylation. For example, growth factor receptors in eukaryotic cells contain intrinsic tyrosine kinase activity and use inter- and intra-molecular interactions to recruit and orient potential protein substrates for phosphorylation. Equally important in determining the magnitude and kinetics of such a response is protein dephosphorylation, catalysed by phosphatase enzymes. A growing body of evidence indicates that certain protein tyrosine phosphatases (PTPs), like tyrosine kinases, are affected by intermolecular interactions that alter the specific activity or localization of their catalytic domains. Using a detailed kinetic modeling framework, we theoretically explore the regulation of PTPs through their association with receptor tyrosine kinases, as noted for the Src homology 2-domain-containing PTPs, SHP-1 and -2. Receptor-PTP binding, in turn, is expected to influence the phosphorylation pattern of those receptors and proteins they associate with, and we show how PTPs might serve to co- or counter-regulate parallel pathways in a signaling network.     

Differential effects of expression of the CD45 tyrosine protein phosphatase on the tyrosine phosphorylation of the lck, fyn, and c-src tyrosine protein kinases.

Expression of the CD45 tyrosine protein phosphatase is required for the response of functional lymphocytes to stimulation through the antigen receptor. One or more of its substrates may therefore be essential for signal transduction during lymphocyte activation. We have studied the phosphorylation of the closely related lck, fyn, and c-src tyrosine protein kinases in leukemic murine T-cell lines that have lost the expression of CD45. The phosphorylation of the lck kinase at an inhibitory site of tyrosine phosphorylation, Tyr-505, was increased by two-, six-, and eightfold in three different cell lines. Phosphorylation of the fyn kinase at the homologous site, Tyr-531, was unaltered in one of these cell lines, but increased by 2.5-fold in the two others. The phosphorylation of p60c-src at the homologous tyrosine was essentially unchanged in the one CD45-negative cell line in which it was examined. The expression of CD45 therefore regulates the phosphorylation and potentially the activity of the lck and fyn tyrosine protein kinases, but the effect on the lck kinase is much greater than on the fyn kinase. This finding and the observation that CD45 had no effect on the phosphorylation of p60c-src suggest that CD45 exhibits polypeptide substrate specificity in vivo. Additionally, these findings are consistent with the hypothesis that the unresponsiveness of CD45-negative lymphoid cells to antigenic stimulation is due largely to hyperphosphorylation of the lck kinase.     

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to approximately 200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.     

On the mechanism of iodination of tyrosine

Existing data contain proof that the iodinating species of tyrosine and its derivatives contained in mixtures of iodine and iodide is hypoiodous acid, HOI. It appears likely that the peroxidase-catalyzed iodination reaction with hydrogen peroxide, tyrosine or a tyrosine derivative and either iodide or iodine as substrates involves enzyme-activated HOI.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.     

Leucine motif-dependent tyrosine autophosphorylation of type III receptor tyrosine kinases

Activation loop tyrosine autophosphorylation is an essential requirement for full kinase activation of receptor tyrosine kinases (RTKs). However, mechanisms involved are not fully understood. In general, kinase domains of RTKs are folded into two main lobes, NH2- and COOH-terminal lobes. The COOH-terminal lobe of vascular endothelial growth factor receptor-2 (VEGFR-2) is folded into seven alpha-helices (alphaD-alphaI). In the studies presented here we demonstrate that leucine residues of helix I (alphaI) regulate tyrosine autophosphorylation and phosphotransferase activity of VEGFR-2. The presence of leucines 1158, 1161, and 1162 are essential for tyrosine autophosphorylation and kinase activation of VEGFR-2 and are involved in helix-helix packing via hydrophobic interactions. The presence of leucine 1158 is critical for kinase activation of VEGFR-2 and appears to interact with alphaE, alphaF, alphaH, and beta7. The analogous residue, leucine 957 on platelet-derived growth factor receptor-beta and leucine 910 on colony stimulating factor-1R are also found to be critical for tyrosine autophosphorylation of these receptors. Leucines 1161 and 1162 are also involved in helix-helix packing but they play a less critical role in VEGFR-2 activation. Thus, we conclude that leucine motif-mediated helix-helix interactions are critical for kinase regulation of type III RTKs. This mechanism is likely to be shared with other kinases and might provide a basis for the design of a novel class of tyrosine kinase inhibitors.     

Insulin stimulates the tyrosine phosphorylation of caveolin

The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin- sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin- associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton- insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an approximately 60 kD protein that cross-reacts with an antibody against the Src-family kinase p59Fyn. Thus, the insulin- dependent tyrosine phosphorylation of caveolin represents a novel, insulin-specific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor.     

Analysis of the immunoadjuvant octadecyl tyrosine hydrochloride

A simple, rapid, high performance liquid chromatographic (HPLC) method for the analysis of the immunoadjuvant octadecyl tyrosine hydrochloride is described. The HPLC procedure can be applied to the direct determination of amino acid reactants present as contaminants in the adjuvant (tyrosine, ethyl tyrosine) and from this information the content of octadecanol reactant can be estimated. Further, these same determinations provide a means of monitoring immunoadjuvant stability in any vaccine preparation.     

Steric course of the tyrosine ammonia-lyase reaction

The tyrosine ammonia-lyase reaction proceeds with loss of the pro-3S and retention of the pro-3R hydrogen from the tyrosine side chain and thus involves anti-periplanar elimination of the elements of ammonia.