NH2-terminal Tyrosine Research Articles

Potential Molecular Mechanism for c-Src Kinase-mediated Regulation of Intestinal Cell Migration

The ubiquitously expressed Src tyrosine kinases (c-Src, c-Yes, and c-Fyn) regulate intestinal cell growth and differentiation. Src activity is also elevated in the majority of malignant and premalignant tumors of the colon. The development of fibroblasts with the three ubiquitously expressed kinases deleted (SYF cells) has identified the role of Src proteins in the regulation of actin dynamics associated with increased cell migration and invasion. Despite this, unexpectedly nothing is known about the role of the individual Src kinases on intestinal cell cytoskeleton and/or cell migration. We have previously reported that villin, an epithelial cell-specific actin-modifying protein that regulates actin reorganization, cell morphology, cell migration, cell invasion, and apoptosis, is tyrosine-phosphorylated. In this report using the SYF cells reconstituted individually with c-Src, c-Yes, c-Fyn, and wild type or phosphorylation site mutants of villin, we demonstrate for the first time the absolute requirement for c-Src in villin-induced regulation of cell migration. The other major finding of our study is that contrary to previous reports, the nonreceptor tyrosine kinase, Jak3 (Janus kinase 3), does not regulate phosphorylation of villin or villin-induced cell migration and is, in fact, not expressed in intestinal epithelial cells. Further, we identify SHP-2 and PTP-PEST (protein-tyrosine phosphatase proline-, glutamate-, serine-, and threonine-rich sequence) as negative regulators of c-Src kinase and demonstrate a new function for these phosphatases in intestinal cell migration. Together, these data suggest that in colorectal carcinogenesis, elevation of c-Src or down-regulation of SHP-2 and/or PTP-PEST may promote cancer metastases and invasion by regulating villin-induced cell migration and cell invasion.

Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells

Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-Tyr 418, FAK-Tyr 397-Tyr 576-Tyr 925, and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 micromol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr 397 and Tyr 576 but not FAK-Tyr 925 or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr 925 phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr 925 phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr 925-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.

Degradation of HER2 by Cbl-Based Chimeric Ubiquitin Ligases

Targeting disease-causing proteins for ubiquitination and degradation by chimeric molecules represents a promising alternative therapeutic strategy in cancer. Here, several Cbl-based chimeric ubiquitin ligases were recombined to achieve effective down-regulation of HER2. These chimeric molecules consisted of the Cbl NH(2)-terminal tyrosine kinase binding domain, linker, and RING domain, with the Src homology 2 domain replaced with that from growth factor receptor binding protein 2 (Grb2), Grb7, p85, or Src. The chimeric proteins not only interacted with HER2 but also enhanced the down-regulation of endogenous overexpressed HER2. After the chimeric proteins were introduced into HER2-overexpressing breast cancer SK-BR-3 cells or ovarian cancer SK-OV-3 cells, they effectively promoted HER2 ubiquitination and degradation in a RING finger domain-dependent manner. Consequently, expression of these chimeric molecules led to an inhibition of colony formation, increased the proportion of cells in the G(1) cycle, and suppressed tumorigenicity. Collectively, our findings suggest that the Cbl-based chimeric ubiquitin ligases designed in the present study may represent a novel approach for the targeted therapy of HER2-overexpressing cancers.

Brain-Specific Aminopeptidase: From Enkephalinase to Protector Against Neurodegeneration

The major breakthrough discovery of enkephalins as endogenous opiates led our attempts to determine their inactivation mechanisms. Because the NH2-terminal tyrosine is absolutely necessary for the neuropeptides to exert analgesic effects, and aminopeptidase activities are extraordinarily high in the brain, a specific "amino-enkephalinase" should exist. Several aminopeptidases were identified in the central nervous system during the search. In fact, our laboratory found two novel neuron-specific aminopeptidases: NAP and NAP-2. NAP is the only functionally active brain-specific enzyme known. Its synaptic location coupled with its limited substrate specificity could constitute a "functional" specificity and contribute to enkephalin-specific functions. In addition, NAP was found to be essential for neuron growth, differentiation, and death. Thus, aminopeptidases are likely important for mental health and neurological diseases. Recently, puromycin-sensitive aminopeptidase (PSA) was identified as a modifier of tau-induced neurodegeneration. Because the enzymatic similarity between PSA and NAP, we believe that the depletion of NAP in Alzheimer's disease (AD) brains plays a causal role in the development of AD pathology. Therefore, use of the puromycin-sensitive neuron-aminopeptidase NAP could provide neuroprotective mechanisms in AD and similar neurodegenerative diseases.

Synthesis of α(1,3) Fucosyltransferases IV- and VII-Dependent Eosinophil Selectin Ligand and Recruitment to the Skin

Selectins mediate the initial adhesion of leukocytes to endothelial cells in many contexts of inflammation-dependent leukocyte recruitment. The glycans that contribute to P- and E-selectin counterreceptor activity arise through glycosylation reactions in which the terminal steps are catalyzed by alpha(1,3) fucosyltransferases (FTs). We examined how selectin ligand activities are controlled in eosinophils by characterizing FT expression profiles and regulatory mechanisms in eosinophils isolated from human blood. We found that FT-IV and FT-VII mRNAs were up-regulated by transforming growth factor-beta1, but the FT-IV transcript consistently predominated in eosinophils. To further define the physiological role of FT-IV and FT-VII in expression of eosinophil selectin ligand, we characterized models of dermal eosinophilia in FT-IV- and/or FT-VII-deficient mice in vivo. FT-IV deficiency yielded a significant decrease in eosinophil recruitment to the skin. Likewise, deficiency of FT-VII also yielded a decrease in eosinophil recruitment. Eosinophil recruitment that remained in the absence of FT-VII was further inhibited by blocking P- or E-selectin and was essentially absent in mice deficient in both enzymes. These observations indicate that FT-IV and FT-VII are both important contributors to selectin-dependent eosinophil recruitment to the skin and may represent therapeutic targets for treating diseases in which eosinophil recruitment contributes to pathophysiology.

Cbl-b positively regulates Btk-mediated activation of phospholipase C-gamma2 in B cells.

Genetic studies have revealed that Cbl-b plays a negative role in the antigen receptor–mediated proliferation of lymphocytes. However, we show that Cbl-b–deficient DT40 B cells display reduced phospholipase C (PLC)-γ2 activation and Ca2+ mobilization upon B cell receptor (BCR) stimulation. In addition, the overexpression of Cbl-b in WEHI-231 mouse B cells resulted in the augmentation of BCR-induced Ca2+ mobilization. Cbl-b interacted with PLC-γ2 and helped the association of PLC-γ2 with Bruton's tyrosine kinase (Btk), as well as B cell linker protein (BLNK). Cbl-b was indispensable for Btk-dependent sustained increase in intracellular Ca2+. Both NH2-terminal tyrosine kinase-binding domain and COOH-terminal half region of Cbl-b were essential for its association with PLC-γ2 and the regulation of Ca2+ mobilization. These results demonstrate that Cbl-b positively regulates BCR-mediated Ca2+ signaling, most likely by influencing the Btk/BLNK/PLC-γ2 complex formation.

Purification and Characterization of a Major Kyotorphin-Hydrolyzing Peptidase of Rat Brain1

We purified a major kyotorphin (L-Tyr-L-Arg)-hydrolyzing peptidase (KTPase) from the rat brain, to electrophoretic homogeneity using conventional chromatographic techniques. KTPase was purified 1,660-fold with a specific activity of 161 mumol/min/mg protein and 6.8% recovery. The purified enzyme was composed of a single polypeptide with a molecular mass of 67 kDa and an isoelectric point (pI) of 5.5. KTPase has the ability to hydrolyze a variety of natural dipeptides. It also liberated NH2-terminal tyrosine from Tyr-Gly-Gly and Tyr-Tyr-Leu. Bestatin and arphamenine B were potent inhibitors of this enzyme, while amastatin and puromycin had little effect. An excess of anti-KTPase antibody raised in a white rabbit precipitated approximately 80% of the kyotorphin-hydrolyzing activity in the cytosol of rat brain. These data suggested that 67 kDa KTPase has a role in the degradation of kyotorphin within neuronal cells of the rat brain.

Structure-function analysis of stimulation of food intake by neuropeptide Y: Effects of receptor agonists

Neuropeptide Y (NPY) is a potent natural orexigenic signal in the rat. In this study, we have compared the effects of several COOH-terminal fragments of NPY and NPY receptor agonists on cumulative food intake in male rats. Rats were implanted with permanent cannulae either into the third cerebroventricle or paraventricular necleus (PVN). NPY 1–36 and various COOH-terminal fragments of NPY, two agonist analogues [Leu 31, Pro 34]NPY and NPY 1-4-Aca (ϵ-amino-caproic acid)-25-36, were administered intracerebroventricularly (ICV) or directly into the PVN, and the cumulative 2-h food intake response was compared. We observed that peptides that were effective by ICV were also effective when administered into the PVN, but smaller amounts of the peptides were required after PVN injection to evoke an equivalent food intake response. Injection of NPY 1–36 induced a dose-dependent increment in food intake. Surprisingly, deletion of NH 2-terminal tyrosine residue did not adversely affect feeding behavior. In fact, NPY 2–36 was consistently more effective than NPY 1–36; the enhancement in feeding by NPY 2–36 was dose-related and was higher than evoked by NPY 1–36 at each dose tested. Further serial deletion of aminoacids at NH 2-terminal resulted in complete loss of activity. In addition, NPY agonist analogue, NPY 1-4-Aca-25-36, failed to stimulate feeding. However, NPY Y 1 receptor agonist, [Leu 31, Pro 34]NPY, but not Y 2 receptor agonist, NPY 13–36, stimulated feeding. These studies show that deletion of NH 2-terminal tyrosine residue renders NPY 2–36 more effective than natural NPY 1–36, and stimulation of feeding by NPY 1–36 and NPY 2–36 is mediated by NPY Y 1 receptor subtypes. Since NPY 1-4-Aca-25-36 and NPY 13–36 were inactive, it suggests 5–12 amino acid residues in NPY 1–36 and NPY 2–36 may be actively engaged in evoking ingestive behavior.

Further characterization of the major forms of reptile beta-endorphin

Biosynthetically labeled reptile intermediate pituitary beta-endorphin-sized material was fractionated by SP-Sephadex ion exchange chromatography into two major opiate-active forms which eluted at 0.28 M NaCl and 0.32 M NaCl, respectively; the 0.32 M form of reptile β-endorphin ( mw=3500 ), serves as the precursor for the 0.28 M form of reptile β-endorphin ( mw=3200 ), (Dores and Surprenant, 1983). Analysis of tryptic digests of these reptile β-endorphins by paper electrophoresis at pH 3.5 and gel filtration on a Sephadex G-15 column indicated that there are two tyrosine residues, two arginine residues and one methionine residue in reptile β-endorphin. Furthermore, the NH 2-terminal tryptic peptide of both reptile β-endorphins is approximately nine amino acids in size and contains tyrosine, methionine and arginine. Analyses of chymotryptic/protease digests of the [ 3H]tyrosine-labeled NH 2-terminal tryptic peptide analyzed by descending paper chromatography revealed that the NH 2-terminal tyrosine of reptile β-endorphin is not α-N-acetylated. A second tyrosine-containing tryptic peptide was detected in the COOH-terminal region of reptile β-endorphin; however this tryptic peptide differs in the two forms of reptile β-endorphin in terms of size and net charge at pH 3.5. These differences account for the apparent molecular weight differences and distinct ion exchange properties of the 0.28 M and 0.32 M forms of reptile β-endorphin. Thus in the reptile intermediate pituitary the principal post-translational mechanism for modifying β-endorphin is COOH-terminal proteolytic cleavage.

“Joining peptide” of pro-opiomelanocortin. I. Radioimmunoassay and extraction of related peptides from pituitary glands

In order to immunoassay the specific region of bovine pituitary pro-opiomelanocortin (POMC) between ACTH and γ-MSH, referred to as “joining peptide,” antisera were prepared against the synthetic amidated decapeptide Val-Ala-Val-Gly-Glu-Gly-Pro-Gly-Pro-Arg-NH 2. The non-amidated peptide represents residues −23 to −14 of bovine POMC [25]. An NH 2-terminal tyrosine analog of the decapeptide was used as the radioligand. Under optimal conditions, immunoassays with selected antisera exhibited a sensitivity (50% displacement of the radioligand) toward the decapeptide in the range of 31–55 pg. Immunoreactivity found in extracts of fresh or lyophilized bovine pituitary glands displaced the iodinated Tyr-decapeptide in the RIA in a parallel manner. The amount of imunoreactive (ir)-material was dependent upon the state of preservation of the tissue, the method of extraction, and the particular antiserum used. Extractable immunoreactivity was separated into low (Mr 1,500) and high (Mr 17,000) molecular weight peptides using gel chromatography (G-75). Additional ir-material appeared in the void volume ( Mr>22,500 ). Thus, these antisera have the capacity to interact not only with a region of the joining peptide but also with its larger, and apparent precursor forms. The immunoassay developed should be valuable in understanding the disposition and processing in this specific region of POMC.

Degradation of dynorphin-(1–13) by membrane-bound rat brain enzymes

Under the incubation conditions of the opioid receptor binding assay, dynorphin-(1–13) is metabolized by membrane-bound exopeptidases which cleave the NH 2-terminal tyrosine and the COOH-terminal lysine. Enzymatic attack, particularly that of carboxypeptidase, is extremely rapid. Following a 5 min incubation at 22°C with wellwashed brain membranes (0.5 ml; 1 g original wet weight per 100 ml), less than 50% of dynorphin-(1–13) (20 nM) remains intact. Various methods of stabilization of dynorphin-(1–13) have been examined. Degradation can be reduced, but not eliminated, by the use of metabolic inhibitors or by lowering the incubation temperature to 0°C. Substitution of an amide or methyl ester function at the carboxyl terminus of the peptide provides complete protection not only from carboxypeptidase attack but also from aminopeptidase attack. Carboxyl-substituted compounds are limited in their usefulness as stable analogues, however, since they are not resistant to hydrolysis in the presence of physiological salts.

Beta-Endorphin: demonstration of a tertiary structure in aqueous solution.

Difference spectra generated during thermolysin digestion of camel beta-endorphin at pH 8.2 or at pH 6.5 indicate rapid blue shifting of the near-UV absorption bands of the NH2-terminal tyrosine. A similar spectral change is not observed for the NH2-terminal tyrosine in [Met]enkephalin when it is digested under similar conditions. These results suggest that enzymatic digestion destroys or alters some structural interaction between the NH2-terminal tyrosyl residue of the endorphin and a residue(s) within the COOH-terminal segment of the molecule. Peptide mapping of the digest as a function of time suggests that cleavage of the bond linking the alanine-21 and isoleucine-22 residues produces most of the observed effect. These data provide evidence for the existence of a tertiary structure for beta-endorphin in aqueous solutions.

On the mechanism of aryl sulfotransferase.

Aryl sulfotransferase IV (EC 2.8.2.1), purified to homogeneity from male rat liver, catalyzes the sulfation of a variety of substituted phenols, including catecholamines, tyrosine esters, and peptides containing NH2-terminal tyrosine residues. An investigation of the mechanism of the enzyme was carried out using 2-chloro-4-nitrophenol as a model substrate. Kinetic, inhibition, and binding studies with aryl sulfotransferase IV are all consistent with a random rapid equilibrium Bi Bi kinetic mechanism with two dead end product inhibitor complexes. Studies of the chemical mechanisms of the enzyme-catalyzed reaction demonstrate that electron-withdrawing substituents decrease the maximal velocity of phenol sulfation. The maximal velocity of the reaction correlates with Hammett sigma p- constants (rho = -0.25). Evidence is presented for the mechanism by which adenosine 3',5'-bisphosphate and aryl sulfotransferase catalyze the transfer of sulfate from 2-chloro-4-nitrophenyl sulfate to other phenols.

Specific receptor for the opioid peptide dynorphin: structure--activity relationships.

The structural features responsible for the high potency and opiate receptor specificity of the opioid peptide dynorphin in the guinea pig ileum myenteric plexus were examined. Successive removal of COOH-terminal amino acids from dynorphin-(1--13) demonstrated important contributions of lysine-13, lysine-11, and arginine-7 to the potency. Removal of the NH2-terminal tyrosine abolished the biologic activity. Several other structural modifications were shown to affect potency: substitution of D-alanine for glycine-2 reduced the potencies of dynorphin-(1--13) amide, -(1--11), and -(1--10); and methyl esterification of the COOH terminus enhanced the potencies of dynorphin-(1--12), -(1--10), -(1--9), -(1--8), and -(1--7). Within the dynorphin sequence, lysine-11 and arginine-7 were found to be important for selectivity of interaction with the dynorphin receptor, which is distinguishable from the mu receptor in this tissue.

Further analysis of post-translational processing of beta-endorphin in rat intermediate pituitary.

Rat intermediate pituitary cells maintained in culture synthesize the same forms of beta-endorphin observed in intermediate pituitary extracts. Biosynthetically labeled intermediate pituitary beta-endorphin-sized material was fractionated by ion exchange chromatography on sulfopropyl-Sephadex and the identities of the major peaks were determined by co-chromatography with synthetic marker peptides, gel filtration, and analysis of pronase, chymotrypsin, and trypsin digests. Peaks of alpha-N-acetyl-beta-endorphin(1-27), alpha-N-acetyl-beta-endorphin(1-31), and beta-endorphin(1-31) were identified and a fourth peak (eluting from the sulfopropyl-Sephadex column at 0.18 M NaCl) was tentatively identified as alpha-N-acetyl-beta-endorphin(1-26). Analysis of beta-endorphin synthesized in the presence of [35S]methionine and [3H]histidine confirmed the absence of His in the material eluting at 0.18 M NaCl. Based on both steady labeling and pulse-chase incubations, beta-endorphin(1-31) was the first form of labeled beta-endorphin-sized material to appear in cell extracts. This molecule was quickly N-acetylated on its NH2-terminal tyrosine residue and was then more slowly converted to alpha-N-acetyl-beta-endorphin(1-27) and then to alpha-N-acetyl-beta-endorphin(1-26).

Isolation, structure, biological characterization, and synthesis of beta-[Tyr9]melanotropin-(9-18) decapeptide from pig hypothalami.

A decapeptide with the amino acid sequence H-Tyr-Phe-Arg-Trp-Gly-Ser-Pro-Pro-Lys-Asp-OH was isolated from acid extracts of porcine hypothalami, structurally and biologically characterized, and synthesized. Except for the NH2-terminal tyrosine, this decapeptide corresponds to the amino acid sequences 9-18 of porcine beta-melanotropin (beta-MSH) and 49-58 of porcine beta-lipotropin (beta-LPH); it also has a tetrapeptide sequence of amino acids (Phe-Arg-Trp-Gly) common to the 7-10 sequences in corticotropin (ACTH) and alpha-MSH. beta-MSH, beta-LPH, alphaMSH, and ACTH from various species all have a histidine residue in the position immediately preceding the common sequence, and the occurrence of a natural peptide with the tyrosine residue in the corresponding site has not been previously reported. This suggests that the beta-[Tyr9]MSH-(9-18) decapeptide might be a fragment of a still larger precursor (prohormone) possibly related to beta-LPH.

Energy conformation study of Met-enkephalin and its D-Ala2 analogue and their resemblance to rigid opiates.

Conformational similarities of Met-enkephalin and its D-Ala2 analogue to rigid opiates were studied by both empirical and quantum mechanical methods. By both methods, conformers with maximum resemblance to rigid opiates have the highest energies. Conformers with the lowest energy had no resemblance to rigid opiates. However, several low and intermediate energy conformers were identified in which at least the NH2-terminal tyrosine residue overlaps with the phenethylamine moiety of rigid opiates and which could equally well accommodate either Gly2 or D-Ala2. The conformer among these with greatest additional resemblance to other binding sites of rigid opiates is proposed as the most likely candidate for an induced fit at the receptor site.

Morphinomimetic activity of synthetic fragments of beta-lipotropin and analogs.

In the myenteric plexus-longitudinal muscle bioassay, beta-endorphin, i.e., beta-lipotropin (beta-LPH)-[61-91], has a potency of 450 with confidence limits of 281-966 when Met5-enkephalin is used as a reference standard with a potency of 100. The primary amide and the ethylamide of Met5-enkephalin have potencies statistically overlapping with that of beta-endorphin. The primary amide of alpha-endorphin has twice the potency of the free acid form of alpha-endorphin. An intact NH2-terminal tyrosine is not necessary for full intrinsic activity. The shortest fragment of beta-LPH with morphinomimetic activity is beta-LPH-[61-64].

Oxidation of tyrosine and of NH2-terminal tyrosine peptides with the Cu2+-H2O2 system.

Oxidation of tyrosine and of NH2-terminal tyrosine peptides with the Cu2+-H2O2 system.