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PubMed1969

D-Tyrosine as a metabolic inhibitor of Bacillus subtilis.

The d-isomer of tyrosine is a potent inhibitor of growth in transformable strain 168 of Bacillus subtilis. A d-tyrosine-resistant mutant of the inhibited strain was isolated which excreted l-tyrosine, had a diminished growth rate, and required l-phenylalanine to attain the growth rate of the wild-type parent. Mapping by deoxyribonucleate transformation located this resistance in the gene coding for prephenate dehydrogenase. This enzyme in the d-tyrosine-resistant mutant was insensitive to the usual feedback inhibition exerted by l-tyrosine in extracts of strain 168. In contrast, the growth of poorly transformable strain 23 of B. subtilis, as well as that of several other Bacillus species, was not affected by the analogue. Transformation mapping demonstrated no linkage of this latter "natural resistance" to several different aromatic markers. Prephenate dehydrogenase in extracts from strain 23 was as sensitive as that from strain 168 to feedback inhibition by l-tyrosine in vitro. The relationships of the latter results to the regulation of tyrosine biosynthesis and the possible nature of strain differences in d-tyrosine sensitivity are discussed.

Bacillus subtilis↗
PubMed1991

Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells.

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.

Animals↗
PubMed1993

Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively.

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.

Animals↗
PubMed1996

Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation.

Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine-phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady-state activated Ras is insufficient to activate B-Raf in vivo.

3T3 Cells↗
PubMed1984

Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells.

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.

Adenosine Triphosphate↗
PubMed1986

Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen.

We characterized the tyrosine phosphorylation sites of free pp60c-src and of pp60c-src associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60c-src were different, both in vitro and in vivo. Free pp60c-src was phosphorylated in vitro at a single site, tyrosine 416. pp60c-src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60c-src in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60c-src associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60c-src associated with mT in transformed cells are identical to those of pp60v-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60c-src associated with mT may play a role in the enhancement of the pp60c-src protein kinase activity and in cell transformation by polyomavirus.

Amino Acids↗
PubMed1998

Randomised controlled trial of tyrosine supplementation on neuropsychological performance in phenylketonuria.

OBJECTIVE: To test the efficacy of tyrosine supplementation, as an adjunct to dietary treatment, on neuropsychological test performance in individuals with phenylketonuria. DESIGN: A randomised controlled trial of tyrosine supplementation using a double blind crossover procedure with three four week phases. SETTING: The Hospital for Sick Children, Toronto. PARTICIPANTS: 21 individuals with phenylketonuria (ages 6 to 28 years, mean 11.3). INTERVENTION: Participants were given 100 mg/kg body weight/d of L-tyrosine or L-alanine (placebo). RESULTS: At baseline, performance on several of the neuropsychological test measures was correlated with tyrosine levels. Dietary supplements of tyrosine increased plasma tyrosine concentrations; however, no change in test performance was found across the tyrosine and placebo phases of the study. CONCLUSIONS: Tyrosine supplementation of this type does not appear to alter neuropsychological performance in individuals with phenylketonuria.

Administration, Oral↗
PubMed1984

Dietary tyrosine supplementation enhances weight gain in streptozotocin-diabetic rats.

The effect of dietary protein concentration and tyrosine supplementation on growth in streptozotocin (65 mg/kg, ip) diabetic rats was evaluated. When rats were fed diets ranging from 15 to 60% protein, weight gain and feed efficiency were greatest in rats fed the 45% protein diet. Adding tyrosine to this diet (8%, incorporated as a percentage of protein) did not promote further weight gain relative to nonsupplemented diabetic animals. In contrast, rats choosing 45% of total calories as protein by selecting from 10 and 60% protein diets supplemented with either 0, 4, or 8% tyrosine demonstrated a 35% (4% tyrosine) to 45% (8% tyrosine) increase in weight gain. Proximate analysis indicated similar body composition in tyrosine supplemented and nonsupplemented diabetic animals. Including tryptophan (1.45%) with tyrosine in the self-selection diet was without effect. Thus, tyrosine supplementation promoted a modest but consistent and specific increase in weight gained by self-selecting diabetic rats.

Animals↗
PubMed1986

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells.

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Protein tyrosine phosphorylation, cellular Ca2+, and Ca2+ sensitivity for contraction of smooth muscle.

Our studies are guided by the novel hypothesis that protein tyrosine phosphorylation is an important mechanism for regulating contraction of smooth muscle. Several lines of evidence are reviewed which suggest that enhanced tyrosine phosphorylation participates in mechanisms that regulate cytosolic Ca2+ and Ca2+ sensitivity for contraction. First, vanadate-induced contraction of guinea-pig taenia coli is functionally linked to enhanced protein tyrosine phosphorylation of at least three substrates, apparently resulting from vanadate-mediated inhibition of protein tyrosine phosphatase activity. Second, vanadate-induced contraction is dependent on extracellular Ca2+. Third, increases in cytosolic Ca2+ resulting from stimulation of alpha 1-adrenergic receptors in cultured canine vascular smooth muscle cells are associated with enhanced tyrosine phosphorylation and are inhibited by genistein, a potent inhibitor of tyrosine kinase activity. Fourth, genistein markedly and reversibly suppresses Ca2+ sensitivity for contraction in ileal longitudinal smooth muscle permeabilized with staphylococcal alpha-toxin. Moreover, the same or similar substrates (e.g., 42-45, 70, 80-85, 95, 100, 110, 116, and 205 kDa) are tyrosine phosphorylated in response to Ca2+ or stimulation of muscarinic or alpha 1-adrenergic receptors. Collectively, these data strongly suggest that tyrosine phosphorylation is an important mechanism for regulation of smooth muscle contraction.

Animals↗
PubMed2002

Cr supplementation decreases tyrosine phosphorylation of the CreaT in skeletal muscle during sepsis.

Myocellular creatine (Cr) uptake is predominantly governed by a sodium-dependent Cr transporter (CreaT) and plays a pivotal role in skeletal muscle energy metabolism. The CreaT belongs to a neurotransmitter transporter family that can be functionally regulated by protein tyrosine kinase-induced tyrosine phosphorylation. The association between myocellular Cr and c-Src-related tyrosine phosphorylation of the CreaT and the influence of oral Cr supplementation on this association were investigated during sepsis. Animals were randomized to receive standard rat chow or standard rat chow with oral Cr supplementation for 4 days followed by cecal ligation and puncture (CLP) or sham operation. Fast-twitch gastrocnemius muscles were harvested 24 h after operation. Myocellular free Cr levels were 70% higher after CLP. Western blotting of the immunoprecipitated CreaT with an anti-phosphotyrosine or anti-phospho-c-Src (Y-416) antibody revealed that tyrosine phosphorylation of the CreaT and tyrosine-phosphorylated c-Src (Tyr(416)) expression in the CreaT-c-Src complex were significantly increased after CLP compared with sham operation. These changes were observed in homogenates and plasma membrane fractions of gastrocnemius muscles. Although oral Cr supplementation increased myocellular free Cr levels equivalently in CLP and sham-operated animals, c-Src-related tyrosine phosphorylation of the CreaT in homogenates and plasma membrane fractions of gastrocnemius muscles was, however, downregulated in Cr-supplemented CLP animals compared with Cr-supplemented sham-operated rats. During sepsis, increased myocellular free Cr levels are associated with enhanced tyrosine phosphorylation of the CreaT, which is likely induced by active c-Src. Oral Cr supplementation downregulates c-Src-related tyrosine phosphorylation of the CreaT. The data suggest that myocellular Cr homeostasis and CreaT activity are tightly regulated and closely related during sepsis.

Administration, Oral↗
PubMed1997

Tyrosine phosphorylation modulates current amplitude and kinetics of a neuronal voltage-gated potassium channel.

The modulation of the Kv1.3 potassium channel by tyrosine phosphorylation was studied. Kv1.3 was expressed in human embryonic kidney (HEK 293) cells, and its activity was measured by cell-attached patch recording. The amplitude of the characteristic C-type inactivating Kv1.3 current is reduced by >95%, in all cells tested, when the channel is co-expressed with the constitutively active nonreceptor tyrosine kinase, v-Src. This v-Src-induced suppression of current is accompanied by a robust tyrosine phosphorylation of the channel protein. No suppression of current or tyrosine phosphorylation of Kv1.3 protein is observed when the channel is co-expressed with R385A v-Src, a mutant with severely impaired tyrosine kinase activity. v-Src-induced suppression of Kv1.3 current is relieved by pretreatment of the HEK 293 cells with two structurally different tyrosine kinase inhibitors, herbimycin A and genistein. Furthermore, Kv1.3 channel protein is processed properly and targeted to the plasma membrane in v-Src cotransfected cells, as demonstrated by confocal microscopy using an antibody directed against an extracellular epitope on the channel. Thus v-Src-induced suppression of Kv1.3 current is not mediated through decreased channel protein expression or interference with its targeting to the plasma membrane. v-Src co-expression also slows the C-type inactivation and speeds the deactivation of the residual Kv1.3 current. Mutational analysis demonstrates that each of these modulatory changes, in current amplitude and kinetics, requires the phosphorylation of Kv1.3 at multiple tyrosine residues. Furthermore, a different combination of tyrosine residues is involved in each of the modulatory changes. These results emphasize the complexity of signal integration at the level of a single ion channel.

DNA, Complementary↗
PubMed1988

Comparison of the tyrosine kinase activity with the proliferation rate in human colon solid tumors and tumor cell lines.

The role of the tyrosine kinase signal transduction pathway was investigated in human colon solid tumors and colon tumor cell lines. A high level of tyrosine kinase activity was found in 7 of the 27 human solid tumors tested (26%). In these cases, a close correlation between the level of tyrosine kinase activity and the high ratio of the S phase cells has been demonstrated (r = 0.8418). High autophosphorylation accompanied by a high proliferation capacity was detected in 8 cases (29.6%). In 12 cases (44%) low tyrosine kinase activity with a lower proliferation rate was found. Seven of the 8 human colon tumor cell lines tested showed tyrosine kinase activity. Differentiation-inducing agents, such as sodium butyrate and retinoic acid, have been applied to influence the rate of cell proliferation. Treatment with 5 mM sodium butyrate (24 h) and 10 microM retinoic acid (48 h) effectively decreased the fraction of S phase cells and 3H-thymidine incorporation. The tyrosine kinase activity fell to 9-22% and to 44-65% of the original value in the case of the sodium butyrate and retinoic acid treatment, respectively. Our results suggest that a significant part of human colon tumors have an active tyrosine kinase signal transduction pathway and that tyrosine kinase plays a role in the process of proliferation rather than in the process of differentiation in these human colon tumor cell lines.

Butyrates↗
PubMed1998

Priming of platelet alphaIIbbeta3 by oxidants is associated with tyrosine phosphorylation of beta3.

Reactive oxygen species play an important role at the site of vascular injuries and arterial thromboses. We studied the mechanism mediating platelet aggregation induced by H2O2, a major cellular oxidant. Exposure to H2O2 triggered platelet aggregation, but only when the platelets were stirred. Strong platelet aggregation induced99032416 required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (NaVO4) and was dependent on the participation of integrin alphaIIbbeta3 (glycoprotein IIb-IIIa). A specific inhibitor of alphaIIbbeta3 blocked platelet aggregation induced by H2O2 and NaVO4, thus confirming that aggregation requires this receptor. In the presence of H2O2 and NaVO4, multiple platelet substrates were phosphorylated on tyrosine. Such tyrosine kinase response was necessary but not sufficient to activate alphaIIbbeta3, as detected by binding of soluble fibrinogen to platelets. Stirring of the platelets exposed to H2O2 and NaVO4 was also needed to allow for binding of fibrinogen to alphaIIbbeta3. The tyrosine kinase inhibitor genistein was able to block platelet aggregation induced by H2O2 and NaVO4, thus confirming that tyrosine kinase activity was needed to trigger alphaIIbbeta3 activation on stirring. N-Acetyl-L-cysteine, a cell-permeant antioxidant, blocked the tyrosine phosphorylation of platelet substrates and also the platelet aggregation induced by H2O2 and NaVO4. We found that beta3 was phosphorylated on tyrosine in platelets exposed to H2O2 and NaVO4, even in the absence of aggregation. Hence, tyrosine phosphorylation of beta3 might contribute to the "priming" of alphaIIbbeta3 induced by H2O2 and NaVO4, whereby the receptor can become activated on stirring of the platelets.

Humans↗
PubMed1996

Tyrosine kinase signaling pathways modulate angiotensin II-induced calcium ([Ca2+]i) transients in vascular smooth muscle cells.

Tyrosine kinases have been implicated in vascular smooth muscle cell proliferation and contraction. Underlying mechanisms may involve C(a2+) -dependent pathways. This study assesses relationships between angiotensin II (Ang II)-stimulated phospholipase C-mediated Ca2+ transients and tyrosine kinase-dependent pathways in vascular smooth muscle cells. Intracellular free Ca2+ concentration ([Ca2+]i) was measured in primary cultured unpassaged vascular smooth muscle cells derived from mesenteric resistance vessels of Wistar-Kyoto rats with the use of fura 2 methodology. [Ca2+]i effects of Ang II (1 nmol/L) were determined in vascular smooth muscle cells in which tyrosine kinase pathways were stimulated by insulin (70 muU/mL; 0.5 nmol/L), insulin-like growth factor-I (1 ng/mL; 0.13 nmol/L), or platelet-derived growth factor-BB (1 ng/mL; 0.04 nmol/L) and in cells in which tyrosine kinase was inhibited by specific inhibitors (1 mumol/L tyrphostin A-23 and genistein). Ang II elicited a rapid and transient [Ca2+]i response (from 94 +/- 8 to 239 +/- 5.8 nmol/L). Activation of the receptor tyrosine kinase by insulin, platelet-derived growth factor, and insulin-like growth factor-I significantly reduced (P < .01) Ang II-induced [Ca2+]i to 161 +/- 7, 189 +/- 3.7, and 183 +/- 5 nmol/L, respectively. In the presence of tyrphostin A-23 and genistein, Ang II-stimulated [Ca2+]i remained persistently elevated and failed to return to basal levels. Tyrphostin A-1, the inactive tyrphostin analogue, had not significant effect on Ang II-induced [Ca2+]i. This study demonstrates that activation of tyrosine kinase pathways reduces Ang II-elicited [Ca2+]i responses, whereas tyrosine kinase inhibition prevents [Ca2+]i recovery after agonist stimulation. Interaction between tyrosine kinase- and phospholipase C-dependent signaling pathways modulates vascular smooth muscle cell [Ca2+]i responses to Ang II.

Angiotensin II↗
PubMed1993

Glucocorticoid regulation of insulin receptor and substrate IRS-1 tyrosine phosphorylation in rat skeletal muscle in vivo.

To test the hypothesis that glucocorticoid-induced insulin resistance might originate from abnormalities in insulin receptor signaling, we investigated the effects of glucocorticoids on in vivo tyrosine phosphorylation of the insulin receptor and the insulin receptor substrate IRS-1 in rat skeletal muscle. Male Sprague-Dawley rats were treated with cortisone (100 mg/kg for 5 d) and compared to pair-fed controls. Cortisone treatment of rats resulted in both hyperglycemia and hyperinsulinemia. Anesthetized animals were injected with 10 U/kg insulin via cardiac puncture and, after 2 min, hindlimb muscles were removed, snap-frozen, and homogenized in SDS. Protein tyrosine phosphorylation was studied by immunoblotting with phosphotyrosine antibody. Insulin receptors and substrate IRS-1 were identified and quantified with specific antibodies. Cortisone treatment increased the amount of insulin receptor protein by 36%, but decreased the total level of receptor tyrosine phosphorylation (69 +/- 4% of control, P < 0.05). The decreased level of receptor phosphorylation was explained by a reduced number of receptors containing phosphorylated tyrosine residues (64.6 +/- 5% of control, P < 0.05). Glucocorticoid excess decreased skeletal muscle IRS-1 content by 50%, but did not significantly alter the total level of IRS-1 tyrosine phosphorylation. The apparent M(r) of IRS-1 was reduced by approximately 10 kD. Treatment with protein phosphatase-2A reduced IRS-1 M(r) in control but not in glucocorticoid-treated muscle indicating that the lower M(r) likely results from lower phosphoserine and/or phosphothreonine content. To investigate the role of hyperinsulinemia in the glucocorticoid response, rats were made insulin-deficient with streptozotocin (100 mg/kg, i.p.). Subsequent treatment with cortisone for 5 d had no effects on insulin levels, tyrosine phosphorylation of insulin receptors or IRS-1, or the M(r) of IRS-1. In conclusion, glucocorticoid-treated skeletal muscle is characterized by: (a) decreased total tyrosine phosphorylation of insulin receptors as a result of a reduction in the pool of receptors undergoing tyrosine phosphorylation; (b) decreased IRS-1 content and reduced serine and/or threonine phosphorylation of IRS-1. Glucocorticoid-induced hyperinsulinemia appears to be essential for the development of these alterations.

Amino Acid Sequence↗
PubMed1993

Crystal-induced neutrophil activation. IV. Specific inhibition of tyrosine phosphorylation by colchicine.

We recently demonstrated that pathologically relevant inflammatory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, potently stimulate a characteristic protein tyrosine phosphorylation pattern in human neutrophils that differed from that observed in response to other soluble or particulate agonists. In this study, the effects of colchicine on protein tyrosine phosphorylation induced by MSU and CPPD crystals in human blood neutrophils were investigated. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that colchicine dose-dependently inhibited the tyrosine phosphorylation of all the proteins phosphorylated in response to MSU and CPPD crystals. Other microtubule-disruptive agents such as vinblastine, nocodazole, and colcemid also inhibited crystal-induced protein tyrosine phosphorylation while lumicolchicine and trimethylcolchicinic acid were without effect. Indomethacin and phenylbutazone were similarly without effect on microcrystal-induced tyrosine phosphorylation. Colchicine, as well as the other active alkaloids, failed to inhibit the protein tyrosine phosphorylation elicited by FMLP, C5a, leukotriene B4, and unopsonized zymosan. Overall, these results demonstrate that colchicine specifically and significantly inhibits the protein tyrosine phosphorylation induced by MSU and CPPD crystals and suggest that its effects are associated, at least in part, with its interaction with microtubules. Furthermore, the use of microtubule-disrupting drugs demonstrate that the mechanisms implicated in the induction of protein tyrosine phosphorylation by microcrystals differed from those involved in response to other soluble or particulate agonists.

Blood Proteins↗
PubMed1998

Phenylalanine and tyrosine metabolism in neonates receiving parenteral nutrition differing in pattern of amino acids.

Tyrosine is considered to be an indispensable dietary amino acid in the neonate, yet achieving adequate parenteral tyrosine intake is difficult due to its poor solubility. Increasing the supply of phenylalanine is the most common means of compensating for low tyrosine levels. Unfortunately, plasma phenylalanine concentrations are sometimes elevated in infants receiving high phenylalanine intake. This led us to study the phenylalanine and tyrosine metabolism in 16 neonates randomized to receive total parenteral nutrition with either a high or a moderate phenylalanine-containing amino acid solution. A primed, 24-h continuous stable isotope infusion of L-[1-13C]phenylalanine and L-[3,3-2H2]tyrosine was given to enable the measurement of phenylalanine and tyrosine kinetics. Results demonstrated that 1) phenylalanine hydroxylation was significantly greater in infants receiving high phenylalanine, 2) phenylalanine oxidation and percent dose oxidized was also significantly greater in infants receiving high phenylalanine, 3) apparent phenylalanine retention was greater in neonates receiving high phenylalanine, and 4) alternate catabolites of phenylalanine and tyrosine metabolism were significantly greater in infants receiving high phenylalanine compared with moderate phenylalanine. We conclude that neonates respond to increased parenteral phenylalanine intake by increasing their hydroxylation and oxidation rates. The greater oxidation of phenylalanine in infants receiving high phenylalanine in conjunction with the urinary excretion of alternate catabolites of phenylalanine and tyrosine suggests that the high phenylalanine intake may be in excess of needs. However, the lower apparent phenylalanine retention observed in infants receiving moderate phenylalanine suggests that the total aromatic amino acid level of moderate phenylalanine may be deficient for neonatal needs.

Amino Acids↗

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