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PubMed1999

The tyrosines in the bidentate motif of the env-sea oncoprotein are essential for cell transformation and are binding sites for Grb2 and the tyrosine phosphatase SHP-2.

The transforming gene product of the S13 avian erythroblastosis virus, the env-sea protein, is a member of the hepatocyte growth factor receptor family of tyrosine kinases comprising Met, Ron, and Sea. Like all three members of this family, the env-sea protein has a so-called bidentate motif (Y557INMAVTY564VNL) composed of two tandemly arranged tyrosines in the carboxyl terminus. To investigate whether the tyrosine residues in this motif are essential for the env-sea-mediated transformation, we generated tyrosine to phenylalanine mutations. Substitutions of both tyrosine residues resulted in complete loss of the transforming activity. In contrast, single mutations at either tyrosine did not inhibit transformation of Rat1 cells, and mutation of tyrosine 564 actually increased transformation of Rat 1 cells. To define signaling pathways activated by the env-sea protein, we looked for protein-protein interactions mediated by these tyrosine residues. We show that the bidentate motif is responsible for interaction with the adapter protein Grb2, phosphatidylinositol 3-kinase, and the tyrosine phosphatase SHP-2. Furthermore, we show that microinjected Src homology 2 domains from either Grb2 or SHP-2 blocked the transforming activity of the env-sea protein. Together, these results suggest that the tyrosines within the bidentate motif are essential for the env-sea transformation.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Growth signal transduction by the human interleukin-2 receptor requires cytoplasmic tyrosines of the beta chain and non-tyrosine residues of the gamma c chain.

To evaluate the possible role for receptor-based tyrosine phosphorylation in growth signaling induced by interleukin-2 (IL-2), a series of substitution tyrosine mutants of the IL-2 receptor beta and gamma c chains was prepared and analyzed. Concurrent mutation of all six of the cytoplasmic tyrosines present in the beta chain markedly inhibited IL-2-induced growth signaling in both pro-B and T cell lines. Growth signaling in a pro-B cell line was substantially reconstituted when either of the two distal tyrosines (Tyr-392, Tyr-510) was selectively restored in the tyrosine-negative beta mutant, whereas reconstitution of the proximal tyrosines (Tyr-338, Tyr-355, Tyr-358, Tyr-361) did not restore this signaling function. Furthermore, at least one of the two cytoplasmic tyrosines that is required for beta chain function was found to serve as a phosphate acceptor site upon induction with IL-2. Studies employing a chimeric receptor system revealed that tyrosine residues of the beta chain likewise were important for growth signaling in T cells. In contrast, although the gamma c subunits is a target for tyrosine phosphorylation in vivo, concurrent substitution of all four cytoplasmic tyrosines of this chain produced no significant effect on growth signaling by chimeric IL-2 receptors. However, deletion of either the Box 1, Box 2, or intervening (V-Box) regions of gamma c abrogated receptor function. Therefore, tyrosine residues of beta but not of gamma c appear to play a pivotal role in regulating growth signal transduction through the IL-2 receptor, either by influencing cytoplasmic domain folding or by serving as sites for phosphorylation and subsequent association with signaling intermediates. These findings thus highlight a fundamental difference in the structural requirements for IL-2R beta and gamma c in receptor-mediated signal transduction.

Amino Acid Sequence↗
PubMed1985

Utilization of N-acetyl-L-tyrosine and glycyl-L-tyrosine during long-term parenteral nutrition in the growing rat.

Utilization of N-acetyl-L-tyrosine and glycyl-L-tyrosine as a source of tyrosine in infusion solutions was tested in rats receiving total parenteral nutrition for 4 wk. The four solutions tested were isonitrogenous and isocaloric. One of the solutions contained an adequate amount of L-phenylalanine; in the other three, two-thirds of the phenylalanine was replaced by a corresponding amount of either glycine, glycyl-L-tyrosine or N-acetyl-L-tyrosine. No differences in weight gain or N-balance could be detected as a result of administering either the solution with glycyl-L-tyrosine or with N-acetyl-L-tyrosine in place of the solution containing an adequate phenylalanine content. The solution in which two-thirds of the L-phenylalanine was replaced by glycine yielded only half of the weight gain and correspondingly reduced values for N-balance. Daily urinary excretion rates for N-acetyl-L-tyrosine and glycyl-L-tyrosine were 11% and 0.5%, respectively, of the infused amount. Plasma amino acid pattern was affected differently by the four solutions. The results indicate that both N-acetyl-L-tyrosine and glycyl-L-tyrosine are efficiently utilized by the rat during total parenteral nutrition.

Amino Acids↗
PubMed1986

Induction of mRNA for tyrosine hydroxylase by cyclic AMP and glucocorticoids in a rat pheochromocytoma cell line: evidence for the regulation of tyrosine hydroxylase synthesis by multiple mechanisms in cells exposed to elevated levels of both inducing agents.

When rat pheochromocytoma PC18 cells are exposed to the cyclic AMP analog, 8-bromocyclic AMP, and/or the synthetic glucocorticoid, dexamethasone, there is a marked increase in the level of a single RNA species that hybridizes to the recombinant plasmid pTH.4, which contains sequences complementary to the RNA coding for tyrosine hydroxylase. This RNA species is 1800-1900 nucleotides in length and is presumably identical to an RNA species of similar size, isolated from rat pheochromocytoma PC8b cells and shown to code for tyrosine hydroxylase. Using RNA dot hybridization to quantitate the relative level of this tyrosine mRNA species, time course studies show that this mRNA increases relatively rapidly in PC18 cells treated with either 8-bromocyclic AMP or dexamethasone. A new steady state level of tyrosine hydroxylase mRNA is achieved after 6 hr or 12-24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, respectively. The changes in the level of the mRNA slightly precede the changes in the rate of synthesis of tyrosine hydroxylase in cells treated with these inducing agents. After 24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, the increases in the level of tyrosine hydroxylase mRNA are identical to the increases in the rate of synthesis of the enzyme in the cells. In cells treated simultaneously with both 8-bromocyclic AMP and dexamethasone, the increases in the enzyme level and rate of synthesis of tyrosine hydroxylase are approximately equal to the sum of the increases in these parameters observed in cells treated with either inducing agent alone. In contrast, there is not an additive increase in the level of tyrosine hydroxylase mRNA in cells treated with both inducing agents. This lack of an additive increase in mRNA for tyrosine hydroxylase is observed in total cellular RNA samples or in cytoplasmic RNA samples. Our results suggest that in cells exposed to elevated levels of either cyclic AMP or glucocorticoids, tyrosine hydroxylase is induced by a mechanism which increases the level of its mRNA, resulting in an increased rate of synthesis of the enzyme. However, in cells exposed to elevated levels of both cyclic AMP and dexamethasone, tyrosine hydroxylase enzyme levels are regulated by multiple mechanisms, one of which regulates the rate of synthesis of the enzyme without affecting the level of its mRNA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Utilization of tyrosine-containing dipeptides and N-acetyl-tyrosine in hepatic failure.

The impact of hepatic dysfunction on the elimination and hydrolysis of three potential tyrosine sources for total parenteral nutrition, the dipeptides L-alanyl-L-tyrosine (Ala-Tyr) and glycyl-L-tyrosine (Gly-Tyr), and N-acetyl-L-tyrosine (Nac-Tyr) were evaluated in six patients with hepatic failure (five chronic, one acute) and seven healthy subjects. In controls, whole-body clearance (Cltot) of Ala-Tyr was higher than of Gly-Tyr (3,169 +/- 214 vs. 1,780 +/- 199 mL/kg/min, P < .01), and both exceeded clearance of Nac-Tyr (309 +/- 29 mL/kg/min, P > .01). Both dipeptides were hydrolyzed and released tyrosine immediately. In hepatic failure, elimination and hydrolysis of Ala-Tyr and Gly-Tyr were comparable to controls, but Cltot of Nac-Tyr was reduced (236 +/- 26 mL/kg/min). Neither in controls nor in patients an increase in plasma tyrosine concentration was seen after Nac-Tyr, and the major part of Nac-Tyr infused was lost in urine. The Cltot of tyrosine as evaluated after Ala-Tyr infusion (with the immediate release of tyrosine) was severely reduced in hepatic failure (152.7 +/- 38.4 vs. 484.4 +/- 41.4 mL/kg/min, P < .001) and half-life (kle) was retarded from 14.4 +/- 1.4 to 90.2 +/- 32.2 minutes (P < .03). The authors conclude that acute and chronic hepatic dysfunction does not affect elimination and hydrolysis of the dipeptides Ala-Tyr and Gly-Tyr and the constituent amino acids are released immediately. Nac-Tyr elimination was not grossly affected by hepatic failure, but neither in healthy subjects nor in hepatic failure patients was an increase of tyrosine seen. Both dipeptides but not Nac-Tyr may serve as a tyrosine source in parenteral nutrition. Moreover, by its rapid hydrolysis, the use of Ala-Tyr, for the first time, enables a simple rapid nonisotope evaluation of tyrosine kinetics for assessment of liver function.

Adult↗
PubMed1998

Asymmetrical phosphorylation and function of immunoreceptor tyrosine-based activation motif tyrosines in B cell antigen receptor signal transduction.

CD79a and CD79b function as transducers of B cell antigen receptor signals via a cytoplasmic sequence, termed the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs contain two conserved tyrosines that may become phosphorylated upon receptor aggregation and bind distinct effectors by virtue of the distinct preference of phosphotyrosyl-containing sequences for SH2 domains. To explore the function of CD79a and CD79b ITAM tyrosines, we created membrane molecules composed of MHC class II I-Ak extracellular and transmembrane domains, and CD79a or CD79b cytoplasmic domains in which one or both of the ITAM tyrosines were mutated to phenylalanine. Functional analysis revealed that both ITAM tyrosines are required for ligand-induced Syk phosphorylation. However CD79a-ITAM and CD79b-ITAM tyrosine phosphorylations were asymmetrical, with >80% of phosphorylation occurring on the N-terminal tyrosine (Y-E-G-L). Thus, these findings suggest that following receptor ligation, only a minor proportion of phosphorylated ITAMs are doubly phosphorylated and thus can engage Syk. Only the N-terminal ITAM tyrosine of CD79a was required for ligand-mediated phosphorylation of the receptor and a subset of downstream substrates, including p62, p110, and Shc, and for Ca2+ mobilization. However, responses mediated through CD79b exhibited a greater dependence on the presence of both tyrosines. Neither tyrosine in CD79a or CD79b appeared absolutely essential for Src family kinase phosphorylation. These results indicate that phosphorylations of the tyrosines in CD79a and CD79b occur with very different stoichiometry, and the respective tyrosyl residues have distinct functions.

Antigens, CD↗
PubMed1998

Regulation of mouse PECAM-1 tyrosine phosphorylation by the Src and Csk families of protein-tyrosine kinases.

PECAM-1 is an adhesion molecule expressed on hemopoietic and endothelial cells. Recently, it was observed that PECAM-1 becomes tyrosine-phosphorylated in response to a variety of physiological stimuli. Furthermore, tyrosine-phosphorylated PECAM-1 was shown to associate with SHP-2, a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase expressed ubiquitously. In light of the significance of tyrosine protein phosphorylation as a regulatory mechanism, we wished to understand better the nature and impact of the protein-tyrosine kinases (PTKs) mediating PECAM-1 tyrosine phosphorylation. Through reconstitution experiments in COS-1 cells, we determined that mouse PECAM-1 could be tyrosine-phosphorylated by Src-related PTKs and Csk-related PTKs, but not by other kinases such as Syk, Itk, and Pyk2. Using site-directed mutagenesis and peptide phosphorylation studies, we found that these PTKs were efficient at phosphorylating Tyr-686, but not Tyr-663, of PECAM-1. Src-related enzymes also phosphorylated mouse PECAM-1 at one or more yet to be identified sites. In other studies, we demonstrated that phosphorylation of PECAM-1 by Src or Csk family kinases was sufficient to trigger its association with SHP-2. Moreover, it was able to promote binding of PECAM-1 to SHP-1, a SHP-2-related protein-tyrosine phosphatase expressed in hemopoietic cells. Taken together, these findings indicated that the Src and Csk families of kinases are strong candidates for mediating tyrosine phosphorylation of PECAM-1 and triggering its association with SH2 domain-containing phosphatases under physiological circumstances.

Amino Acid Sequence↗
PubMed2000

Essential functions of protein tyrosine phosphatases PTP2 and PTP3 and RIM11 tyrosine phosphorylation in Saccharomyces cerevisiae meiosis and sporulation.

Tyrosine phosphorylation plays a central role in eukaryotic signal transduction. In yeast, MAP kinase pathways are regulated by tyrosine phosphorylation, and it has been speculated that other biochemical processes may also be regulated by tyrosine phosphorylation. Previous genetic and biochemical studies demonstrate that protein tyrosine phosphatases (PTPases) negatively regulate yeast MAP kinases. Here we report that deletion of PTP2 and PTP3 results in a sporulation defect, suggesting that tyrosine phosphorylation is involved in regulation of meiosis and sporulation. Deletion of PTP2 and PTP3 blocks cells at an early stage of sporulation before premeiotic DNA synthesis and induction of meiotic-specific genes. We observed that tyrosine phosphorylation of several proteins, including 52-, 43-, and 42-kDa proteins, was changed in ptp2Deltaptp3Delta homozygous deletion cells under sporulation conditions. The 42-kDa tyrosine-phosphorylated protein was identified as Mck1, which is a member of the GSK3 family of protein kinases and previously known to be phosphorylated on tyrosine. Mutation of MCK1 decreases sporulation efficiency, whereas mutation of RIM11, another GSK3 member, specifically abolishes sporulation; therefore, we investigated regulation of Rim11 by Tyr phosphorylation during sporulation. We demonstrated that Rim11 is phosphorylated on Tyr-199, and the Tyr phosphorylation is essential for its in vivo function, although Rim11 appears not to be directly regulated by Ptp2 and Ptp3. Biochemical characterizations indicate that tyrosine phosphorylation of Rim11 is essential for the activity of Rim11 to phosphorylate substrates. Our data demonstrate important roles of protein tyrosine phosphorylation in meiosis and sporulation

DNA, Fungal↗
PubMed1997

Analysis of CD28 cytoplasmic tail tyrosine residues as regulators and substrates for the protein tyrosine kinases, EMT and LCK.

The CD28 cell surface receptor provides an important costimulatory signal for T cells necessary for their response to Ag. Early events in CD28 signaling include recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase) and activation of the protein tyrosine kinases (PTKs), LCK and EMT. Recruitment and activation of PI3-kinase is known to be dependent upon phosphorylation of tyrosine 173 of the CD28 cytoplasmic tail contained within a YMNM motif. By contrast, little is known of which residues of the CD28 tail, including tyrosines, are required for the activation of PTKs. To address this we studied the ability of truncation mutants and tyrosine to phenylalanine substitution mutants of the CD28 cytoplasmic tail to activate LCK and EMT in Jurkat T leukemia cells. Our results indicate that 1) activation of EMT is partially dependent upon tyrosine 173 of the CD28 tail, although it does not require PI3-kinase activation; 2) activation of LCK is independent of CD28 cytoplasmic tail tyrosine residues; and 3) elements sufficient for the activation of both kinases are contained within the first half of the tail. In addition we studied the CD28 tail as a substrate for both PTKs in in vitro kinase assays. We demonstrate that EMT can phosphorylate all four tyrosines of the CD28 tail, in contrast to LCK, which phosphorylates only tyrosine 173. Together with evidence that in vivo, tyrosines other than tyrosine 173 become phosphorylated following CD28 stimulation, this finding suggests that, like LCK, one function of EMT during CD28 signaling is phosphorylation of the receptor.

CD28 Antigens↗
PubMed1991

Changes in insulin-receptor tyrosine, serine and threonine phosphorylation as a result of substitution of tyrosine-1162 with phenylalanine.

Previous studies, by ourselves and others, have shown that tyrosine residues 1158, 1162 and 1163 are very rapidly autophosphorylated on the human insulin receptor after insulin binding and that this is followed by the autophosphorylation of tyrosine residues 1328 and 1334. The autophosphorylation of these tyrosine residues, and their role in transmembrane signalling, were examined by using Chinese-hamster ovary cells transfected with either normal intact insulin receptors or receptors in which tyrosine residues 1162 or 1162/1163 were substituted with phenylalanine. These studies show the following. (1) Tyrosine-1158 could still be autophosphorylated when tyrosine-1162 and -1163 were substituted with phenylalanine. (2) Insulin-stimulated insulin-receptor tyrosine phosphorylation in intact cells was complete within 30 s and was accompanied, after a lag of 2-5 min, by a rise in serine and threonine phosphorylation the beta-subunit. (3) Replacement of tyrosine-1162 with phenylalanine blocked insulin-stimulated threonine phosphorylation of the insulin receptor in intact cells. (4) Insulin-stimulated serine phosphorylation of the beta-subunit was found in both intact cells and partially purified receptor preparations incubated with [gamma-32P]ATP and was still apparent after the replacement of tyrosine-1162 with phenylalanine. (5) Our data strongly suggest that insulin-stimulated insulin-receptor serine and threonine phosphorylations are initiated through two distinct pathways, with only the latter showing a strict dependence on autophosphorylation of tyrosine-1162.

Amino Acid Sequence↗
PubMed1997

Paxillin is tyrosine-phosphorylated by and preferentially associates with the calcium-dependent tyrosine kinase in rat liver epithelial cells.

We and others have recently cloned a non-receptor, calcium-dependent tyrosine kinase (CADTK; also known as PYK2, CAKbeta, and RAFTK) that shares both overall domain structure and 45% amino acid identity with p125(FAK). We have studied the signaling, activation, and potential function of these related enzymes in GN4 rat liver epithelial cells that express CADTK and p125(FAK) at roughly similar levels. p125(FAK) is nearly fully tyrosine-phosphorylated in resting GN4 cells. In contrast, while CADTK is not tyrosine-autophosphorylated in untreated cells, angiotensin II increases CADTK Tyr(P) by 5-10-fold. With regard to signaling, CADTK activation is correlated with stimulation of c-Jun N-terminal kinase and p70(S6K) pathways but not with the stimulation of mitogen-activated protein kinase or p90(RSK). In this report we assessed the contribution of CADTK and p125(FAK) to tyrosine phosphorylation of focal contact proteins. In adherent GN4 cells, the constitutive activity of p125(FAK) was correlated with basal paxillin, tensin, and p130(CAS) tyrosine phosphorylation. A rapid increase in the tyrosine phosphorylation of each protein was detected after treatment with angiotensin II or other agonists that stimulate CADTK; the prolonged 3-4-fold increase in paxillin tyrosine phosphorylation was the most substantial change. In the WB cell line that expresses 3-fold less CADTK than GN4 cell line agonist-dependent paxillin tyrosine phosphorylation is similarly reduced. Immunoprecipitation of CADTK from GN4 cells revealed CADTK. paxillin complexes that persisted in 500 mM NaCl but not in 0.1% SDS cell lysis buffer. The complexes were largely independent of the tyrosine phosphorylation state of either protein. Surprisingly, we did not detect p125(FAK).paxillin complexes in immunoprecipitates using either of two p125(FAK) antibodies. When CADTK and p125(FAK) were transiently overexpressed in 293(T) cells, both enzymes associated with paxillin, but the avidity of CADTK appeared to be greater. In addition, in transfected 293(T) cells, complexes between CADTK and another potential substrate, p130(CAS), were detected. In summary, in GN4 rat liver epithelial cells stimulation of CADTK was highly correlated with paxillin tyrosine phosphorylation; in addition, CADTK but not p125(FAK) was complexed to paxillin at detectable levels. This suggests that agonist-dependent cytoskeletal changes in epithelial cells might proceed, in part, by CADTK-dependent mechanisms.

Amino Acid Sequence↗
PubMed1991

Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated.

To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), AJ-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti-phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at AJ, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated.

Age Factors↗
PubMed1985

Use of tyrosine-containing polymers to characterize the substrate specificity of insulin and other hormone-stimulated tyrosine kinases.

Synthetic copolymers containing tyrosine residues were used to characterize the substrate specificity of the insulin receptor kinase and compare it to tyrosine kinases stimulated by epidermal growth factor, insulin-like growth factor-1 and phorbol ester. In partially purified receptor preparations from eight different tissues insulin best stimulated (highest V) phosphorylation of a random copolymer composed of glutamic and tyrosine residues at a 4:1 ratio (Glu/Tyr, 4:1). The insulin-stimulated phosphorylation of this polymer was highly significant also in receptor preparations from fresh human monocytes, where insulin binding and autophosphorylation were difficult to detect. Other tyrosine-containing polymers Ala/Glu/Lys/Tyr (6:2:5:1) and Glu/Ala/Tyr (6:3:1) were also phosphorylated by the insulin-stimulated kinase but to a lower extent. A tyrosine kinase stimulated by insulin-like growth factor-1, and one stimulated by phorbol ester also best phosphorylated the polymer Glu/Tyr (4:1). The three kinases differed only in their capability to phosphorylate Glu/Ala/Tyr (6:3:1) or Ala/Glu/Lys/Tyr (6:2:5:1). Glu/Tyr (4:1) was a poor substrate for the epidermal growth factor receptor kinase which best phosphorylated the polymer Glu/Ala/Tyr (6:3:1). Three additional polymers: Glu/Tyr (1:1), Glu/Ala/Tyr (1:1:1), and Lys/Tyr (1:1) failed to serve as substrates for all four tyrosine kinases tested. Taken together these findings suggest that. Hormone-sensitive tyrosine kinases have similar yet distinct substrate specificity and are likely to phosphorylate their native substrates on tyrosines adjacent to acidic (glutamic) residues. Tyrosine-containing polymer substrates are highly sensitive and convenient tools to study (hormone-sensitive) tyrosine kinases whose native substrates are unknown or present at low concentrations.

Animals↗
PubMed1993

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.

Animals↗
PubMed1992

Regulation of basal tyrosine phosphorylation of the B cell antigen receptor complex by the protein tyrosine phosphatase, CD45.

Signal transduction via the B cell AgR complex has recently been shown to be dependent on the activation of one or more protein tyrosine kinases. Similarly, it has been found that signal transduction requires the expression of the protein tyrosine phosphatase CD45. Thus, transduction of a signal after AgR cross-linking must involve the coordinate interaction of these two enzymatic activities. It is therefore logical to hypothesize that the competence of the B cell to respond to ligands that bind the AgR may be dependent on the maintenance of an equilibrium between the tyrosine phosphorylation and dephosphorylation of specific signal transduction components. We have demonstrated in the present study that in resting B cells, the basal level of AgR complex tyrosine phosphorylation is regulated by cellular protein tyrosine phosphatases. Treatment of cells with the protein tyrosine phosphatase inhibitor, Na3VO4, resulted in rapid hyperphosphorylation of the receptor complex. Based on this observation, experiments were designed to examine the role of CD45 in regulation of AgR complex phosphorylation. Treatment of B cells with anti-CD45 mAb alone was found to have no effect on cytoskeletal association of CD45 or on its distribution within the membrane. Addition of a secondary cross-linking reagent, however, induced the association of CD45 with the cytoskeleton and caused capping. Subsequent studies demonstrated that increased tyrosine phosphorylation of the mIg-associated proteins MB-1 and B29 could be induced after incubating cells with anti-CD45 mAb and a secondary cross-linker, but not after the addition of anti-CD45 mAb alone. Changes in tyrosine phosphorylation of MB-1 and B29 were found to correlate with the cytoskeletal association of CD45. Interestingly, although cross-linking CD45 induced alterations in its association with the cytoskeleton and in its distribution within the membrane, no significant change in the level of protein tyrosine phosphatase activity could be detected under these conditions. These findings support the possibility that ligand binding to CD45 can induce biochemical and/or physical alterations in the molecule that presumably inhibit its ability to interact with specific substrates in the cell, thereby shifting the established equilibrium between tyrosine-specific phosphorylation and dephosphorylation.

Animals↗
PubMed1997

Cross-talk between phorbol ester-mediated signaling and tyrosine kinase proto-oncogenes. I. Activation of protein kinase C stimulates tyrosine phosphorylation and activation of ErbB2 and ErbB3.

The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), acutely stimulates the tyrosine phosphorylation of proteins of approximately 190, 120, and 70 kDa in the well differentiated Fao rat hepatoma cell line. This phosphorylation is dependent on protein kinase C (PKC) and is abolished by down-regulation of PKC or pretreatment with a PKC inhibitor. Purification of the 190-kDa tyrosine-phosphorylated protein revealed that it consists of both ErbB2 and ErbB3. Following PMA-induced tyrosine phosphorylation, ErbB2 and ErbB3 were able to associate with the SH2 domains of several signaling proteins including the p85alpha subunit of phosphatidylinositol 3-kinase, Syp, and Grb2. The 120-kDa protein phosphorylated in response to PMA consists of at least two proteins: focal adhesion kinase that exhibits a minor increase in tyrosine phosphorylation following treatment with PMA, and a major 120-kDa tyrosine-phosphorylated species in PMA-stimulated Fao cells which as yet is unidentified. Similarly, the 70-kDa tyrosine-phosphorylated protein also appears to represent more than one protein, including paxillin and a second protein of similar mobility which appears to be the major tyrosine phosphorylation in response to PMA. Both ErbB2 and paxillin also exhibit reduced migration on SDS-polyacrylamide gel electrophoresis following PMA treatment, suggesting that they are also phosphorylated on serine/threonine residues. The mobility shift of both of these proteins is abolished by treatment with inhibitors of PKC or mitogen-activated protein kinase/extracellular signal-related kinase kinase. These results suggest a novel mechanism of cross-talk between the serine/threonine kinase PKC and tyrosine phosphorylation pathways. The activation of ErbB2 and ErbB3 that is initiated by PMA may contribute to the tumor promoting activity of these compounds.

3T3 Cells↗
PubMed1982

The conversion of phenylalanine to tyrosine in man. Direct measurement by continuous intravenous tracer infusions of L-[ring-2H5]phenylalanine and L-[1-13C] tyrosine in the postabsorptive state.

Steady state phenylalanine and tyrosine turnover and the rate of conversion of phenylalanine of tyrosine in vivo were determined in 6 healthy postabsorptive adult volunteers. Continuous infusions of tracer amounts of L-[ring-2H5]phenylalanine were determined intravenously for 13-14 hr. After 9-10 hr, a priming dose followed by a continuous infusion of L-[1-13C]tyrosine was added and maintained, along with the [2H5]phenylalanine infusion, for 4 hr. Venous plasma samples were obtained before the initiation of each infusion and every 30 min during the course of the combined [2H5]phenylalanine and [13C]tyrosine infusion for determination of isotopic enrichments of [2H5]phenylalanine, [13C]tyrosine, and [2H4]tyrosine by gas chromatograph-mass spectrometric analysis of the N-trifluoroacetyl-, methyl ester derivatives of the amino acids. Calculated from the observed enrichments, free phenylalanine and tyrosine turnover rates were 36.1 +/- 5.1 mumole . kg-1 . h-1 and 39.8 +/- 3.5 mumole . kg-1 . h-1, respectively. Phenylalanine was converted to tyrosine at the rate of 5.83 +/- 0.59 mumole . kg-1 . h-1, accounting for approximately 16% of either the phenylalanine or the tyrosine flux. The results indicate that the normal basal steady state phenylalanine hydroxylase activity in vivo in man is lower than that obtained from phenylalanine loading studies. This supports the existence of some type of substance activation of the enzyme as reflected in the previously reported exponential relationship between phenylalanine concentration and phenylalanine hydroxylase activity in vitro. The use of continuous simultaneous infusions of tracer amounts of stable isotope-labeled phenylalanine and tyrosine provides a direct means for studying physiological regulation of phenylalanine hydroxylase activity in vivo.

Absorption↗
PubMed1984

Tyrosine and tyrosinate fluorescence of bovine testes calmodulin: calcium and pH dependence.

At physiological pH, bovine testes calmodulin (t-CaM) upon excitation at 278 nm shows typical tyrosine fluorescence at 305 nm and a spectral band characteristic of emission from tyrosinate , at 330-350 nm. In addition, a new band at 312-320 nm appears upon excitation at 288 nm. The pH dependence of the excitation spectra demonstrates that the intense tyrosinate emission at 330-355 nm originates from direct excitation of ground-state tyrosinate . The tyrosinate emission shows complex pH dependence and reaches its highest intensities at pH 7.0 and 8.5, in both apo (Ca free) and holo (Ca saturated) t-CaM. The evidence suggests that the major contribution to the tyrosinate emission at 330-350 nm originates from Tyr-99. In holo t-CaM, the tyrosine emission at 305 nm is quenched at basic pH values and exhibits a sigmoidal pH titration curve with pK(app) 7.0. The tyrosine emission in apo t-CaM is weaker and is almost insensitive to changes in pH. The pH dependence of the emission at 316 nm is the same as the pH dependence of the tyrosine emission in both apo and holo t-CaM. The differences between the fluorescence of apo and holo t-CaM are attributed to a Ca2+-induced shift in the pKa of carboxylic side chains located in the immediate vicinity of the tyrosine residues. The enhancement of the fluorescence by Ca2+ is pH dependent and is maximal at pH 6.5. Above pH 8.0, there is almost no Ca2+ effect on the fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗