3-Fluorotyrosine as a Complementary Probe of Hemoglobin Structure and Dynamics: A (19) F-NMR Study of Synechococcus sp. PCC 7002 GlbN

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 (GlbN) contains three tyrosines (Tyr5, Tyr22, and Tyr53), each of which undergoes a structural rearrangement when the protein binds an exogenous ligand such as cyanide. We explored the use of 3-fluorotyrosine and (19) F-NMR spectroscopy for the characterization of GlbN. Assignment of (19) F resonances in fluorinated GlbN (GlbN*) was achieved with individual Tyr5Phe and Tyr53Phe replacements. We observed marked variations in chemical shift and linewidth reflecting the dependence of structural and dynamic properties on oxidation state, ligation state, and covalent attachment of the heme group. The isoelectronic complexes of ferric GlbN* with cyanide and ferrous GlbN* with carbon monoxide gave contrasting spectra, the latter exhibiting heterogeneity and enhanced internal motions on a microsecond-to-millisecond time scale. The strength of the H-bond network involving Tyr22 (B10) and bound cyanide was tested at high pH. 3-Fluorotyrosine at position 22 had a pK(a) value at least 3 units higher than its intrinsic value, 8.5. In addition, evidence was found for long-range communication among the tyrosine sites. These observations demonstrated the utility of the 3-fluorotyrosine approach to gain insight in hemoglobin properties.     

The importance of three membrane-distal tyrosines in the adaptor protein NTAL/LAB

NTAL (non-T cell activation linker)/LAB (linker for activation of B cells) is a LAT (linker for activation of T cells)-like molecule that is expressed in B cells, mast cells, natural killer cells, and monocytes. Upon engagement of the B cell receptor or Fc receptors, it is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT(-/-) mice. In this study, we utilized various LAB Tyr to Phe mutants to map the phosphorylation and Grb2-binding sites of LAB. We also examined the function of these mutants by investigating their ability to rescue signaling defects in LAT-deficient Jurkat cells and thymocyte development in LAT(-/-) mice. Our results indicated that human LAB was primarily phosphorylated on three membrane-distal tyrosines, Tyr(136), Tyr(193), and Tyr(233). Mutation of these three tyrosines abolished Grb2 binding and LAB function. Our data suggested that these tyrosines are the most important tyrosines for LAB function.     

Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase

Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr(465) and Tyr(381) act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr(465) and Tyr(381) are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr(465) and Tyr(381) decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr(465) is conserved in microsomal EHs. The substitution of analogous Tyr(374) with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.     

Phosphorylation sites in the PDGF receptor with different specificities for binding GAP and PI3 kinase in vivo.

Tyrosine residues have been identified in the human platelet-derived growth factor (PDGF) receptor beta-subunit whose phosphorylation is stimulated by PDGF. These sites are also in vitro autophosphorylation sites. There are a total of three phosphorylation sites in the kinase insert region, tyrosines 740, 751 and 771. Mutagenesis studies show that Tyr740 and 751 are involved in the PDGF-stimulated binding of phosphatidylinositol (PI) 3 kinase, and Tyr771 is required for efficient binding of GAP, the GTPase activator of Ras. The requirement for Tyr751 is only detected at low PDGF receptor levels, suggesting that it increases the affinity of binding of PI3 kinase but is not absolutely required. Small deletions in the kinase insert only 10 residues from Tyr740 and Tyr771 do not significantly reduce binding of PI3 kinase or GAP, indicating that distant sequences are probably unimportant for recognition. The data suggest that the receptor signals to different pathways via different phosphorylated tyrosines, and that certain proteins, such as PI3 kinase, can recognize two phosphorylated tyrosines in a single receptor.     

Identification of reelin-induced sites of tyrosyl phosphorylation on disabled 1

The study of mice with spontaneous and targeted mutations has uncovered a signaling pathway that controls neuronal positioning during mammalian brain development. Mice with disruptions in reelin, dab1, or both vldlr and apoER2 are ataxic, and they exhibit severe lamination defects within several brain structures. Reelin is a secreted extracellular protein that binds to the very low density lipoprotein receptor and the apolipoprotein E receptor 2 on the surface of neurons. Disabled-1 (Dab1), an intracellular adapter protein containing a PTB (phosphotyrosine binding) domain, is tyrosyl-phosphorylated during embryogenesis, but it accumulates in a hypophosphorylated form in mice lacking Reelin or both very low density lipoprotein receptor and apolipoprotein E receptor 2. Dab1 is rapidly phosphorylated when neurons isolated from embryonic brains are stimulated with Reelin, and several tyrosines have been implicated in this response. Mice with phenylalanine substitutions of all five tyrosines (Tyr(185), Tyr(198), Tyr(200), Tyr(220), and Tyr(232)) exhibit a reeler phenotype, implying that tyrosine phosphorylation is critical for Dab1 function. Here we report that, although Src can phosphorylate all five tyrosines in vitro, Tyr(198) and Tyr(220) represent the major sites of Reelin-induced Dab1 phosphorylation in embryonic neurons.     

Genetic engineering of Escherichia coli inorganic pyrophosphatase. Tyr55 and Tyr141 are important for the structural integrity.

Two tyrosines are supposed to be essential for the activity and to participate in the stabilization of Escherichia coli inorganic pyrophosphatase (PPiase) against heat denaturation [Samejima, T., Tamagawa, Y., Kondo, Y., Hachimori, A., Kaji, H., Takeda, A. and Shiroya, Y. (1988) J. Biochem. (Tokyo) 103, 766-772]. To locate these two tyrosines in the amino acid sequence, we substituted all the eight tyrosines of E. coli PPiase with phenylalanine and studied the properties of these YF mutant PPiases. Interestingly, substitution of the tyrosines (Tyr51, Tyr55 and Tyr141) conserved with the amino acid sequence of yeast PPiase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K. and Cooperman, B. (1990) Biochim. Biophys. Acta 1038, 338-345] exerted the most drastic effects on the structure and activity of E. coli PPiase. PPiase variants YF51, YF55 and YF141 had 64%, 7% and 22% of the wild-type PPiase activity, respectively. Furthermore, PPiase variant YF141 had an increased sensitivity to heat denaturation, whereas mutant PPiase YF55 displayed a profound conformational change, as demonstrated by the binding of the fluorescent dye 9-(diethylamino)-5H-benzo(alpha) phenoxazine-5-one (Nile red) that monitors the hydrophobicity of protein surfaces. None of the tyrosines of E. coli PPiase seem to be essential for catalysis, but Tyr55 and Tyr141 are important for the structural integrity of E. coli PPiase.     

Site-Directed Mutagenesis of BmK AGP-SYPU1: The Role of Two Conserved Tyr (Tyr5 and Tyr42) in Analgesic Activity

In this study, the role of two conversed tyrosines (Tyr5 and Tyr42) from the scorpion toxin BmK AGP-SYPU1 was investigated with an effective Escherichia coli expression system. Site-directed mutagenesis was used to individually substitute Tyr5 and Tyr42 with hydrophobic or hydrophilic amino acids, and the extent to which these scorpion toxin BmK AGP-SYPU1 tyrosines contribute to analgesic activity was evaluated. The results of the mouse-twisting test showed that Tyr5 and Tyr42 are associated with the analgesic activity of the toxin because the analgesic activities of Y5F and Y42F were significantly increased compared with the rBmK AGP-SYPU1; however, the Y5W had decreased activity. The results of molecular simulation reveal the following: (1) for analgesic activity, the core domain of the scorpion toxin BmK AGP-SYPU1 is key and (2) for pharmacological function, Tyr42 is most likely involved when the core domain conformation is altered. These studies identify a new relationship between the structure and analgesic activity of the scorpion toxin BmK AGP-SYPU1 and are significant for further research and the application of analgesic peptides.     

The role of tyrosine residues in fibroblast growth factor receptor 1 signaling in PC12 cells. Systematic site-directed mutagenesis in the endodomain.

To assess the contribution of the intracellular domain tyrosine residues to the signaling capacity of fibroblast growth factor receptor 1 (FGFR1), stably transfected chimeras bearing the ectodomain of the platelet-derived growth factor receptor (PDGFR) and the endodomain of FGFR1 were systematically altered by a tyrosine to phenylalanine bloc and individual conversions. The 15 tyrosine residues of the endodomain of this construct (PFR1) were divided into four linear segments (labeled A, B, C, and D) that contained 4, 4, 2, and 5 tyrosine residues, respectively. When stimulated by platelet-derived growth factor, derivatives in which the A, B, or A + B blocs of tyrosines were mutated were about two-thirds as active as the unmodified chimera at 48 h but achieved full activity by 96 h in a neurite outgrowth assay in transfected PC12 cells. Elimination of only the two activation loop tyrosines (C bloc) also inactivated the receptor. All derivatives in which 4 (or 5) of the D bloc tyrosines were mutated were inactive in producing differentiation but showed low levels of kinase activity in in vitro assays. Derivatives in which 1, 2, or 3 tyrosines of the D bloc in different combinations were systematically changed demonstrated that 2 residues (Tyr(677) and Tyr(701), using hFGFR1 numbering) were essential for bioactivity, but the remaining 3 residues, including Tyr(766), the previously identified site for phospholipase C gamma (PLC gamma) activation, were not. Differentiation activity was paralleled by the activation (phosphorylation) of FRS2, SOS, and ERK1/2. PLC gamma activity was dependent on the presence of Tyr(766) but also required Tyr(677) and/or Tyr(701). Although fully active chimeras did not require PLC gamma, the responses of chimeras showing reduced activation of FRS2 were significantly enhanced by this activity. These results establish that PFR1 does not utilize any tyrosine residues, phosphorylated or not, to activate FRS2. However, it does require Tyr(677) and/or Tyr(701), which may function to stabilize the active conformation directly or indirectly.     

Coordinated activation of autophosphorylation sites in the RET receptor tyrosine kinase: importance of tyrosine 1062 for GDNF mediated neuronal differentiation and survival.

The catalytic and signaling activities of RET, a tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF), are controlled by the autophosphorylation of several tyrosine residues in the RET cytoplasmic domain. To analyze the phosphorylation state of individual tyrosines, we generated antibodies recognizing specific phosphotyrosine sites involved in the catalytic (Tyr(905)) and downstream signaling (Tyr(1015), Tyr(1062), and Tyr(1096)) activities of this receptor. Stimulation with GDNF induced coordinated phosphorylation of the 4 tyrosine residues in neuronal cell lines and in primary cultures of sympathetic neurons isolated from rat superior cervical ganglia. Neurturin and artemin, two other members of the GDNF ligand family, also induced synchronized phosphorylation of RET tyrosines with kinetics comparable to those observed with GDNF. Tyrosine phosphorylation was maximal 15 min after ligand stimulation, decaying thereafter with similar kinetics in all 4 residues. Co-stimulation with a soluble form of the GFRalpha1 co-receptor potentiated ligand-dependent phosphorylation of different intracellular tyrosines to a similar extent and increased the survival of superior cervical ganglion neurons compared with treatment with GDNF alone. In vivo, high levels of phosphorylated Tyr(905), Tyr(1015), and Tyr(1062) were detected in embryonic mouse dorsal root ganglia, with a sharp decline at early postnatal stages. Protein transduction of anti-Tyr(P)(1062) antibodies into cultured cells reduced activation of MAPKs ERK1 and ERK2 and the AKT kinase in response to GDNF and diminished GDNF-dependent neuronal differentiation and survival of embryonic sensory neurons from the nodose ganglion. These results demonstrate synchronized utilization of individual RET tyrosine residues in neurons in vivo and reveal an important role for RET Tyr(1062) in mediating neuronal survival by GDNF.     

Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA

We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.     

3-Nitrotyrosine modification of SERCA2a in the aging heart: a distinct signature of the cellular redox environment

In the aging heart, decreased rates of calcium transport mediated by the SERCA2a isoform of the sarcoplasmic reticulum (SR) Ca-ATPase are responsible for the slower sequestration of cytosolic calcium and consequent prolonged muscle relaxation times. We report a 60% decrease in Ca-ATPase activity in the senescent Fischer 344 rat heart relative to that of young adult hearts; this functional decrease can be attributed, in part, to the 18% lower abundance of SERCA2a protein. Here, we show that the additional loss of activity is a result of increased 3-nitrotyrosine modification of the Ca-ATPase. Age-dependent increases in nitration of cardiac SERCA2a are identified using multiple analytical methods. In the young (adult) heart 1 molar equivalent of nitrotyrosine is distributed over at least five tyrosines within the Ca-ATPase, identified as Tyr(122), Tyr(130), Tyr(497), Tyr(586), and Tyr(990). In the senescent heart, the stoichiometry of nitration increases by more than two nitrotyrosines per Ca-ATPase, coinciding with the appearance of nitrated Tyr(294), Tyr(295), and Tyr(753). The abundant recovery of native analogues for each of the nitrated peptides indicates partial modification of multiple tyrosines within cardiac SERCA2a. In contrast, within skeletal muscle SERCA2a, a homogeneous pattern of nitration appears, with full site (1 mol/mol) nitration of Tyr(753), in young, with additional nitration of Tyr(294) and Tyr(295), in senescent muscle. The nitration of these latter vicinal sites correlates with diminished transport function in both striated muscle types, suggesting that these sites provide a mechanism for downregulation of ATP utilization by the Ca-ATPase under conditions of nitrative stress.     

Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling

The linker for activation of T cells (LAT) is a critical adaptor molecule required for T cell antigen receptor (TCR)-mediated signaling and thymocyte development. Upon T cell activation, LAT becomes highly phosphorylated on tyrosine residues, and Grb2, Gads, and phospholipase C (PLC)-gamma1 bind LAT via Src homology-2 domains. In LAT-deficient mutant Jurkat cells, TCR engagement fails to induce ERK activation, Ca(2+) flux, and activation of AP-1 and NF-AT. We mapped the tyrosine residues in LAT responsible for interaction with these specific signaling molecules by expressing LAT mutants with tyrosine to phenylalanine mutations in LAT-deficient cells. Our results showed that three distal tyrosines, Tyr(171), Tyr(191), and Tyr(226), are responsible for Grb2-binding; Tyr(171), and Tyr(191), but not Tyr(226), are necessary for Gads binding. Mutation of Tyr(132) alone abolished PLC-gamma1 binding. Mutation of all three distal tyrosines also abolished PLC-gamma1 binding, suggesting there might be multiple binding sites for PLC-gamma1. Mutation of Tyr(132) affected calcium flux and blocked Erk and NF-AT activation. Since Grb2 binding is not affected by this mutation, these results strongly suggest that PLC-gamma activation regulates Ras activation in these cells. Mutation of individual Grb2 binding sites had no functional effect, but mutation of two or three of these sites, in combination, also affected Erk and NF-AT activation.     

The long and short isoforms of Ret function as independent signaling complexes

Ret, the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family ligands (GFLs), is alternatively spliced to yield at least two isoforms, Ret9 and Ret51, which differ only in their C termini. To identify tyrosines in Ret that are autophosphorylation sites in neurons, we generated antibodies specific to phosphorylated Y905Ret, Y1015Ret, Y1062Ret, and Y1096Ret, all of which are autophosphorylated in cell lines. All four of these tyrosines in Ret became phosphorylated rapidly upon activation by GFLs in sympathetic neurons. These tyrosines remained phosphorylated in sympathetic neurons in the continued presence of GFLs, albeit at a lower level than immediately after GFL treatment. Comparison of GFL activation of Ret9 and Ret51 revealed that phosphorylation of Tyr(905) and Tyr(1062) was greater and more sustained in Ret9 as compared with Ret51. In contrast, Tyr(1015) was more highly phosphorylated over time in Ret51 than in Ret9. Surprisingly, Ret9 and Ret51 did not associate with each other in sympathetic neurons after glial cell line-derived neurotrophic factor stimulation, even though they share identical extracellular domains. Furthermore, the signaling complex associated with Ret9 was markedly different from the Ret51-associated signaling complex. Taken together, these data provide a biochemical basis for the dramatic functional differences between Ret9 and Ret 51 in vivo.     

Phosphorylation of Tyr342 in the linker region of Syk is critical for Fc epsilon RI signaling in mast cells

The linker region of Syk and ZAP70 tyrosine kinases plays an important role in regulating their function. There are three conserved tyrosines in this linker region; Tyr317 of Syk and its equivalent residue in ZAP70 were previously shown to negatively regulate the function of Syk and ZAP70. Here we studied the roles of the other two tyrosines, Tyr342 and Tyr346 of Syk, in Fc epsilon RI-mediated signaling. Antigen stimulation resulted in Tyr342 phosphorylation in mast cells. Syk with Y342F mutation failed to reconstitute Fc epsilon RI-initiated histamine release. In the Syk Y342F-expressing cells there was dramatically impaired receptor-induced phosphorylation of multiple signaling molecules, including LAT, SLP-76, phospholipase C-gamma2, but not Vav. Compared to wild-type Syk, Y342F Syk had decreased binding to phosphorylated immunoreceptor tyrosine-based activation motifs and reduced kinase activity. Surprisingly, mutation of Tyr346 had much less effect on Fc epsilon RI-dependent mast cell degranulation. An anti-Syk-phospho-346 tyrosine antibody indicated that antigen stimulation induced only a very minor increase in the phosphorylation of this tyrosine. Therefore, Tyr342, but not Tyr346, is critical for regulating Syk in mast cells and the function of these tyrosines in immune receptor signaling appears to be different from what has been previously reported for the equivalent residues of ZAP70.     

The cytoplasmic domain of the polymeric immunoglobulin receptor contains two internalization signals that are distinct from its basolateral sorting signal.

The C-terminal cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) contains two tyrosine residues, Tyr668 and Tyr734. Previous work identifying Tyr734 as a critical residue in the endocytosis of the pIgR in Madin-Darby canine kidney (MDCK) cells also suggested that a second functional internalization signal was present (Breitfeld, P. P., Casanova, J. E., McKinnon, W. C., and Mostov, K. E. (1990) J. Biol. Chem. 265, 13750-13757). To test this hypothesis, Tyr668 and Tyr734 were mutated singly or together by oligonucleotide-directed mutagenesis of pIgR cDNA, and the mutants were expressed in MDCK cells. The amount of ligand internalized within 5 min from the basolateral membrane by the pIgR in which cytoplasmic tyrosines were mutated separately to Cys668 or Ser734 or together to Cys668, Ser734 was 58, 39, and 20%, respectively, of the internalized by the wild-type pIgR. The cytoplasmic and transmembrane domains of the pIgR, when joined to the external domain of the influenza virus hemagglutinin, retained the capacity to mediate rapid internalization. As with the full-length pIgR, mutation of either tyrosine in the chimera resulted in impairment of endocytosis, with mutation of Tyr734 having a significantly greater effect than mutation on Tyr668 on the initial rate of endocytosis (3 and 44% of control values, respectively). However, unlike the full-length pIgR, mutation of both tyrosines together in the chimera did not reduce internalization further. The two tyrosines in the cytoplasmic sequence of the pIgR, although widely separated in the linear amino acid sequence, both contribute to internalization of the protein, suggesting that both can function as internalization signals. In addition, the correlation between endocytosis and basolateral targeting of the pIgR in MDCK cells was investigated. Neither tyrosine of the cytoplasmic domain was necessary for basolateral targeting of the pIgR.     

Modulation of Lck function through multisite docking to T cell-specific adapter protein

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr(280), Tyr(290), and Tyr(305) were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)(280) and Tyr(P)(290) phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)(505) phosphopeptide, whereas the TSAd Tyr(P)(305) peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20-30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is approximately 1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.     

Tyrosine phosphorylation of the integrin beta 3 subunit regulates beta 3 cleavage by calpain

Outside-in signaling of beta(3) integrins induces and requires phosphorylation at tyrosine 747 (Tyr(747)) and tyrosine 759 (Tyr(759)) of the beta(3) subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of beta(3) cytoplasmic domain. Here we show that beta(3) tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes beta(3) to calpain cleavage. Furthermore, phosphorylation at Tyr(747) and Tyr(759) of beta(3) in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr(759) is associated with calpain cleavage at Tyr(759). Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the beta(3) cytoplasmic domain.     

Vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans: FTIR and electrochemical studies on Tyr-D4-labeled and on Tyr280His and Tyr35Phe mutant enzymes

A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer. Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines. Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes. The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1). A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine. Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra. Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276. The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine. The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding. The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center. Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected. In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed. Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B). Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV. Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase.     

Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.     

Phosphorylation sites at the C-terminus of the platelet-derived growth factor receptor bind phospholipase C gamma 1.

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.