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PubMed1997

SHP1 and SHP2 protein-tyrosine phosphatases associate with betac after interleukin-3-induced receptor tyrosine phosphorylation. Identification of potential binding sites and substrates.

The cytoplasmic tyrosine phosphatases, SHP1 and SHP2, are implicated in the control of cellular proliferation and survival. Here we demonstrate that both SHP1 and SHP2 associate with the betac subunit of the human interleukin-3 (IL-3) receptor following IL-3 stimulation and that the src homology region 2 (SH2) domains of these phosphatases mediate this interaction. Sequential immunoprecipitation analyses suggest this interaction is direct. Competition studies, using phosphotyrosine-containing peptides based on sequences surrounding key tyrosine residues within betac, suggest that phosphorylation of tyrosine 612 is the key event mediating the association of betac with SHP1 and SHP2. However, inhibition of SHP2 binding to betac, did not prevent tyrosine phosphorylation of SHP2. Interestingly, this same phosphopeptide served as a substrate for the tyrosine phosphatase activity of both SHP1 and SHP2. Binding of these protein-tyrosine phosphatases to the IL-3 receptor may regulate IL-3 signal transduction pathways, both through their catalytic activity and through the recruitment of other molecules to the receptor complex.

Binding Sites↗

PubMed2001

A highly acidic tyrosine 9 and a normally titrating tyrosine 212 contribute to the catalytic mechanism of human glutathione transferase A4-4.

Human glutathione transferase A4-4 is an enzyme catalyzing the detoxication of intracellularly produced electrophiles such as 4-hydroxynonenal and other alkenal products of lipid peroxidation. Two tyrosines in the active site of the enzyme have been studied with help of UV difference spectroscopy and site-directed mutagenesis. The titration curve of GST A4-4 shows a pK(a) of 6.7 attributable to tyrosine 9, which in the Y212F mutant was shifted to pK(a) 7.1. In both cases the pK(a) was independent of the absence or presence of GSH. Thus, the active-site tyrosine 9 of this isoenzyme is more than one unit more acidic than the corresponding tyrosine of other Alpha class glutathione transferases. The tyrosines remaining in the Y9F mutant titrate like free tyrosine with pK(a) values > or = 10. A mechanism involving a tyrosine-9-bound water molecule acting as a proton shuttle is proposed for the Michael additions catalyzed by GST A4-4.

Base Sequence↗

PubMed1986

Kinetic properties of the insulin receptor tyrosine protein kinase: activation through an insulin-stimulated tyrosine-specific, intramolecular autophosphorylation.

The insulin receptor is an insulin-activated, tyrosine-specific protein kinase. Previous studies have shown that autophosphorylation of tyrosine residues on the Mr 95,000 is associated with an activation of the protein kinase activity toward exogenous protein substrates. We have employed the highly purified insulin receptor, immobilized on insulin-Sepharose or eluted in an active form, to define the metal/ATP requirements for kinase activation, the relationship of receptor autophosphorylation to activation, and the kinetic properties of the autophosphorylated, activated receptor kinase. Prior incubation of the immobilized receptor with 2 mM ATP, 10 mM Mg (or 10 mM Mn), followed by removal of these reactants, served to abolish the upward curvilinearity in the rate of histone 2b (tyrosine) phosphorylation measured subsequently. This treatment also markedly increased the rate of histone 2b phosphorylation as compared to that observed with the unmodified, immobilized receptor, as estimated under conditions that per se minimized further activation. The extents of maximal activation of receptor histone 2b (tyrosine) kinase obtained on preincubation with MgATP or MnATP are identical; however, the affinity of the receptor for MnATP is approximately 10-fold higher than that for MgATP. The higher affinity of the receptor for MnATP is observed for both autophosphorylation/autoactivation and histone 2b tyrosine kinase activity (Km MnATP approximately 0.01 mM; Km MgATP approximately 0.1 mM). Autophosphorylation/autoactivation per se does not significantly alter the apparent affinity for MeATP (or protein substrate, as previously reported) but increases Vmax. Activation of receptor histone 2b (tyrosine) kinase is due to tyrosine-specific autophosphorylation of the Mr 95,000 (beta) subunit; thus the extent of total 32P incorporation into the beta subunit correlates precisely with the extent of kinase activation, both over time and at a wide variety of Me2+ ATP concentrations. Sequential treatment of the autophosphorylated receptor with elastase and trypsin yields a single, basically charged 32P-peptide, Mr less than 2000. The functional properties of the unphosphorylated and fully phosphorylated receptor were compared after elution from insulin-Sepharose. The insulin binding characteristics of the two forms of the receptor were indistinguishable; the kinase properties differed greatly; whereas the histone 2b activity of the unphosphorylated receptor was low in the basal state, and activated 10-fold by insulin, the fully autophosphorylated receptor exhibits maximal histone 2b kinase in the basal state and is unaffected by insulin addition.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1985

Chemiluminescence in L-tyrosine-H2O2-horseradish peroxidase system: possible formation of tyrosine cation radical.

Tyrosine-H2O2-horseradish peroxidase system at pH 7.4 emitted light in visible region. Phenolic compounds other than tyrosine were also emissive, whereas methoxy phenylalanine and phenyl compounds were not, in H2O2-peroxidase systems. Chemiluminescence spectrum of tyrosine of tyrosine-H2O2-horseradish peroxidase system showed two prominent peaks at 478 nm and 500 nm (Luminescence 1) and additional two or three peaks near 550 and 610 nm (Luminescence 2). Luminescence 1 is quite similar to the phosphorescence originated from an excited tyrosine in triplet state, while Luminescence 2 is quite similar to the phosphorescence originated from an indole in triplet state. Possible formation of tyrosine cation radical (a precursor of the excited tyrosine) and indole cation radical in the enzyme protein (a precursor of the excited tryptophan residue) were discussed.

Free Radicals↗

PubMed1995

Effect of chronic protein ingestion on rat central nervous system tyrosine levels and in vivo tyrosine hydroxylation rate.

Groups of young adult male rats ingested ad libitum for two weeks diets containing 2%, 5%, 10% or 20% protein. They were then killed early in the daily dark period, 30 min after receiving m-hydroxybenzylhydrazine (100 mg/kg i.p., to allow measurement of in vivo tyrosine hydroxylation rate). Tyrosine levels in retina, hypothalamus and cerebral cortex were lowest in rats ingesting 2% protein, and rose progressively to a plateau in rats ingesting 10% protein. No consistent increase occurred between 10% and 20% protein. Tyrosine hydroxylation rate in retina and hypothalamus, but not prefrontal cortex rose in parallel with the increments in tyrosine level between 2% and 10% protein, showing no further increase between 10% and 20% protein. These changes were found to be related to the level of protein, not caloric intake. And, the low rates of hydroxylation observed in rats consuming low protein (2%) were found not to be attributable to low endogenous tyrosine hydroxylase activity. Together, the results indicate that the differences in tyrosine levels in some regions of the central nervous system (retina, hypothalamus) produced by chronic variations in protein intake may influence directly the rate of tyrosine hydroxylation, and thus perhaps the overall rate of catecholamine synthesis. This relationship might provide the hypothalamus (a region important in food intake control) with a signal for monitoring and ultimately modulating the chronic level of protein intake.

Amino Acids↗

PubMed1996

Tyrosine phosphorylation blocks tyrosine free radical formation and, hence, the hormonogenic iodination reaction.

It is known that a tyrosine free radical is produced during the hormonogenic iodination reaction of tyrosine residues on thyroglobulin. In the hormonogenic region of thyroglobulin, phosphorylated tyrosine residues have been detected. Using an vitro tyrosine iodinating system we report that the hormonogenic reaction cannot go off if tyrosine becomes phosphorylated. Enzymatic dephosphorylation of the modified amino acid restored the ability of the molecule to become iodinated. Considering the mechanism of the tyrosine free radical formation, these observations are due to the inability of the phosphorylated amino acid to form a free radical. Our data may suggest a putative regulatory mechanism in thyroid hormone synthesis by phosphorylation of hormonogenic tyrosine residues on thyroglobulin.

Dimerization↗

PubMed1998

Stimulation of G-protein coupled receptors in vascular smooth muscle cells induces tyrosine kinase dependent increases in calcium without tyrosine phosphorylation of phospholipase C gamma-1.

It is often believed that increases in intracellular Ca2+ ([Ca2+]i) resulting from stimulation of G-protein coupled receptors in vascular smooth muscle cells (VSMC) require activation of the beta1 isoform of phospholipase C (PLC). However, recent studies showed that rat aortic VSMC do not express PLC beta-1 and that stimulation with angiotensin-II induces tyrosine kinase dependent increases in [Ca2+]i and tyrosine phosphorylation of PLC gamma-1. Whether this pathway is activated by other vasoactive agents that stimulate G-protein coupled receptors is unknown. Here, we show that A10 VSMC express PLC beta-2, PLC beta-3, PLC delta-1, and PLC gamma-1. The cells also expressed Galpha(q/11). However, neither PLC beta-1 nor PLC beta-4 was detected. Stimulation with angiotensin-II, vasopressin, serotonin, or endothelin induced tyrosine kinase dependent increases in [Ca2+]i. However, tyrosine phosphorylation of PLC gamma-1 did not occur. In contrast, stimulation with platelet derived growth factor increased [Ca2+]i and tyrosine phosphorylation of PLC gamma-1. The results show that tyrosine phosphorylation of PLC gamma-1 is not required for tyrosine kinase dependent increases in [Ca2+]i resulting from stimulation of diverse G-protein coupled receptors in VSMC.

Angiotensin II↗

PubMed2000

Mutation of tyrosine 318 (Y318F) in the delta-opioid receptor attenuates tyrosine phosphorylation, agonist-dependent receptor internalization, and mitogen-activated protein kinase activation.

Opioid receptors are known for their ability to activate diverse second messenger systems. Previously, we showed that selective delta-opioid agonists were able to induce the rapid tyrosine phosphorylation of delta-opioid receptors (delta-ORs) through Src. Src-dependent tyrosine phosphorylation of delta-ORs appears to be important for activation of the mitogen-activated protein kinase cascade and for receptor sequestration into clathrin-coated endosomes, as the Src antagonist, PP1, inhibited both. In an attempt to clarify the role of tyrosine phosphorylation in delta-OR signalling and regulation, we constructed a mutant receptor in which the tyrosine located in the conserved NPXXY motif of the C-terminus was replaced by a phenylalanine (Y318F-delta-OR). Mutation of Y318 resulted in a receptor that was comparable to the wild type in its expression level in HEK-293 cells and in its affinity for opioid ligands. Both receptors showed effective coupling to G proteins and were capable of inhibiting forskolin-stimulated cAMP accumulation with similar potencies. However, the mutant receptor was able to stimulate (35)S-GTPgammaS binding with a lower EC(50) than the wild type receptor. The stimulation of tyrosine phosphorylation in delta-ORs by [D-Thr(2)]-Leu-enkephalin-Thr (DTLET) was significantly less in cells expressing the Y318F-delta-OR than in cells expressing the wild type. In addition, both rapid receptor internalization and down-regulation were markedly attenuated in the mutant. Finally, the mutant receptor was unable to induce a robust activation of the MAPK pathway, suggesting that tyrosine phosphorylation of the delta-OR protein is important for this signalling pathway. These findings implicate tyrosine phosphorylation of Y318 in receptor signalling and agonist-mediated regulation.

Amino Acid Sequence↗

PubMed1987

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosines-26 and -64.

Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR570----K630 or BR570----M412 transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K630 and the deprotonation of a tyrosine by M412 [Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M412 as concluded previously [Scherrer, P., & Stoeckenius, W. (1985) Biochemistry 24, 7733-7740]. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm-1 in the BR570----M412 FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue [Engelhard, M., Gerwert, K., Hess, B., Kreutz, W., & Siebert, F. (1985) Biochemistry 24, 400-407; Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group.

Bacteriorhodopsins↗

PubMed1991

Polarized secretion of tyrosine-sulphated proteins and free tyrosine O-sulphate by filter-grown Madin-Darby canine kidney (MDCK) cells.

Filter-grown Madin-Darby canine kidney (MDCK) cells labelled for 24 h with [35S]sulphate were found to secrete macromolecules [35S]sulphated on their carbohydrate moieties predominantly into the basolateral medium, whereas the tyrosine-[35S]sulphated proteins synthesized were predominantly secreted into the apical medium. In contrast with the predominant apical secretin of tyrosine-[35S]sulphated proteins, the free tyrosine O-[35S]sulphate (Tyr[35S]) was released mostly into the basolateral medium. A time-lapse study using prelabelled MDCK cells incubated in fresh medium revealed that, during the 48 h time course monitored, the release of tyrosine-[35S]sulphated proteins into the apical medium was faster and quantitatively greater than that into the basolateral medium. During the same time there was a concomitant release, predominantly into the basolateral medium, of the free Tyr[35S] derived from the degradation of tyrosine-[35S]sulphated proteins. An endocytotic degradation experiment was performed to demonstrate the endocytosis of tyrosine-sulphated proteins and their degradation to generate free TyrS. It was found that free Tyr[35S] was generated and released when an apically secreted (or basolaterally secreted) tyrosine-[35S]sulphated protein preparation was added to the apical medium (or the basolateral medium) of unlabelled filter-grown MDCK cells. In both cases, the free Tyr[35S] generated was predominantly released into the basolateral medium similar to the results obtained in the time-lapse study.

Animals↗

PubMed1996

Association of Lyn tyrosine kinase with the nuclear matrix and cell-cycle-dependent changes in matrix-associated tyrosine kinase activity.

The nuclear matrix isolated from HeLa cells and Rat2 fibroblasts harbors tyrosine kinase and tyrosine phosphatase activities. Polypeptides of 53, 56 and 60 kDa, associated with this subnuclear structure, were phosphorylated at tyrosine in vivo. By immunoblot and immunolabelling experiments, we identified one of the nuclear-matrix-associated tyrosine kinases as Lyn, a Src family member. Lyn was distributed as foci throughout the matrix. The p56 and p53 isoforms of Lyn remained firmly associated with the nuclear matrix after a variety of matrix preparation procedures, and were not detectable in the chromatin fraction of the nucleus. The tyrosine kinase activity associated with the nuclear matrix showed cell-cycle-dependent changes, maximum activity being observed at the G1/S transition phase. Polyoma-virus-transformed rat fibroblast cells showed sixfold higher tyrosine kinase activity in the nuclear matrix preparations compared to that in untransformed cells. These observations are consistent with the suggestion that tyrosine kinase activity associated with the nuclear matrix may be an important determinant of cellular proliferation.

Animals↗

PubMedDate not supplied

Tyrosine and tyrosinate fluorescence of S-100b. A time-resolved nanosecond fluorescence study. The effect of pH, Ca(II), and Zn(II).

The properties of the tyrosine and tyrosinate emissions from brain S-100b have been studied by nanosecond time-resolved fluorescence at emission wavelengths in the range 305 to 365 nm. The effect of pH on the fluorescence has been studied at pH 6.5, 7.5, and 8.5 for the Ca(II) apo and holo forms of the protein, and for the apo and holo forms in the presence and absence of Zn(II) at pH 7.5. The fluorescence decay is biexponential at pH 8.5 and triexponential at pH 6.5 and 7.5. The three components of the decay have wavelength and metal ion dependent lifetimes in the ranges 0.06 to 1.05 ns, 0.49 to 3.76 ns, and 3.60 to 14.5 ns. The observation of a long lifetime component at wavelengths characteristic of emission from tyrosinate suggests that in class A proteins this may be a useful diagnostic of the environment of tyrosine in their native structures. The time-resolved emission spectra provide evidence for efficient, subnanosecond protolysis of the excited state of the single tyrosine (Tyr17) under all conditions studied except in 6 M guanidium chloride in which the protein shows only emission from tyrosine (lambda em 305 nm), suggesting that the tyrosinate emission is a property of the tertiary structure of the native protein. The Zn(II)-dependence of the fluorescence is fully consistent with the earlier suggestion that Tyr17 is near the Zn(II) binding site and remote from the high affinity Ca(II) binding site.

Animals↗

PubMed1999

Comparison of the effects of alpha-methyl-p-tyrosine and a tyrosine-free amino acid load on extracellular noradrenaline in the rat hippocampus in vivo.

Peripheral administration of an amino acid load lacking tyrosine and its precursor, phenylalanine, causes a lowering of central tyrosine levels. The aim of the present study was to examine the effects of tyrosine depletion on extracellular noradrenaline using microdialysis. Extracellular noradrenaline was measured in hippocampus of the anaesthetized rat under both baseline conditions (with reuptake inhibitor, desipramine, in the perfusion medium) and following administration of the alpha2-adrenoreceptor antagonist, idazoxan. The tyrosine free amino acid load did not alter either baseline noradrenaline or the twofold rise in noradrenaline evoked by idazoxan compared with saline controls. In contrast, the catecholamine synthesis inhibitor, alpha-methyl-p-tyrosine, caused a marked reduction in baseline extracellular noradrenaline and abolished the rise induced by idazoxan. In conclusion, the present data indicate that under the conditions used, a tyrosine-free amino acid mixture may not be an effective means to interfere with central noradrenaline function. This contrasts with recent findings demonstrating that the tyrosine-depletion approach can be used to decrease presynaptic dopamine function.

Adrenergic alpha-2 Receptor Antagonists↗

PubMed1992

Mutagenesis of T cell antigen receptor zeta chain tyrosine residues. Effects on tyrosine phosphorylation and lymphokine production.

Occupancy of the T cell antigen receptor triggers a complex set of events that culminate in cellular activation. It is clear that tyrosine kinases play important roles in this process. The zeta subunit of the T cell antigen receptor is a 16-kDa transmembrane structure that exists primarily as a disulfide-linked homodimer. On receptor activation, a subset of zeta molecules undergo tyrosine phosphorylation. To evaluate this process and the role of zeta phosphorylation in T cell activation, site-specific mutagenesis of the intracytoplasmic tyrosines of zeta has been carried out. Analysis of cells expressing these mutant zeta subunits demonstrated that multiple tyrosines underwent phosphorylation in response to receptor engagement, and that the four most carboxyl tyrosines were most crucial to this process. Despite abnormalities in phosphorylation induced by the mutations, lymphokine production in these transfectants was unaffected. Hence, although zeta is a prominent substrate for a receptor-activated tyrosine kinase, neither the mutation of individual tyrosines nor the alteration of the phosphorylation state of the molecule substantively affected the coupling of T cell receptor activation to lymphokine production. These findings raise questions regarding the role of zeta phosphorylation in T cell activation.

Animals↗

PubMed1994

Tyrosine phosphorylation of the alpha and beta subunits of the type I interferon receptor. Interferon-beta selectively induces tyrosine phosphorylation of an alpha subunit-associated protein.

We studied the phosphorylation of the alpha and beta subunits of the Type I interferon (IFN) receptor induced by Type I IFNs in the human U-266 and MOLT-4 cell lines. Both IFN-alpha and IFN-beta induced tyrosine phosphorylation of the beta subunit of the receptor. The Type I IFN-induced tyrosine phosphorylation of the beta subunit was rapid and transient, being detectable within 1 min of Type I IFN treatment and gradually diminishing to almost base-line levels by 60 min. All Type I IFNs studied were found to induce tyrosine phosphorylation of the alpha subunit of the Type I IFN receptor, the p135tyk2 and JAK-1 tyrosine kinases, and the ISGF3 alpha components. Interestingly, IFN-beta, but not IFN-alpha or IFN-omega, induced tyrosine phosphorylation of an alpha subunit-associated protein with an apparent molecular mass of approximately 100 kDa (p100). These data suggest the existence of a common signaling pathway(s) for Type I IFNs involving the alpha and beta subunits of the receptor, the tyrosine kinases p135tyk2 and JAK-1, and the ISGF3 alpha components. However, differences between the signaling pathways of different Type I IFNs exist, as suggested by tyrosine phosphorylation of an alpha subunit-associated protein only in response to IFN-beta.

Humans↗

PubMed1996

Regulation of tyrosine phosphorylation and protein tyrosine phosphatases during oligodendrocyte differentiation.

Tyrosine phosphorylation is key to the differentiation of oligodendrocytes, as the FGF2 and PDGF receptor tyrosine kinases are known to mediate the proliferation and maintenance of their precursors. Marked changes in the levels and localization of tyrosine-phosphorylated proteins were found to accompany differentiation in the CG4 rat oligodendrocyte cell line. These alterations in phosphorylation as well as other differentiation-specific changes were found to be sensitive to inhibition by a tyrosine phosphatase inhibitor. This suggested that at some point early in the differentiation process, tyrosine phosphatases are important. A differential display strategy revealed 11 distinct tyrosine phosphatases in the oligodendrocyte lineage, with both precursor cells and oligodendrocytes expressing four major phosphatase transcripts: PTP alpha, PTP zeta, PTP sigma, and PTP gamma. A majority of the phosphatases examined show an increase in their mRNA levels during differentiation, with a striking upregulation observed for PTP epsilon. Our results suggest a significant role for this class of signal transducers in oligodendrocyte differentiation.

Animals↗

PubMed2005

Fyn is a downstream target of the pleiotrophin/receptor protein tyrosine phosphatase beta/zeta-signaling pathway: regulation of tyrosine phosphorylation of Fyn by pleiotrophin.

Pleiotrophin (PTN the protein, Ptn the gene) signals downstream targets through inactivation of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, disrupting the balanced activity of RPTPbeta/zeta and the activity of a constitutively active tyrosine kinase. As a consequence of the inactivation of RPTPbeta/zeta, PTN stimulates a sharp increase in the levels of tyrosine phosphorylation of the substrates of RPTPbeta/zeta in PTN-stimulated cells. We now report that the Src family member Fyn interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system. We further demonstrate that Fyn is a substrate of RPTPbeta/zeta, and that tyrosine phosphorylation of Fyn is sharply increased in PTN-stimulated cells. In previous studies, we demonstrated that beta-catenin and beta-adducin are targets of the PTN/RPTPbeta/zeta-signaling pathway and defined the mechanisms through which tyrosine phosphorylation of beta-catenin and beta-adducin disrupts cytoskeletal protein complexes. We conclude that Fyn is a downstream target of the PTN/RPTPbeta/zeta-signaling pathway and suggest that PTN coordinately regulates tyrosine phosphorylation of beta-catenin, beta-adducin, and Fyn through the PTN/RPTPbeta/zeta-signaling pathway and that together Fyn, beta-adducin, and beta-catenin may be effectors of the previously described PTN-stimulated disruption of cytoskeletal stability, increased cell plasticity, and loss of cell-cell adhesion that are characteristic of PTN-stimulated cells and a feature of many human malignant cells in which mutations have established constitutive expression of the Ptn gene.

Binding Sites↗

PubMed2000

Tyrosine analogues as alternative substrates for protein tyrosine kinase Csk: insights into substrate selectivity and catalytic mechanism.

Protein tyrosine kinases are critical enzymes in cell signal transduction but relatively little is known about the molecular recognition of the tyrosine substrate by these enzymes. Details of tyrosine substrate specificity within the context of a short peptide were investigated for protein tyrosine kinase Csk. It was found that aryl ring functional group substitutions the size of methyl group or smaller were generally well tolerated by the protein tyrosine kinase Csk whereas larger groups caused a decline in substrate efficiency. Extension of the phenol from the peptide backbone by a single methylene was acceptable for phosphorylation whereas removal of a methylene nearly abolished reactivity. Only the L-tyrosine derivative was processed. A negative charge ortho to the phenol hydroxyl was incompatible with substrate reactivity, consistent with previous pH rate profiles which indicated the importance of the neutral phenol. Overall, these studies confirmed the interpretation of a previous linear free energy relationship analysis which suggested that the enzyme followed a dissociative transition state mechanism.

CSK Tyrosine-Protein Kinase↗