Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.26 (GSK)
 6,788 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Polyclonal antibodies have been raised against two synthetic peptides reproducing the 48-64 and 353-369 sequences of CSK, a protein tyrosine kinase implicated in the down-regulation of src-related protein kinases. Both antibodies specifically recognize recombinant CSK and a CSK-related 49 kDa protein tyrosine kinase present in spleen but they do not cross-react with purified TPK-IIB, a spleen protein tyrosine kinase sharing with CSK catalytic activity toward src kinases and incapability to autophosphorylate. CSK and TPK-IIB once resolved from each other by heparin-Sepharose affinity chromatography, display opposite specificities toward synthetic peptides reproducing the sequences around the main phosphoacceptor residues of pp60c-src, namely Tyr-416 and Tyr-527. These data support the view that TPK-IIB and CSK may exert opposite effects on the activity of src-related protein tyrosine kinases.
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PMID:Spleen protein tyrosine kinases TPK-IIB and CSK display different immunoreactivity and opposite specificities toward c-src-derived peptides. 128 Feb 30

A tyrosine protein kinase (TPK-I), isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and recently identified as the product of the lyn oncogene [Brunati, A. M., Donella-Deana, A., Ralph, S., Marchiori, F., Borin, G., Fischer, S. & Pinna, L. A. (1991) Biochim. Biophys. Acta 1901, 123-126], is stimulated by a variety of effectors, including poly(lysine), heparin and very high NaCl concentrations. The efficacy of these compounds is variably dependent on the nature of the phosphoacceptor peptide substrates. Here we show that poly(lysine), but neither NaCl nor heparin, specifically enhances the phosphorylation efficiency of lyn TPK for the peptide EDNEYTA (src peptide). It reproduces the main autophosphorylation site of pp60c-src (Tyr416), which is entirely conserved in lyn TPK. The favourable effect of poly(lysine) is accounted for by both a dramatic drop of the Km value (70 microM versus 670 microM) and more than a threefold increase in Vmax. The effect is not so evident with a variety of different peptides, either unrelated to the src peptide (e.g. angiotensin II, AAYAA) or derived from the src peptide by single or multiple substitutions of the residues located on the N-terminal side of tyrosine. In particular, the responsiveness to poly(lysine) is dramatically reduced whenever alanine is replaced for either asparagine at position -2 or glutamic acid at position -1, either in the src heptapeptide or in its shorter derivative, the pentapeptide NEYTA. In sharp contrast, the favourable effect of 2 M NaCl, which is invariably accounted for only by an increased Vmax, is especially evident with peptides like angiotensin II and AAYAA, whose phosphorylation is less sensitive to poly(lysine) stimulation. The phosphorylation of the src peptides are actually inhibited rather than stimulated by 2 M NaCl. Consistent with this, lyn TPK autophosphorylation is also dramatically stimulated by poly(lysine) while being inhibited by 2 M NaCl. These data show that poly(lysine) deeply alters the selectivity of lyn TPK by conferring to it an enhanced activity and an especially high affinity toward peptides that reproduce the conserved autophosphorylation site of the TPK of the src family. It is suggested that endogenous compound, whose effect is mimicked by poly(lysine) in vitro, may play a relevant role in determining the specificity of lyn TPK in vivo and possibly of other TPK of the src family.
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PMID:Selective effect of poly(lysine) on the enhancement of the lyn tyrosine protein kinase activity. Increased specificity toward src peptides. 137 54

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [gamma-32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purposes, the ELISAs would be an attractive alternative to radioactive assays.
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PMID:Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity. 138 73

We have isolated the full-length sequence for a unique human kinase, designated TTK. TTK was initially identified by screening of a T cell expression library with anti-phosphotyrosine antibodies. The kinases most closely related to TTK are the SPK1 serine, threonine and tyrosine kinase, the Pim1, PBS2, and CDC2 serine/threonine kinases, and the TIK kinase which was also identified through screening of an expression library with anti-phosphotyrosine antibodies. However, the relationships are distant with less than 25% identity. Nevertheless, TTK is highly conserved throughout phylogeny with hybridizing sequences being detected in mammals, fish, and yeast. TTK mRNA is present at relatively high levels in testis and thymus, tissues which contain a large number of proliferating cells, but is not detected in most other benign tissues. Freshly isolated cells from most malignant tumors assessed expressed TTK mRNA. As well, all rapidly proliferating cell lines tested expressed TTK mRNA. Escherichia coli expressing the complete kinase domain of TTK contain markedly elevated levels of phosphoserine and phosphothreonine as well as slightly increased levels of phosphotyrosine. Taken together, these findings suggest that expression of TTK, a previously unidentified member of the family of kinases which can phosphorylate serine, threonine, and tyrosine hydroxyamino acids, is associated with cell proliferation.
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PMID:Expression of TTK, a novel human protein kinase, is associated with cell proliferation. 163 25

TPK-IIB, a spleen tyrosine protein kinase devoid of autophosphorylation activity (Brunati, A. M., and Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457), has been purified to near homogeneity and assayed for its ability to phosphorylate the synthetic peptides EDNEYTA and EPQYQPA reproducing the two conserved phosphoacceptor sites of pp60c-src (Tyr-416 and Tyr-527). While EPQYQPA was phosphorylated with low efficiency (Km = 16.7 mM, Kcat = 14.4), EDNEYTA is an excellent substrate displaying a Km value of 58 microM and a Kcat value of 31.2. The single substitution, in the latter peptide, of the glutamic acid adjacent to the tyrosine by alanine to give EDNAYTA caused a 6-fold increase in the Km. The positive influence on the phosphorylation of the acidic residues at -3 and -4 relative to the tyrosine is indicated by comparison of the kinetic constants for peptides EDAAYAA (Kcat = 4.6, Km 0.325 mM) and QNAAYAA (Kcat 2.4, Km 1.7 mM). Furthermore, when residues in the peptide NEYTA were replaced by alanine, the phosphorylation of the peptides NAYTA and AAYAA, was almost negligible (in terms of Kcat/Km ratio). However, AEYTA, NEYAA and AEYAA were still phosphorylated, albeit less efficiently than NEYTA. The probability that these peptides will adopt a beta-turn is EDNAYTA = EDNEYTA, NAYTA greater than NEYTA, and no predicted beta-turn for AEYTA, NEYAA, and AEYAA. Therefore these results support the concept that an amino-terminal acidic residue(s) is strictly required by TPK-IIB, irrespective of peptide conformation, although a beta-turn may enhance the phosphorylation of those peptides that satisfy this requirement. Two other spleen tyrosine kinases, TPK-I/lyn and TPK-III, both related to the src family, also have a far greater preference for the peptide EDNEYTA over EPQYQPA. However, they can be distinguished from TPK-IIB by their lower affinity for the peptides EDNEYTA and NEYTA and by their different specificity towards the substituted derivatives of NEYTA. TPK-I/lyn, accepts most of the substitutions that are detrimental to TPK-IIB, the triply substituted peptide AAYAA being actually preferred over the parent peptide NEYTA. The substitution of glutamic acid by alanine is also tolerated by TPK-III, although, in contrast to TPK-IIB, the phosphorylation efficiency is drastically decreased by the substitution of the asparagine at position -2.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Peptides reproducing the phosphoacceptor sites of pp60c-src as substrates for TPK-IIB, a splenic tyrosine kinase devoid of autophosphorylation activity. 171 42

The peptide SAEEEDQYN, corresponding to the carboxyl-terminal tryptic fragment of rat progastrin, whose penultimate tyrosyl residue is sulphated in the native peptide, is phosphorylated with Km values of 120 and 180 microM by two spleen tyrosine protein kinases, termed TPK-IIB and TPK-III, respectively. Another spleen tyrosine protein kinase related to the src family (TPK-I/lyn) is poorly active toward this peptide, displaying a Km 6.5 mM. The Tyr-phosphorylated peptide is recognized by an antibody (L304), which reacts with both sulphated and unmodified peptides, while it is not recognized by a second antibody (L303), which reacts with unmodified peptide yet not with the sulphated derivative. These data, in conjunction with previous observations (Hofsteenge, J., Stone, S.R., Donella-Deana, A. and Pinna, L.A. (1990) Eur. J. Biochem. 188, 55-59) support the view that phosphotyrosine is an effective surrogate for sulphotyrosine in a wide spectrum of biological activities.
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PMID:Substitution of phosphotyrosine for sulphotyrosine in biologically active peptides. Enzymatic phosphorylation of a progastrin peptide confers immunoreactivity reminiscent of the sulphated derivative. 171 35

Hirudin, the powerful anticoagulant agent of leech (Hirudo medicinalis) saliva, was readily phosphorylated by two spleen tyrosine protein kinases (TPK-IIB and TPK-III) at Tyr63 with Km values of 238 microM and 74 microM, respectively. The synthetic tridecapeptide DGDFEEIPEEYLQ, corresponding to the hirudin 53-65 C-terminal fragment, was phosphorylated even more efficiently than hirudin itself. Four hirudin mutants, in which one or more of the glutamic acids at positions 57, 58, 61 and 62 have been replaced by glutamines, were poorer substrates than hirudin. The mutant in which all four glutamates were substituted, [Gln57,58,61,62]hirudin, was virtually not phosphorylated by either TPK-IIB and TPK-III. Substitution of Glu57 and Glu58 was less deleterious than substitution of the two glutamic acids adjacent to Tyr63: [Gln61,62]hirudin exhibited a 20-fold lower phosphorylation efficiency with TPK-IIB. With TPK-III, however, the Km value of [Gln61,62]hirudin was slightly lower, while the Vmax decreased sixfold. The substitution of Glu62 alone was also more detrimental with TPK-IIB than with TPK-III. The behaviour of a third spleen TPK, named lyn TPK-I and belonging to the src family, was markedly different in that it did not phosphorylate hirudin but exhibited significant activity towards [Gln57,58,61,62]hirudin. Taken together, these data confirm and extend with a protein substrate the results obtained with short model peptides which indicated the stringent substrate requirements of TPK-IIB (and of TPK-III to a lesser extent) for N-terminal acidic residues. In contrast, such residues are deleterious with lyn TPK-I. These observations also support the concept that tyrosine protein kinases recognize specificity determinants situated in the vicinity of the target residue rather than requiring higher-order structural features.
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PMID:Specificity determinants for tyrosine protein kinase. A study with recombinant hirudin mutants. 193 46

The previously isolated spleen tyrosine protein kinase, conventionally termed TPK-IIA, displaying activation by either positively or negatively charged polyelectrolytes has been further characterized. TPK-IIA is immunologically related with the tyrosine protein kinase encoded by the lyn gene, a member of src subfamily and is dramatically activated by very high NaCl concentration. The stimulatory effects of NaCl and polylysine, which are not additive, are accounted for by increased Vmax values, the Km being virtually unchanged, suggesting that both effectors probably interact with the same site(s). Stimulation of TPK-IIA by heparin appears to be partially additive to that promoted by NaCl and possibly occurring through a different mechanism. The NaCl activatory effect correlates with the electrolytic nature of synthetic peptides used as substrates, being much more consistent with neutral peptides as compared with acidic ones. Of the other three spleen tyrosine protein kinases, TPK-I shows similar biochemical and immunological features, suggestive of close relatedness with TPK-IIA, while TPK-IIB and TPK-III are neither related with the lyn protein nor with the products of three other oncogenes of the src subfamily, namely lck, hck and fyn.
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PMID:Stimulation by NaCl, polylysine and heparin of two forms of spleen tyrosine protein kinase immunologically related with the protein expressed by lyn oncogene. 199 63

20 synthetic peptides, each of which includes a tyrosyl residue flanked by either neutral or acidic amino acids in different proportions and at variable positions, have been employed as model substrates for investigation of the site specificity of three tyrosine protein kinases previously isolated from spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and conventionally termed TPK-I, TPK-IIB and TPK-III. Comparison of the phosphorylation efficiencies shows that each tyrosine protein kinase is considerably different from the others in both the stringency and the nature of its specificity determinants. By considering, in particular, the kinetic constants obtained with the pentapeptides AAYAA, EEYAA, AEYAA, EAYAA, with the tetrapeptides AYAA and EYAA and with the tripeptides AYA and EYA, it turns out that N-terminal acidic residue(s) are only essential with TPK-IIB for efficient phosphorylation with multiple residues displaying a synergistic effect. The very similar Km (130 microM) but 14-fold-different Vmax values with YEEEEE vs. EEEEEY indicate that an N-terminal rather than C-terminal location of acidic residues is required for a high phosphorylation rate with, though not for binding to TPK-IIB. Acidic residues decrease the phosphorylation rate with TPK-I, a kinase related to the src family which is immunologically indistinguishable from the lyn TPK; they are nearly ineffective, however, with TPK-III, the least specific of the tyrosine protein kinases, which exhibits appreciable activity toward tripeptides and dipeptides like GAY and AY which are not significantly affected by TPK-I and TPK-IIB. While the peptide substrate specificity of TPK-I is similar to that of TPK-IIA, a spleen tyrosine protein kinase previously considered [Brunati, A. M., Marchiori, F., Ruzza, P., Calderan, A., Borin, G. & Pinna, L. A. (1989) FEBS Lett. 254, 145-149], the remarkable requirement of TPK-IIB alone for acidic peptides may suggest the involvement of this enzyme, which is also unique in its failure to autophosphorylate, in the phosphorylation of the highly conserved and quite acidic phosphoacceptor sites of the src family protein kinases.
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PMID:Different specificities of spleen tyrosine protein kinases for synthetic peptide substrates. 226 99

T lymphocytes express a tyrosine protein kinase (TPK; protein-tyrosine kinase; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112), pp56lck that is encoded by the lck protooncogene. This TPK was recently found to be associated with the intracellular domain of the T-cell surface glycoproteins, CD4 and CD8, suggesting that it plays an important role in T-cell development and activation. We have studied the regulation of pp56lck and found that this kinase can be rapidly activated by an endogenous mechanism present in T-lymphocyte membranes. This activation was sensitive to sodium orthovanadate and O-phosphotyrosine, consistent with the involvement of a phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphatase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) in pp56lck activation. Based on a recent report demonstrating that CD45, the leukocyte common antigen, is a membrane-bound PTPase, we analyzed its role in pp56lck activation. CD45 was found to be the major (greater than 90%) PTPase in membranes of the murine T-lymphoma line BW5147. Moreover, activation of pp56lck was undetectable in a mutant BW5147 line lacking CD45 expression (and the associated PTPase activity). In contrast, activation of pp56lck was readily detected in the wild-type lymphoma line. More important, when immunoprecipitated CD45 was added to pp56lck, the TPK activity of the latter increased greater than 2-fold within minutes. This effect of CD45 was completely blocked by sodium orthovanadate. These findings indicate an important role for the CD45 PTPase in pp56lck activation. This role could be mediated by direct dephosphorylation of a regulatory tyrosine residue in pp56lck.
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PMID:Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase. 254 4


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