Spleen protein tyrosine kinases TPK‐IIB and CSK display different immunoreactivity and opposite specificities toward c‐<i>src</i>‐derived peptides

Brunati, Anna Maria; Allée, Guillaume; Marin, Oriano; Donella‐Deana, Arianna; Cesaro, Luca; Bougeret, Cécile; Fagard, Rémi; Bénarous, Richard; Fischer, Siegmund; Pinna, Lorenzo A.

doi:10.1016/0014-5793(92)81212-5

Cited by 32 publications

(23 citation statements)

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“…2A) and after conversion to the Fmoc derivative (17) incorporated into the Src autophosphorylation peptide EDNE(F 3 Y)TA by solid phase peptide synthesis. EDNEYTA has previously been reported to be a Csk substrate (18), and we confirmed that here (k cat ϭ 14 Ϯ 0.4 min…”

Section: Resultssupporting

confidence: 79%

The Role of the Catalytic Base in the Protein Tyrosine Kinase Csk

Cole

Grace

Phillips

et al. 1995

Journal of Biological Chemistry

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A potential distinguishing feature between protein tyrosine kinases and homologous serine/threonine kinases is the function of the catalytic base in these enzymes. In this study, we show that a peptide containing the unnatural amino acid trifluorotyrosine shows remarkably similar efficiency as a substrate of the tyrosine kinase Csk (C-terminal Src kinase) compared with the corresponding tyrosine-containing peptide despite a 4-unit change in the phenolic pK a . These results argue against the importance of early tyrosine deprotonation by a catalytic base in Csk. To further explore the role of the proposed catalytic base, the Csk mutant protein D314E was produced. This mutant displayed a significant reduction in k cat (approximately 10 4 ) but relatively little effect on substrate K m values compared with wildtype Csk. Examination of the thio effect (k cat -ATP/k catadenosine 5-O-(thiotriphosphate)) for D314E Csk led to the suggestion that a role of aspartate 314 may be to enhance the reactivity of the ␥-phosphate of ATP toward electrophilic attack. These results may have significant impact on protein tyrosine kinase inhibitor design.Protein tyrosine kinases play central roles in cell proliferation, cell differentiation, and signaling processes (1). While their enzymatic activity, transfer of a phosphoryl group from ATP to tyrosines in proteins, was first described over 15 years ago, details of tyrosine kinase catalytic mechanism have largely remained elusive. In contrast, their homologous family member protein serine/threonine kinases have been more intensively studied and are better characterized enzymologically (2-8). In order to rationally develop specific inhibitors for these enzymes (9) as well as to delineate the biochemical consequences of their in vivo regulation, a more complete understanding of tyrosine kinase catalysis is needed.Recently, we carried out a preliminary mechanistic evaluation of the protein tyrosine kinase Csk (C-terminal Src kinase) from humans (10), overproduced in and purified from Escherichia coli (11). It was shown that, like the better studied serine/threonine kinase protein kinase A, the Csk-catalyzed kinase reaction obeys a ternary complex mechanism, most likely with rapid and random binding of the substrates ATP and 4:1 poly(Glu,Tyr). This argued against a covalent phosphoenzyme intermediate. Moreover, using ATP␥S 1 and microviscosity effects, it was shown that in the Csk ATP reaction, product ADP diffusional release is partially rate-determining for the overall reaction (10). The lack of a large deuterium solvent kinetic isotope effect (actually a slightly inverse, k cat H/ k cat D ϭ 0.74, was seen) in the ATP␥S reaction (where the chemical step appeared fully rate-determining) suggested that tyrosine hydroxyl deprotonation is asymmetric in the reaction transition state. Because a tetrafluorotyrosine-containing peptide was previously reported to be a tight binding inhibitor (and not detectably a substrate) for insulin receptor tyrosine kinase (12), a phenoxide anion was reasoned...

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Section: Resultssupporting

confidence: 79%

The Role of the Catalytic Base in the Protein Tyrosine Kinase Csk

Cole

Grace

Phillips

et al. 1995

Journal of Biological Chemistry

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“…The Klngse Assays. Inhibition of substrate phosphorylation by the purified recombinant EGF-R intracellular domain (ICD) (9), v-abl (10), c-src (11), c-lyn (12), c-fgr (13), TPK-IIB (14), cAMP-dependent protein kinase, phosphorylase kinase (15), and casein kinases 1 and-2 (16,17) was assayed as 4escribed.…”

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confidence: 99%

4,5-Dianilinophthalimide: a protein-tyrosine kinase inhibitor with selectivity for the epidermal growth factor receptor signal transduction pathway and potent in vivo antitumor activity.

Buchdunger

Trinks

Mett

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…The activity of the other protein-tyrosine kinases was assayed under the same conditions described for c-Fgr, using the following as substrates: 0.1 mM cdc2 peptide for Lyn, Fyn, and Lck, 2 mM angiotensin I1 for Syk, and 0.6 mM C-terminal Src-(514-533)-peptide for CSK. In the latter case, the assay was performed as described by Brunati et al (1992). Tyrosine kinase autophosphorylation activity was assayed at 30°C in the same medium described above, except that the peptide substrate was omitted.…”

Section: Methodsmentioning

confidence: 99%

“…CSK, c-Fgr, Fyn, Syk, and Lyn were purified to near homogeneity from rat spleen as described by Brunati et al (1992Brunati et al ( , 1993Brunati et al ( , 1995b and DonellaDeana et al (1992). Lck (recombinant glutathione S-transferaseLck) was a generous gift of Dr Siegmund Fischer (Paris, France).…”

Section: Methodsmentioning

confidence: 99%

Specific Stimulation of c‐Fgr Kinase by Tyrosine‐Phosphorylated (Poly)Peptides

Ruzzene¹,

Brunati²,

Donella‐Deana³

et al. 1997

European Journal of Biochemistry

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Hematopoietic lineage cell-specific HSI protein is converted into a substrate for c-Fgr by previous Syk-mediated phosphorylation, at site(s) that bind to the SH2 domain of c-Fgr [Ruzzene, M., Biochemistry 35, 5327-53321. Here we show that a phosphopeptide derived from one such site, HS1-(320-329)-phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, in a dose-dependent manner, the catalytic activity of c-Fgr either assayed with peptide substrates or evaluated as intermolecular autophosphorylation of c-Fgr itself. The dephosphorylated HS1-(320-329)-peptide is totally ineffective, while the stimulatory efficacy of other phosphopeptides derived from the polyoma virus middle T antigen-(393 -402) sequence, c-Src, and c-Fgr autophosphorylation sites, and the C-terminal c-Src site (Tyr527) is variable and correlates reasonably well with the predicted affinity for the c-Fgr SH2 domain. Stimulation of c-Fgr catalytic activity is also promoted by the full-length HSI protein, previously tyrosine phosphorylated by Syk, and is accounted for by an increased V,,,,, while the K,,, values are unchanged. If the normal activator of c-Fgr kinase, Mg", is replaced by MnZ+, stimulation by HS 1 -(320-329)-phosphopeptide is still observable with peptide substrates, while autophosphorylation is, in contrast, inhibited by the phosphopeptide. These findings, in conjunction with the ability of previously autophosphorylated c-Fgr to be stimulated by HS L(320-329)-phosphopeptide, support the view that stimulation of c-Fgr by phosphopeptide is not or is not entirely a consequence of increased autophosphorylation. Interestingly, neither Syk and C-terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation by phosphopeptide, as observed with c-Fgr. These data support the notion that c-Fgr undergoes a unique mechanism of activation promoted by tyrosine-phosphorylated polypeptide that binds to its SH2 domain. This suggests that such a mode of regulation is peculiar of protein-tyrosine kinases committed to the secondary phosphorylation of sequentially phosphorylated proteins.Keywords: protein-tyrosine kinase : c-Fgr: Src homology domain 2 ; phosphotyrosine peptide; HS1 protein.Src-related tyrosine kinases are members of a family of nonreceptor enzymes that includes not less than ten proteins, which all display a remarkable homology and share a common structure: a poorly conserved N-terminal segment (unique domain), followed by a Src homology 3 (SH3) and a Src homology 2 (SH2) domain, a catalytic domain (SHl), and a C-terminal tail. Two highly conserved in vivo tyrosine phosphorylation sites are known: an autophosphorylation site (Tyr416 in chicken c-Src) and a C-terminal phosphorylation site (Tyr527). This latter site, once phosphorylated by C-terminal Src kinase (CSK) (Okada and Nakagawa, 1989;Okada et al., 1991), plays a negative regulatory role through its interaction with the SH2 domain (Cantley et al

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Spleen protein tyrosine kinases TPK‐IIB and CSK display different immunoreactivity and opposite specificities toward c‐src‐derived peptides

Cited by 32 publications

References 10 publications

The Role of the Catalytic Base in the Protein Tyrosine Kinase Csk

The Role of the Catalytic Base in the Protein Tyrosine Kinase Csk

4,5-Dianilinophthalimide: a protein-tyrosine kinase inhibitor with selectivity for the epidermal growth factor receptor signal transduction pathway and potent in vivo antitumor activity.

Specific Stimulation of c‐Fgr Kinase by Tyrosine‐Phosphorylated (Poly)Peptides

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