Raymond J.A. Budde

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

Csk phosphorylates Src family protein tyrosine kinases on a tyrosine residue near their C-terminus and down-regulates their activity. We previously observed that this regulation requires a stoichiometric ratio of Csk : Src in a time-independent manner. In this report we examined this unusual kinetic behavior and found it to be caused by Src autophosphorylation. First, pre-incubation of Src with ATP-Mg led to time-dependent autophosphorylation of Src, activation of its kinase activity and loss of its ability to be inactivated by Csk. However, the autophosphorylated Src can still be phosphorylated by Csk. The SH2 binding site for phospho-Tyr of this hyperactive and doubly phosphorylated form of Src is not accessible. Second, dephosphorylation of autophosphorylated Src by protein tyrosine phosphatase 1B allowed Src to be inactivated by Csk. Third, protein tyrosine phosphatase 1B preferentially dephosphorylates the Src autophosphorylation site and allows for Src regulation by Csk. Finally, Yes, another member of the Src family, was also only partially inactivated when a sub-stoichiometric amount of Csk was used. Mutation of the tyrosine autophosphorylation site of Yes to a phenylalanine resulted in a mutant Yes enzyme that can be fully inactivated by a sub-stoichiometric amount of Csk in a time-dependent manner. These results demonstrate that Csk phosphorylation inactivates Src and Yes only when they are not previously autophosphorylated and Src autophosphorylation can block the inactivation by Csk phosphorylation. This conclusion suggests a dynamic model for the regulation of the Src family protein tyrosine kinases, which is discussed in the context of previously reported observations on the regulation of Src family protein tyrosine kinases.

Tight-binding inhibitory sequences against pp60(c-src) identified using a random 15-amino-acid peptide library

A bacteriophage peptide library containing a random 15‐amino‐acid insert was screened for identification of peptide sequence(s) that bind pp60 c−src . Sequencing the random insert from more than 100 virions indicated that more than 60% of the phage virions that bound to this enzyme contained a GXXG sequence motif in which X was frequently a hydrophobic residue. The GXXG sequence was often repeated as GXXGXXG. Two nonameric peptides were synthesized to determine whether or not the peptide inhibits pp60 c−src tyrosine kinase activity and the importance of the glycine residues within this sequence. The peptide containing glycine had a K i of μM, whereas replacing the glycines with proline increased the K i value to 3.1 mM.

Recombinant pp60c−src from baculovirus-infected insect cells: purification and characterization

A simple and effective method has been developed to purify the recombinant protein tyrosine kinase pp60c-src from a baculovirus-insect cell expression system. The procedure includes affinity chromatography and HPLC. Milligram quantities of protein have been isolated with an activity of 3.9 mumol/min/mg protein using the substrate poly E4Y. This specific activity is many times higher than any published protocol. The enzyme is stable for months when stored in buffered 10% glycerol at -70 degrees C. This purification technique is compared to the immuno-affinity technique which is widely used for this enzyme. Enzyme kinetics were characterized with respect to substrate specificity, the effect of temperature, ionic strength, pH, and Mg+2 versus Mn+2 ions. Similar to the enzyme expressed in human cells, the recombinant enzyme demonstrated a higher Vmax and substrate specificity for poly E4Y over 5V-Agt-II. An activation energy of 14.2 kcal/mol was determined. Inhibition by increasing ionic strength is mostly due to an increase in Km for the poly E4Y substrate and hence was substrate dependent. The Km(ATP) was pH dependent while the Km(poly E4Y) was pH independent. For the phosphorylation of poly E4Y, free Mg+2 was stimulatory while Mn+2 was inhibitory. In contrast, Mn+2 stimulated the phosphorylation of 5V-Agt-II.

Use of synthetic peptides and copolymers to study the substrate specificity and inhibition of the protein tyrosine kinase pp60c-src

The ability of synthetic peptides and polypeptides to act as substrates and/or inhibitors of pp60c-src was examined. The random copolymer, poly(K4Y) had a threefold lower specificity than poly(E4Y). Peptides containing lysine vs. glutamate were also found to have a lower substrate specificity (Vmax:Km ratio). In order to assess the substrate specificity of acidic peptides, an assay protocol using DEAE-membranes was developed. Peptides containing a (YXE)5YXD motif (X = G, A, V, P, or norvaline) were tested as inhibitors and substrates of pp60c-src. The glycine-containing peptide was the best substrate having a specificity 16,000-fold higher than 5Val-angiotensin II, the most commonly used peptide substrate. Most of the peptides, except for the proline containing peptide, had Ki values of 20-100 microM. In a series of (XGE)5XGD peptides, where X = Y or F, tyrosine at position 10 was found to be the preferred site for accepting a phosphate. Analogs in which the glycine was replaced with alanine indicated that loss of flexibility around position 10 was detrimental to substrate specificity. Results suggest that conformational requirements of the peptides tested was important and substrate specificity was a more sensitive parameter than binding as measured by Ki values.

pp60ν-src kinase overexpression leads to cellular resistance to the antiproliferative effects of tumor necrosis factor

While some tumor cells are sensitive to the antiproliferative effects of tumor necrosis factor (TNF), others are resistant. The molecular basis for cellular resistance to TNF is not completely understood. Previously we have shown that transfection of cells with an oncogene HER2/neu/erb B2, a receptor tyrosine kinase, leads to resistance to the anticellular effects of TNF [(1988) Proc. Natl. Acad. Sci. USA 85, 5102‐5106]. In the present study, we demonstrate that the overexpression of another oncogenic tyrosine kinase, pp60 v‐src also induces resistance to TNF. In contrast to HER2, however, pp60 v‐src transfection of cells did not lead to down‐modulation of TNF receptors but rather to decreased intracellular glutathione levels. The pp60 v‐src ‐induced cellular resistance to TNF could be abrogated by interferon‐γ. Thus, these results indicate that the resistance of certain tumors to TNF may also be due in part to the overexpression of pp60 v‐src oncogene.

An assay for acidic peptide substrates of protein kinases

The assay of acidic peptides as substrates for protein kinases has not been as easy to perform as testing basic peptides or polypeptides. We have developed a simple, rapid, and cost-effective procedure that allows the design and testing of potential peptide substrates without the constraints imposed by the phosphocellulose filter paper method (the need to incorporate positively charged residues into the peptide sequence). The technique combines the chelation of 32Pi by acid molybdate with PEI-cellulose chromatography. In this way the migration of 32P-labeled Pi, ATP, and protein are impeded while phosphopeptide is eluted in 1.5 ml from a 0.25-ml disposable column. In order to validate the assay we used two angiotensin II analogues as peptide substrates for the protein tyrosine kinase pp60c-src. The assay results using the new procedure were compared to those of the phosphocellulose filter paper technique. We also demonstrated the use of this method to test linear and cyclic peptides that could not be assayed with the phosphocellulose paper technique. This assay will aid those who are attempting to determine the substrate specificity of protein kinases.

Discovery, development, and testing of substrates and inhibitors of pp60c-SRC

AbstractCurrently, there are no specific protein tyrosine kinase inhibitors available. This review summarizes our efforts to develop an active-site-directed inhibitor of pp60c-srv Initial efforts are directed at determining substrate specificity with synthetic peptides and at developing a biological system to test the potential of pp60c-src inhibitors to effectively inhibit the growth of pp60c-src activated cell lines. To meet these goals, we have developed new methods for purifying recombinant pp60C-SRC, assaying tyrosine kinase activity, synthesizing cyclic peptides, and generating random peptide libraries. In addition, we discuss the generation of potential artifacts while using polyhydroxy aromatic compounds as tyrosine kinase inhibitors.

Cloning, expression, purification and characterization of the alternate splice Src variants for drug discovery

Abstract Protein tyrosine kinases (PTKs) are key members of intra- and extra-cellular signaling pathways. Aberrant signaling pathways are responsible for many human diseases, making these enzymes targets for drug development programs. The difficulty in PCR-amplification of Src due to the high G-C content was overcome using a commercial “G-C melt” reagent. The N06 Src was cloned along with the N12 and N23 neuronal variants. Neuronal variants of Src occur due to splicing within the N-loop of the SH3 domain. These variants have greater catalytic activity. Affinity purification methodologies were established that takes advantage of binding sites within the SH1 and SH2 domains. The purified enzyme is stable, without loss of activity for >1 year when frozen and more than 1 week at 4°C. A 96-well solution phase assay was developed and validated that overcomes many of the false positives and negatives generated by other assays. Studies of the catalytic mechanism have indicated that a second metal ion is essential for catalysis. Some transition metals can be substituted for the second metal ion and maintain activity while others act as dead-end inhibitors with binding constants in the sub-micromolar range. The precise role of this second metal ion is being studied.

Phosphotyrosyl peptides targeted to the SH2 domain are potent inhibitors of the kinase activity of pp60c-src

(Src) is the prototype enzyme of the src-family of protein tyrosine kinases (PTKs). Src participates in several signal transduction cascades, growth signaling, and because of its elevated activity in a wide variety of human cancers is a target for the development of inhibitors for chemotherapeutics [1]. Src is composed of an N-terminal myristoyl site, a unique region of approximately 85 amino acids, an SH3 domain, an SH2 domain, and the catalytic or SH1 domain. The SH2 domain binds phosphotyrosine residues which is proposed to mediate the localization of this PTK to its signal transduction partners. Src kinase activity is down-regulated by intra-molecular binding of the SH2 domain to phosphorylated Tyr, located in the C-terminal tail of the catalytic domain [2]. The crystal structure of the inactivated enzyme [3] illustrates the interactions between the linker region and the SH3 domain with the catalytic domain and the resulting distortions in the latter. Src is activated by phosphorylation on Tyr in the so-called activation loop [2]. The phosphotyrosine peptide from the middle T antigen, EQpYEEIPIYL, has been shown to be a high affinity ligand selective for Src family SH2 domains [4]. This peptide was also shown to activate Src that was autoinhibited by phosphorylation on Tyr [5], presumably by disrupting the intramolecular interaction between the SH2 domain and the phosphorylated C-terminal tail, causing the linker region and SH3 domain to dissociate from the catalytic domain resulting the refolding of the latter to an active conformation. In this study, we show that EQpYEEIPIYL can also be a potent inhibitor of the kinase activity of Src.