Gongqin Sun

Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation

Accumulating evidence suggests that protein tyrosine phosphorylation-based signaling pathways are under the regulation of reactive oxygen species. Although protein tyrosine phosphatases are directly regulated by reversible oxidation, it is not clear whether protein tyrosine kinases (PTKs) are also directly regulated by reduction/oxidation (redox). In this study we report a mechanism of direct oxidative inactivation specific for the PTKs in the Src and fibroblast growth factor receptor (FGFR) families, key enzymes in mammalian signal transduction. Src is fully active when reduced and retains 8–25% of the full activity toward various substrates when oxidized. This inactivation is caused by oxidation of a specific cysteine residue (Cys-277), which results in homodimerization of Src linked by a disulfide bridge. Cys-277 is located in the Gly loop in the catalytic domain. This cysteine residue is conserved only in 8 of the >90 PTKs in the human kinome, including 3 of the 10 Src family kinases and all 4 kinases of the FGFR family. FGFR1 is also reversibly regulated by redox because of this cysteine residue, whereas Csk, a PTK that lacks a cysteine residue at the corresponding position, is not similarly regulated. These results demonstrate a mechanism of direct redox regulation conserved in certain specific PTKs.

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.

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTK–substrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substrate-docking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 = 21 μM) Csk phosphorylation of Src but only moderately inhibited (IC50 = 422 μM) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.

Conformationally constrained peptide analogues of pTyr-Glu-Glu-Ile as inhibitors of the Src SH2 domain binding.

A series of conformationally constrained peptides were designed and synthesized as the Src SH2 domain ligands based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI). In general, the constrained peptides such as compounds 6, 7, and 11 (IC(50) = 1.1-1.5 microM) showed higher binding affinities to the Src SH2 domain relative to the corresponding linear peptides 8a, 9a, and 13a, respectively (IC(50) > 100 microM), and pYEEI (IC(50) = 6.5 microM), as evaluated by a fluorescence polarization assay. Molecular modeling studies revealed that in constrained peptides, the isoleucine side chain penetrates very deeply into the hydrophobic binding pocket (P + 3 site) of the Src SH2 domain. These constrained peptides can serve as novel templates for the design of small and nonpeptidic inhibitors of the Src SH2 domain.

Recent advances in the discovery of Src kinase inhibitors

Src family kinases are involved in the regulation of a wide variety of normal cellular signal transduction pathways, such as cell growth, differentiation, survival, adhesion and migration. Considerable evidence implicates elevated expression and/or activity of Src kinases in many human cancers, osteoporosis, cardiovascular disorders and immune system dysfunction; thus, this family of protein tyrosine kinases now exists as intriguing targets for both basic research and drug discovery. Herein, a number of examples of currently developed Src family kinase inhibitors in selected patents from 2002 – 2005 will be described. Special attention will be made to the chemical diversity of ATP binding site inhibitors, potency, selectivity and therapeutic application of the compounds.

Docking-based Substrate Recognition by the Catalytic Domain of a Protein Tyrosine Kinase, C-terminal Src Kinase (Csk)

Protein tyrosine kinases are key enzymes of mammalian signal transduction. Substrate specificity is a fundamental property that determines the specificity and fidelity of signaling by protein tyrosine kinases. However, how protein tyrosine kinases recognize the protein substrates is not well understood. C-terminal Src kinase (Csk) specifically phosphorylates Src family kinases on a C-terminal Tyr residue, which down-regulates their activities. We have previously determined that Csk recognizes Src using a substrate-docking site away from the active site. In the current study, we identified the docking determinants in Src recognized by the Csk substrate-docking site and demonstrated an interaction between the docking determinants of Src and the Csk substrate-docking site for this recognition. A similar mechanism was confirmed for Csk recognition of another Src family kinase, Yes. Although both Csk and MAP kinases used docking sites for substrate recognition, their docking sites consisted of different substructures in the catalytic domain. These results helped establish a docking-based substrate recognition mechanism for Csk. This model may provide a framework for understanding substrate recognition and specificity of other protein tyrosine kinases.

Functions of the Activation Loop in Csk Protein-tyrosine Kinase

Autophosphorylation in the activation loop is a common mechanism regulating the activities of protein-tyrosine kinases (PTKs). PTKs in the Csk family, Csk and Chk, are rare exceptions for lacking Tyr residues in this loop. We probed the function of this loop in Csk by extensive site-specific mutagenesis and kinetic studies using physiological and artificial substrates. These studies led to several surprising conclusions. First, specific residues in Csk activation loop had little discernable functions in phosphorylation of its physiological substrate Src, as Ala scanning and loop replacement mutations decreased Csk activity toward Src less than 40%. Second, some activation loop mutants, such as a single residue deletion or replacing all residues with Gly, exhibited 1–2% of wild type (wt) activity toward artificial substrates, but significantly higher activity toward Src. Third, introduction of a thrombin cleavage site to the activation loop also resulted in loss of 98% of wt activity for poly(E4Y) and loss of 95% of wt activity toward Src, but digestion with thrombin to cut the activation loop, resulted in full recovery of wt activity toward both substrates. This suggested that the catalytic machinery is fully functional without the activation loop, implying an inhibitory role by the activation loop as a regulatory structure. Fourth, Arg313, although universally conserved in protein kinases, and essential for the activity of other PTKs so far tested, is not important for Csk activity. These findings provide new perspectives for understanding autophosphorylation as a regulatory mechanism and imply key differences in Csk recognition of artificial and physiological substrates.

Conformational basis for SH2-tyr(P)527 binding in Src inactivation

Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr527. Phosphorylation of Tyr527 triggers Src inactivation, caused by Tyr(P)527 binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)527 binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr(P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)527 binding is largely independent of the amino acid sequence surrounding Tyr(P)527, and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)527 binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)527 binding is attributed to a conformational contribution that requires residues Trp260 and Leu255. Although the SH3 domain is essential for Src inactivation by Tyr(P)527, it does not contribute to the SH2-Tyr(P)527 binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.

To C or not to C: direct and indirect redox regulation of Src protein tyrosine kinase.

Src protein tyrosine kinase is a master regulator of cell proliferation by modulating cell metabolism, division, survival and migration, thus the mechanisms that regulate Src function are of great interest to cancer research. One emerging mode of Src regulation is its response to reactive oxygen species (ROS). ROS have historically been viewed as damaging agents in cells under oxidative stress, but recent studies establish H2O2 as a secondary messenger to growth signals. A large number of cellular events respond to ROS, and many responses require the activity of Src, suggesting that Src may be a primary target of ROS. How Src kinase responds to ROS has not been established, as conflicting reports of Src activation or inactivation in response to increased concentration of ROS in the cells have been published. To determine how Src directly responds to oxidation, we investigated the effect of the redox environment on purified Src enzyme in vitro. The study reveals that Src is active in the reducing environment, and retains only 8-25% of activity in the absence of reducing agents. The inactivation is mediated by oxidation of Cys277, which leads to Src homodimers linked by a disulfide bond between the Cys277 residues of two Src monomers. A similar inactivation mechanism appears to be conserved in eight of more than 90 PTKs, including three Src family kinases and all four members of the FGFR family. The finding contradicts the view that Src is activated by oxidation, and suggests a complex response by Src to redox regulation. In this Extra View, we examine the conflicting observations in the context of complex mechanisms of Src regulation.

Synthesis and evaluation of 3-phenylpyrazolo[3,4-d]pyrimidine-peptide conjugates as Src kinase inhibitors.

3‐Phenylpyrazolo[3,4‐d]pyrimidine (PhPP) derivatives substituted with an alkyl or aryl carboxylic acid at the N1‐endocyclic amine, such as PhPP‐CH2COOH (IC50=250 μM), and peptides Ac‐CIYKYY (IC50=400 μM) and Ac‐YIYGSFK (IC50=570 μM) were weak inhibitors of polyE4Y phosphorylation by active c‐Src. A series of PhPP–peptide conjugates were synthesized using PhPP as an ATP mimic and CIYKYY or YIYGSFK as a peptide substrate to improve the inhibitory potency against active c‐Src kinase. PhPP derivatives were attached to the N terminus or the side chain of amino acids in the peptide template. Two N‐terminal substituted conjugates, PhPP‐CH2CO‐CIYKYY (IC50=0.38 μM) and PhPP‐CH2CO‐YIYGSFK (IC50=2.7 μM), inhibited the polyE4Y phosphorylation by active c‐Src significantly higher than that of the parent compounds. The conjugation of PhPP with the peptides produced a synergistic inhibition effect possibly through creation of favorable interactions between the conjugate and the kinase domain as shown by molecular modeling studies.