Anomalous systemic arterial supply to the basal segment of the left lung with huge pseudoaneurysm.
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Sungsoo Lee
Publications and source records attributed to Sungsoo Lee.
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.
Amino Acid Sequence↗
Probing the communication between the regulatory and catalytic domains of a protein tyrosine kinase, Csk.
Protein tyrosine kinases (PTKs) are important regulators of mammalian cell function and their own activities are tightly regulated. Underlying their tight regulation, all PTKs contain multiple regulatory domains in addition to a catalytic domain. C-terminal Src kinase (Csk) contains a catalytic domain and a regulatory region, consisting of an SH3 and an SH2 domain. In this study, we probed the communication between the regulatory and catalytic domains of Csk. First, kinetic characterization of SH3 and SH2 domain deletion mutants demonstrated that the SH3 and SH2 domains were crucial in maintaining the full activity of Csk, but were not directly involved in Csk recognition of its physiological substrate, Src. Second, highly conserved Trp188, corresponding to a key residue in domain-domain communication in other PTKs, was found to be important for maintaining the active structure of Csk by the presence of the regulatory region, but not required for Csk activation triggered by a phosphopeptide binding to the SH2 domain. Third, structural alignment indicated that the presence of the regulatory domains modulated the conformation of multiple substructures in the catalytic domain, some directly and others remotely. Mutagenic and kinetic studies supported this assignment. This report extended previous studies of Csk domain-domain communication, and provided a foundation for further detailed investigation of this communication.
Alanine↗
Phosphoryl transfer step in the C-terminal Src kinase controls Src recognition.
All members of the Src family of nonreceptor protein tyrosine kinases are phosphorylated and subsequently down-regulated by the C-terminal Src kinase, Csk. Although the recognition of Src protein substrates is essential for a diverse set of signaling events linked to cellular growth and differentiation, the factors controlling this critical protein-protein interaction are not well known. To understand how Csk recognizes Src, the chemical/physical events that modulate apparent substrate affinity and turnover were investigated. Src is phosphorylated in a biphasic manner in rapid quench flow experiments, suggesting that the phosphoryl transfer step is fast and highly favorable and does not limit overall turnover. As opposed to other kinase-substrate pairs, turnover is not limited by the physical release of ADP based on stopped-flow fluorescence and catalytic trapping experiments, suggesting that other steps control net phosphorylation. The K(d) for Src is considerably larger than the K(m) based on single turnover kinetic and equilibrium sedimentation experiments. Taken together, the data are consistent with a mechanism whereby Csk achieves a low K(m) for the substrate Src, not by stabilizing protein-protein interactions but rather by facilitating a fast phosphoryl transfer step. In this manner, the phosphoryl transfer step functions as a chemical clamp facilitating substrate recognition.
Adenosine Diphosphate↗
ATP-phosphopeptide conjugates as inhibitors of Src tyrosine kinases.
A number of Src SH2 domain inhibitors enhance the kinase catalytic activity by switching the closed inactive to the open active conformation. ATP-phosphopeptide conjugates were designed and synthesized as Src tyrosine kinase inhibitors based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI) and ATP to block the SH2 domain signaling and substrate phosphorylation by ATP, respectively. In general, ATP-phosphopeptide conjugates with optimal linkers such as compounds 5 and 7 (K(i) = 1.7-2.6 microM) showed higher binding affinities to the ATP-binding site relative to the other ATP-phosphopeptide conjugates having short or long linkers, 1-4 and 6, (K(i) = 10.1-16.1 microM) and ATP (K(m) = 74 microM). These ATP-phosphopeptide conjugates may serve as novel templates for designing protein tyrosine kinase inhibitors to block SH2 mediated protein-protein interactions and to counter the activation of enzyme that resulted from the SH2 inhibition.
Adenosine Triphosphate↗
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 microM) Csk phosphorylation of Src but only moderately inhibited (IC50 = 422 microM) 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.
Alanine↗
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.
Amino Acid Sequence↗
Expression, purification, and biochemical characterization of Chk, a soluble protein tyrosine kinase.
CSK family contains two protein tyrosine kinases: Csk (C-terminal Src kinase) and Chk (Csk homologous kinase). They are responsible for phosphorylating Src family protein tyrosine kinases on a C-terminal Tyr (Tyr527) and negatively regulating their activities. However, Chk and Csk have different expression patterns, mechanisms of regulation, and different biological functions, and appear to play different roles in the development of breast cancer. To obtain pure human Chk for biochemical characterization, its coding region was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The enzyme was highly expressed but unusually prone to proteolytic degradation during purification. Expression of the enzyme as a dual fusion protein with glutathione S-transferase on N-terminus and streptag, a 10 amino acid peptide, on C-terminus allowed purification of the full-length fusion protein. The purified enzyme was able to phosphorylate and inactivate Src. Chk (no inhibition up to 18.5 microM) and Csk (IC(50)= 1 microM) were differentially inhibited by PP2, probably due to the size difference of one residue (Thr265 in Csk versus Met304 in Chk) in the ATP-binding domain. The expression, purification, and initial characterizations of Chk provided an important step toward full characterization of Chk and Csk, two important enzymes in cellular regulation.
Amino Acid Sequence↗
Differential changes of calcium binding proteins in the rat striatum after kainic acid-induced seizure.
It has been suggested that calcium binding proteins protect against Ca2+ overload, thus rendering neurons more resistant against excitotoxicity. The influence of kainic acid, which induces status epilepticus, on the expressions of calbindin D28k, parvalbumin and calretinin was examined in the rat striatum by immunohistochemistry and microdensitometry. At 1, 3 and 6 days after kainic acid-induced seizure, the number of calretinin-positive neurons in the striatum was significantly lower than in control rats. However, no significant difference was observed in the number of calbindin D28k- and parvalbumin-positive neurons in control and seizure rats. At 1, 3 and 6 days after seizure the optical densities of calretinin- and parvalbumin-positive neurons in the striatum were significantly lower than in control rats. Our finding concerning the selective loss of calretinin-positive neurons in seizure groups suggests that calcium binding proteins in the striatum have differential vulnerabilities to kainic acid-induced seizure.
Animals↗
Contribution of an active site cation-pi interaction to the spectroscopic properties and catalytic function of protein tyrosine kinase Csk.
Csk is a soluble protein tyrosine kinase that phosphorylates and negatively regulates protein tyrosine kinases of the Src family. The spectral properties of the intrinsic Trp fluorescence of Csk and their underlying structural features were investigated in combination with urea denaturation and site-specific mutagenesis. It was found that W352 contributed approximately 35% of the total Trp fluorescence of Csk even though seven other Trp residues were present. The enhanced contribution by W352 to Csk fluorescence was due to an interaction between its indole ring and the positively charged guanidino group of R318. W352 is located on the peptide substrate binding P+1 loop, and R318 is located on the catalytic loop. The W352-R318 interaction, called a cation-pi interaction, uniquely couples the two loops in the active site. Mutations that disrupted this coupling resulted in varying levels of decreases in Csk activity, and consistent and significant increases in K(m) values for its physiological substrate, Src protein tyrosine kinase. These results indicated that structural coupling between the two loops by the cation-pi interaction played an important role in Csk substrate binding. Since both R318 and W352 are highly conserved among protein tyrosine kinases, this cation-pi interaction is likely a signature structural feature of most, if not all, PTKs. These studies elucidated the roles of two conserved signature residues in Csk and formed a baseline for further structure-function studies of Csk and other PTKs.
Animals↗