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In this study, the authors determined a substrate-docking site on Csk that …


Biology Articles » Biochemistry » Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase » Discussion

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

PTKs of the Csk family specifically phosphorylate SFKs on a C-terminal tail Tyr residue and regulate their activity. This system is chosen to investigate PTK substrate specificity because the exclusive PTK–substrate relationship is well established in vivo and in vitro, and extensive structural and biological data are available on Csk and SFKs to assist such studies. One unique feature of this system that was central to our strategy was that Csk and Chk shared functional identity but relatively low sequence identity (54%). This feature allowed us to locate some key residues in the docking site by evaluating uniquely conserved residues in Csk family.

The identified substrate-docking site of Csk is composed of six residues, Ser-273, Arg-279, Ser-280, Arg-281, Arg-283, and Phe-382. This identification of the substrate-docking site is supported by several lines of evidence. First, several of the key residues of the docking site are uniquely conserved in Csk and Chk. The unique conservation is consistent with Csk and Chk being the only two kinases able to phosphorylate SFKs on Ytail. Second, mutations of the residues within the docking site abolished Csk activity toward Src without significantly affecting its general kinase activity. Mutation of many residues outside this region did not preferentially affect Csk's ability to phosphorylate the physiological substrate. Third, Csk mutants containing multiple point mutations in the docking site resulted in a >95% loss of Csk activity toward kdSrc but only a modest decrease in activity toward artificial substrate, effectively converting Csk into a generic PTK unable to phosphorylate Src. Correspondingly, the Csk mutants were unable to regulate Src activity. Fourth, the loss of activity toward kdSrc correlates to the loss of ability to physically bind to kdSrc, indicating that the mutated site is indeed critical for Csk–Src interaction. Fifth, a peptide mimicking part of the docking site potently inhibited Csk phosphorylation of kdSrc but only moderately inhibited Csk activity toward an artificial substrate. These results demonstrate that the substructure consisting of the α-helix D and Phe-382 is indeed the docking site that specifically interacts with SFKs for efficient phosphorylation.

At present, it is difficult to assign quantitative contributions to the individual residues. The identified residues (Fig. 2B), containing three positively charged residues, two polar residues, and one hydrophobic residue, appear well suited to provide a complex surface for highly specific and unique interaction with the substrate. The docking site is located near the active site (Fig. 6) and appears well positioned for Src docking. Crystallized IRK–peptide substrate complexes (38) and mutagenic studies of other PTKs (39) indicate that the P + 1 loop provides the main platform for peptide substrate binding to PTKs. It is likely that the P + 1 loop in Csk performs the same function in binding to SFK C-terminal tail and presents Ytail to the active site. The docking site is located on the same side of the kinase molecule as the P + 1 loop. It can be envisioned that substrate-docking site would interact with the docking determinants on SFKs, which would bring the C-terminal tail peptide to Csk active site. Because Src binding and phosphorylation are both abolished by docking site mutations, it appears that the docking interaction may be primarily responsible for SFKs recognition by Csk.

This work raises several important questions that remain to be answered. First, what are the specificity determinants on SFKs that allow them to be specifically recognized by Csk and Chk? We reason that such determinants would be a surface area complementary to the Csk substrate-docking site. The identification of the docking site on Csk will facilitate the identification of such determinants on SFKs. Second, it is unclear whether other PTKs will use a similar mechanism for recognizing their physiological substrates. Csk family PTKs have unusually strict substrate specificity in that they only phosphorylate SFKs. Other PTKs may phosphorylate multiple families of protein substrates. Alignments of primary and tertiary structures of PTKs indicate that the structures of α-helix D are highly variable between PTK families, consistent with the possibility of its being a key structure for substrate specificity determination. Third, it is interesting that mutations of the docking site only moderately affected the Km of Csk for kdSrc but more dramatically affected the kcat. We started this work with the expectation that the docking site would mainly affect the complementarity between the enzyme and the substrate, and thus perturbation to the docking site would more significantly affect the Km. The large decrease in kcat caused by the mutations suggest that the docking interaction is critical for Csk transition state complementarity with SFKs instead.


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