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.
Proc Natl Acad Sci U S A. 2003 December 9; 100(25): 14707–14712.
Protein tyrosine kinases (PTK) are a large family of enzymes that transfer the γ-phosphate of ATP to Tyr hydroxyl group in proteins (1). This covalent modification is a fundamental mechanism in cellular regulation and signal transduction (2). Activation of specific PTKs is associated with various types of human cancer and other proliferative diseases, making many PTKs targets for drug discovery (3, 4). Although all PTKs share a highly conserved catalytic domain (5), each PTK phosphorylates only one or a few protein substrates on specific tyrosine residues, enabling them to transduce regulatory signals to specific targets. The tertiary structures of a dozen PTKs have been determined (5), but the mechanisms by which PTKs recognize their physiological substrates are still poorly understood.
One of the best understood PTK regulatory systems is the regulation of Src family PTKs (SFKs) (6). There are nine kinases in the Src family. They share an overall structural organization, containing a myristoylation motif, a unique region, an Src homology (SH)3 domain, an SH2 domain, a catalytic domain, and a regulatory C-terminal tail. SFKs are regulated by phosphorylation on two Tyr residues, one located on the activation loop (Yact) and the other on the C-terminal tail (Ytail). Yact is the site of autophosphorylation catalyzed by SFKs through an intermolecular mechanism, which activates SFKs (7). Ytail is phosphorylated by another family of PTKs containing two members, C-terminal Src kinase (Csk) (8) and Csk-homologous kinase (Chk) (9, 10). Phosphorylated Ytail binds to the SH2 domain intramolecularly (11, 12), which leads to inactivation of SFKs (13). Additionally, phosphorylation of Yact blocks the inactivation of SFKs by Ytail phosphorylation (14). Because the phosphorylation of Yact and Ytail has opposite effects on SFK function, it is critical that they are specifically phosphorylated by respective kinase activities. The phosphorylation of Ytail by Csk and Chk is indeed highly specific and exclusive (15). Only Csk and Chk phosphorylate the Ytail of SFKs, which are the only physiological substrates for Csk and Chk. It is not clear whether Csk and Chk have identical or different specificity among SFKs.
The C-terminal tails of SFKs have a consensus sequence of TATEXQYtailQXQ/G, where the X's are variable residues. Early efforts to understand Csk substrate specificity used peptides mimicking this phosphorylation site (8, 16). However, such peptides are ≈1,000 times less efficient than SFKs as substrates for Csk (8, 16, 17). By screening a random peptide library, Cole and coworkers (17) identified an optimal peptide substrate for Csk with the sequence of EEEIYFFF. The optimal peptide is 500 times better as a substrate than are peptides mimicking the Src C-terminal tail and bears little resemblance to the physiological phosphorylation site (17). These studies demonstrate that the C-terminal tail does not contain sufficient determinants for Csk recognition. Recent mutagenic studies of Src indicated that, although the local sequence surrounding the phosphorylation site played important roles, additional determinants residing outside the C-terminal tail are required for efficient Ytail phosphorylation by Csk (18). These observations suggest that Csk recognition of Ytail of SFKs involves two types of interactions: docking interactions between Csk and SFKs and local interactions between the active site of Csk and the tail peptide sequence. Such bivalent interactions would allow Csk to specifically recognize SFKs and position the Ytail into Csk active site for phosphorylation. The docking site on Csk and the docking determinants on SFKs have not been determined.
The tertiary structures of Csk (19, 20) and several SFKs (11, 12, 21) have been determined, but the structures do not provide clear clues to how Csk recognition of SFKs may be achieved. In this current study, using structure-guided site-specific mutagenesis, we determined a substrate-docking site on Csk that is critical for its ability to bind to, phosphorylate, and regulate SFKs but is not important for the general kinase activity. Furthermore, a peptide mimicking the substrate-docking site potently inhibited Csk phosphorylation of Src but only moderately inhibited the general kinase activity of Csk. To our knowledge, this is the first report of a substrate-docking site on any PTK.