Determinants of substrate recognition in nonreceptor tyrosine kinases

Abstract

Cytoplasmic tyrosine kinases do not occur as isolated catalytic domains. Instead, each kinase family possesses a characteristic array of additional domains that are appended to the catalytic domain. The combination and the arrangement of these modular domains are important in kinase regulation and function. This Account describes how the noncatalytic regions of Src family tyrosine kinases are involved in enzyme regulation, substrate selection, and multisite phosphorylation.

Figure 1

Top: domain structure of Src-family tyrosine kinases. Bottom: the three-dimensional structure of the Src family kinase Hck. The SH3 domain is yellow, the SH2 domain is green, and the catalytic domain is blue. Phosphorylation sites at Tyr416 (in the activation loop) and Tyr527 (in the C-terminal tail) are indicated.

Figure 2

Mechanisms for Src kinase activation. The inactive form of the kinase is depicted at the upper left. Three processes can lead to increased autophosphorylation at Tyr416 and enhanced catalytic activity: Tyr527 dephosphorylation, SH2 ligation, or SH3 ligation. pY represents phosphorylated tyrosine.

Figure 3

Coupling of Src-family kinase activation to substrate recognition. The catalytic domain (blue) is inhibited by intramolecular interactions between the SH3 domain (yellow) and the linker region and between the SH2 domain (green) and phosphorylated Tyr527. Ligands for the SH3 or SH2 domains can disrupt the intramolecular interactions and activate the kinase. Many of the best Src substrates contain ligands for the SH3 and/or SH2 domain. In this case, the ligands concomitantly activate the kinase and tether the substrate to the enzyme, facilitating phosphorylation.

Figure 4

Multisite phosphorylation of Cas by Src can follow either a processive (shaded) mechanism or a nonprocessive mechanism. Unphosphorylated tyrosines on Cas are represented by open circles, and phosphorylated sites are represented by red circles. For simplicity, only three tyrosines are shown, although Cas contains approximately 15 potential Src phosphorylation sites.

Figure 5

Enhanced phosphorylation of substrates containing SH2 ligands (panel A) and SH3 ligands (panel B) by Hck. The sequences of the peptides are given above the curves. Red type indicates the substrate sequence, and blue type indicates the SH3 or SH2 ligand sequence. Residues changed for control peptides are indicated in magenta. The rates of phosphorylation were determined using the phosphocellulose paper assay in panel A and with the spectrophotometric assay in panel B.

Figure 6

Peptide model for processive phosphorylation by Src. The synthetic peptide contains the SH2 binding sequence pYEEI and two copies of a substrate sequence derived from Cas. Substrate sequence 2 is phosphorylated first by Hck, followed by substrate sequence 1, as pictured. In this figure, phosphotyrosine is indicated with a red circle.

Figure 7

Phosphorylation of the model substrate (pictured in Figure 6) follows a processive mechanism. The peptide was incubated with Hck and [³²P]-labeled ATP in a pulse reaction to allow phosphorylation of substrate sequence 2. This was followed by a chase reaction carried out in the presence of excess unlabeled ATP. The rate of conversion to the final product was measured by HPLC and scintillation counting. The data points are shown in closed circles (with standard deviation), and the predictions for processive and nonprocessive mechanisms are shown by dashed lines.

Figure 8

Phosphorylation of Cas. Top: the domain structure of Cas. The SH3- and SH2-binding sequences at the C-terminus of Cas are indicated. The mutated SH3 binding sequence is shown above the figure. Bottom: a pulse-chase reaction shows that Src phosphorylates Cas at multiple sites. The polyPro mutant form of Cas shows reduced phosphorylation.