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3.4 The functional domains of Src

To illustrate some of the principles of multidomain protein function, we will use as an example, the Src protein, a very well-characterised tyrosine kinase. As described earlier, Src contains four domains: two kinase domains, which together comprise the catalytic component of this protein, and two distinct binding/regulatory domains. The binding domains are of the SH2 and SH3 types. The identification of domains in other proteins, homologous to those in Src, led to the ‘Src homology’ nomenclature. If we represent the amino acid sequence of Src in a linear form, the domains can be mapped as indicated in Figure 28.

Figure 28 Representation of the tyrosine kinase Src, showing the linear map of its domains. The N-terminus is fatty acylated. The locations of intramolecular interactions (described in the text) are indicated.
  • Note that the N-terminus of Src has a fatty acyl chain attached. Where do you think Src is located in the cell?

  • Src is anchored to the cytosolic surface of the plasma membrane.

The general domain structure depicted in Figure 28 is common to all members of the Src family of tyrosine kinases. The conformation of the protein kinase fold of Src differs between ‘on’ (catalytically active) and ‘off’ (inactive) forms. The protein kinase domain is extremely well conserved, not only among tyrosine kinases, but also among the family of serine/threonine kinases. A marked similarity in the general three-dimensional structure of the kinase fold in its ‘on’ state in these enzymes is reflected in their common catalytic activity.

In the three-dimensional schematic representation of Src (Figure 29), the kinase domain can be seen to comprise two distinct lobes or subdomains. The smaller N-terminal lobe (N lobe) is composed of a five-stranded β sheet and one prominent α helix (called helix αC). The larger C lobe is predominantly helical. Figure 29b also shows the substrates for the kinase, namely ATP and a stretch of peptide sequence containing a tyrosine residue, in their appropriate binding sites. ATP binds in a deep cleft between the two lobes and the peptide binds across the front of the ATP binding pocket, close to the terminal (or gamma, γ) phosphate of the ATP. Close to the peptide substrate is a loop termed the ‘activation loop’.

Figure 29 A schematic showing the regulation of Src tyrosine kinase between active and inactive states. (a) The inactive conformation is stabilised by intramolecular interactions between the SH2 domain and phosphorylated Tyr 527 in the C-terminal tail and between the SH3 domain and a specific sequence in the link between the SH2 domain and the kinase domain. (b) In the switch to the active conformation, these interactions are disrupted and the SH2 and SH3 domains dissociate and bind to other specific ligands. The inhibitory phosphate group on Tyr 527 is removed and Tyr 416 is phosphorylated. As a result, the activation loop adopts an open conformation and the peptide substrate can bind to the C lobe of the kinase domain. (Adapted from Huse and Kuriyan, 2002.)

How do the SH2 and SH3 domains regulate Src's kinase activity? The basis for the regulatory activity of these domains is their specificity for particular binding motifs (Table 5). In the inactive form of the enzyme, a tyrosine near the C-terminus (Tyr 527) is phosphorylated and is bound to the SH2 domain (Figure 29). This interaction positions the SH3 domain so that it binds to an internal proline-rich motif in the region linking SH2 to the kinase domains. These intramolecular interactions stabilise an inactive conformation of the kinase.

Src is activated when the SH2 and SH3 domains bind specific ligands. These ligands, components of a signalling cascade in which Src participates, bear the specific motifs recognised by the SH2 and SH3 domains and compete with the internal binding sites. Thus the SH2 domain binds to a ligand containing a phosphorylated tyrosine residue and the SH3 domain recognises and binds to a proline-rich domain in another signalling protein. Binding of the SH2 and SH3 ligands disrupts intramolecular interactions and permits removal of the inhibitory phosphate from Tyr 527. With phosphorylation of a tyrosine residue in the activation loop (Tyr 416), this loop is stabilised in an open extended form that permits binding of the peptide substrate, and the enzyme is fully activated.


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