br Substrate Interactions Outside of the Catalytic Cleft As

Substrate Interactions Outside of the Catalytic Cleft As for other types of protein–protein interactions, kinase–substrate docking interactions can occur through large binding interfaces or through recognition of short linear sequence motifs. For kinases with a large number of substrates, the use of short motifs for substrate targeting makes sense from an evolutionary standpoint, as substrates can be lost or gained through a small number of point mutations (Box 1). Recent studies of mitogen-activated protein kinases (MAPKs) have provided insight into how linear docking motifs can mediate selective targeting by closely related members of a kinase family. MAPKs share a common phosphorylation site consensus (S/T-P), but members of the different subfamilies (ERK, p38, and JNK) target a largely distinct set of substrates [6]. Two different regions of MAPK catalytic domains are known to interact with substrate docking motifs (Figure 3). DEF (docking site for ERK, FxF) motifs (also called F-sites) are generally Ω-x-Ω sequences (where Ω is an aromatic residue) that engage a small Ethacrynic acid - d5 pocket proximal to the catalytic cleft of ERK and p38 family MAPKs 20, 21. Distinct DEF-site sequence preferences among p38 isoforms have been rationalized based primarily on the size of this pocket. D-site motifs consist of a basic patch separated by a short linker from a ϕ-x-ϕ sequence (where ϕ is a hydrophobic residue [5]). The D-site motif, which is found in both substrates and regulators of MAPKs, interacts with a groove within the kinase C-lobe that is located on the opposite face from the active site (Figure 3). Because this groove is structurally similar among the various MAPKs, how it might bind to specific sequences to mediate selective targeting has remained obscure. The Reményi group recently reported a series of X-ray crystal structures of MAPKs in complex with D-site peptides. Comparison with previously reported structures revealed that variations in the linker sequence gave rise to distinct conformations that promoted selective targeting of JNK versus the p38 and ERK MAPKs [22]. By contrast, the Bardwell group reported that the identity of the hydrophobic residues drove selective interactions with JNK MAPK [23]. By leveraging unique structural or sequence features of the D-site, both groups have been able to computationally predict and verify new MAPK substrates 24, 25. Intriguingly, a number of the predicted and established JNK D-sites overlap with a docking site for the phosphatase calcineurin, suggesting a mechanism for coordinate regulation of substrate phosphorylation and dephosphorylation through a common motif [26]. Other recent work has indicated that in some cases MAPK regulators, including the ERK5 activator MEK5 [27] and the p38 phosphatase HePTP [28], make additional contacts with the catalytic domain outside of the canonical D-site interaction groove. These additional interactions serve to enhance binding affinity and likely mediate precise targeting of specific MAPKs to prevent potentially deleterious crosstalk between pathways. In contrast to their less selective counterparts, kinases that phosphorylate only a small number of proteins tend to have larger interaction surfaces that confer stringent specificity 29, 30. A recently studied example is the interaction between LIM kinase (LIMK) and its major substrate, the actin depolymerizing factor cofilin. LIMK phosphorylates cofilin family proteins at a single site near their N termini (Ser2). The recently solved structure of the LIMK1 catalytic domain in complex with cofilin revealed an extensive binding interface involving a region of the kinase C-terminal lobe adjacent to the catalytic cleft [31] (Figure 2D). This region includes a conserved structural feature of the kinase (helix αG) that has previously been observed to engage in kinase docking interactions [29]. The docking interaction appears to guide the phosphorylation site residue into the catalytic center without making canonical main chain interactions with the activation loop [31]. Instead, the N-terminal region binds in a manner perpendicular to the typical substrate binding mode. Presumably the additional binding energy provided by the interaction surface can compensate for the loss of main chain hydrogen bonds typically thought to be essential for substrate binding.