Tumor Suppressor INK4:  Quantitative Structure−Function Analyses of p18^INK4C as an Inhibitor of Cyclin-Dependent Kinase 4

Abstract

We report the first detailed structure-function analyses of p18 INK4C (p18), which is a homologue of the important tumor suppressor p16 INK4A (p16). Twenty-four mutants were designed rationally. The global conformations of the mutants were characterized by NMR, while the function was assayed by inhibition of cyclin-dependent kinase 4 (CDK4). Most of these mutants have unperturbed global structures, thus the changes in their inhibitory abilities can be attributed to the mutated residues. The important results are summarized as follows: (a) some residues at loops 1 and 2, but not 3, are important for the inhibitory function of p18, similar to the results for p16; (b) two residues at the first helix-turn-helix motif and two at the third are important for inhibition; (c) while the results generally agree with the prediction based on the crystal structures of p16-CDK6 and p19-CDK6 binary complexes, there are significant differences in a few residues, suggesting that the interactions in the binary complexes may not accurately represent the interactions in the ternary complexes (in the presence of cyclin D2); (d) most importantly, the extra loop of p18 appears to contribute to the function of p18, even though the crystal structure of the p19 INK4D -CDK6 complex indicates no interactions involving this loop; (e) detailed analyses of the crystal structures and the functional results suggest that there are notable differences in the interactions between different members of the INK4 family and CDKs. INK41 proteins are a group of ankyrin repeat-containing proteins, which specifically bind to and inactivate cyclindependent kinases 4 and 6 (1-6). Currently, there are four known members of the INK4 family, p16INK4A , p15 INK4B , p18 INK4C , and p19 INK4D (which are abbreviated as p16, p15, p18, and p19, respectively, in this paper) (7-13). While p15 and p16 consist of four ankyrin repeats (ankyrin I-IV), p18 and p19 have five (ankyrin I-V). An ankyrin repeat is a motif of about 34 amino acid residues and is present in a large number of proteins. Ankyrin repeats exist in groups of four or more and are often involved in protein-protein interaction (14,15). Since 1997, the tertiary structures of p19 (16), p18 (17, 18), p16 (19), and p15 (20) (15,16,19,20). Figure 1 shows the correlation between p16, p15, p18, and p19 in terms of residue numbers and the locations of helices (e.g., helix IA stands for the first helix of the first ankyrin) and loops (e.g., loop 1 stands for the first loop, connecting ankyrin I and ankyrin II). Another major advance in the field is the recent solution of the crystal structures of p16-CDK6 (21) and p19-CDK6 (22) binary complexes. In the crystal structure of the p16-CDK6 complex, p16 binds to one side of the catalytic cleft of CDK6, opposite the cyclin-binding site. The concave surface of p16 contacts the N lobe of CDK6, while the loops between neighboring ankyrin repeats interact with the C lobe. As a result, the interactions between p16 and CDK6 involve multiple regions in both proteins. M...

Dissection of Protein–Protein Interaction and CDK4 Inhibition in the Oncogenic versus Tumor Suppressing Functions of Gankyrin and P16

“…As revealed in the crystal structure of P16/CDK6 15 (a close homolog of CDK4), and other biochemical studies [16][17][18] (Figure 1), contacts between P16 (or other INK4 proteins, in general) and CDK4 occur in discontinuous patches, and a number of residues located in both loop and helical regions of P16 contribute to CDK4 binding through electrostatic, hydrogen bonding, and van der Waals interactions 3 . While mutations of most of CDK4-interacting P16 residues only lead to mild decrease in its inhibitory activity, the D84H mutant of P16 (a mutant found in human cancers), or the corresponding mutant of P18, D76A, loses all inhibitory activity even though these two mutants are still able to bind to CDK4 16,18 . On the other hand, it has been reported that CDK4 R24C, a mutant frequently found in human cancers, retains its kinase activity but is resistant to P16 inhibition 19,20 .…”

Ankyrin Repeat: A Unique Motif Mediating Protein−Protein Interactions

“…On one hand, ARs do not recognize specific sequences of targets, unlike SH2 that recognizes sequences with phosphorylated Tyr (70) and SH3 that binds to sequences that are usually proline rich and exhibit a polyproline II helix conformation (71). On the other hand, AR binding involves discontinuous patches of residues dispersed in the whole molecules of both the AR protein and its target (18,57,72), while SH2 or SH3 binding usually involves a localized region of SH2 or SH3 and a very short motif in the target. From this point of view, binding of AR proteins to targets is similar to the association of Fv and Fab fragments with antigens.…”

An NF-κB-Specific Inhibitor, IκBα, Binds to and Inhibits Cyclin-Dependent Kinase 4

“…Error bars represent standard deviations. their targets (23,27). On the basis of the results of competition experiments, the IκBR67-302 binding site of CDK4 should overlap, at least partially, with the INK4 binding site of CDK4.…”

“…GST-tagged and nontagged p16, p18, p18 D76A, and Yar-1 proteins were expressed and purified as described previously (3,23,24).…”