Tumor suppressor INK4: quantitative structure-function analyses of p18INK4C as an inhibitor of cyclin-dependent kinase 4.
We report the first detailed structure-function analyses of p18INK4C (p18), which is a homologue of the important tumor suppressor p16INK4A (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 p19INK4D-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.
Li
J
J
Department of Biochemistry, Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.
Poi
M J
MJ
Qin
D
D
Selby
T L
TL
Byeon
I J
IJ
Tsai
M D
MD
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018797:Cell Cycle Proteins; D018360:Crystallography, X-Ray; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D050763:Cyclin-Dependent Kinase Inhibitor p18; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D016147:Genes, Tumor Suppressor; D019077:Helix-Turn-Helix Motifs; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D025521:Tumor Suppressor Proteins
C493971:CDKN2C protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050763:Cyclin-Dependent Kinase Inhibitor p18; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D017346:Protein Serine-Threonine Kinases; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases