Biologic and biochemical analyses of p16(INK4a) mutations from primary tumors.
BACKGROUND
Point mutations in the tumor suppressor gene p16(INK4a) (also known as p16, CDKN2, MTS1, and INK4a) are found in many tumor types. Because the function of the products of these naturally occurring mutants has not been fully explored, we investigated the functional activities of a wide range of naturally occurring p16 mutant proteins.


METHODS
Sixteen cancer-associated p16 mutant proteins, resulting from missense mutations, were characterized for their ability to bind and inhibit the cyclin-dependent kinases (CDK4 and CDK6) and to induce cell cycle arrest in G(1) phase.


CONCLUSIONS
Among 16 mutants analyzed, nine had detectable functional defects. Three mutants (D84V, D84G, and R87P) had defects in CDK binding, kinase inhibition, and cell cycle arrest. The corresponding mutations are located in the third ankyrin repeat in a highly conserved region believed to form the CDK binding cleft. Three mutants (P48L, D74N, and R87L) had defects in kinase inhibition and cell cycle arrest. Among the 10 mutants with normal CDK binding and inhibitory activity, three mutants (N71S, R80L, and H83Y) had defects only in their ability to induce cell cycle arrest. Thus, p16 mutant proteins that retain CDK4 and CDK6 binding may have more subtle functional defects. All nine mutations leading to functional impairments mapped to the central portion of the p16 protein. Ankyrin repeats II and III appear more critical to p16 function, and mutations in ankyrin repeats I and IV are less likely to disrupt p16 function.
Yarbrough
W G
WG
Department of Surgery, Division of Otolaryngology/Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599-7295, USA. wgy@med.unc.edu
Buckmire
R A
RA
Bessho
M
M
Liu
E T
ET
D000595:Amino Acid Sequence; D017487:Ankyrins / Q000235:genetics*; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016193:G1 Phase / Q000235:genetics*; D019942:Genes, p16; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins
D017487:Ankyrins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D011518:Proto-Oncogene Proteins; D005982:Glutathione Transferase; 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