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Biologic and biochemical analyses of p16(INK4a) mutations from primary tumors
W G Yarbrough1, R A Buckmire, M Bessho
1Department of Surgery, Division of Otolaryngology/Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599-7295, USA. wgy@med.unc.edu
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.
Results/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.
Insights
Point mutations in the p16 tumor suppressor gene can impair its function. Nine of sixteen analyzed p16 mutants showed defects in binding cyclin-dependent kinases (CDK4/6), inhibiting them, or causing cell cycle arrest.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Point mutations in the p16 tumor suppressor gene are common in various cancers.
- The functional consequences of naturally occurring p16 mutants are not fully understood.
Purpose of the Study:
- To investigate the functional activities of naturally occurring p16 mutant proteins.
- To characterize the impact of mutations on p16's ability to bind and inhibit CDK4/6 and induce cell cycle arrest.
Main Methods:
- Characterized sixteen cancer-associated p16 missense mutants.
- Assessed binding and inhibition of cyclin-dependent kinases (CDK4 and CDK6).
- Evaluated induction of G(1) cell cycle arrest.
Main Results:
- Nine out of sixteen p16 mutants exhibited functional defects.
- Three mutants (D84V, D84G, R87P) showed defects in CDK binding, kinase inhibition, and cell cycle arrest.
- Three mutants (P48L, D74N, R87L) had defects in kinase inhibition and cell cycle arrest.
- Three mutants (N71S, R80L, H83Y) retained CDK binding and inhibition but failed to induce cell cycle arrest.
Conclusions:
- p16 mutant proteins retaining CDK4/6 binding can still have subtle functional defects.
- Mutations affecting p16 function map to the central portion of the protein.
- Ankyrin repeats II and III are critical for p16 function; mutations in repeats I and IV are less disruptive.