野生型p16INK4A能够与CDK4或CDK6形成复合物，并阻止由CDK4或CDK6和d型细胞周期蛋白(CCND)组成的催化活性CDK复合物的形成。在癌症中发现的几个CDKN2A错义突变导致p16INK4A的氨基酸替换，削弱了p16INK4A突变与CDK4和CDK6的结合。p16INK4A的错义突变是隐性的，通常与其他CDKN2A等位基因的基因组缺失或表观遗传沉默结合发现(Kamb et al. 1994, Castellano et al. 1997, Liew et al. 1999)。p16INK4A编码序列的失活突变也可能伴随着杂合性(LOH)的缺失(Castellano et al. 1997, Kumar et al. 1999, Liew et al. 1999)。功能测试的p16INK4A突变体不能结合CDK4或CDK6或显示很少的残余结合，不能抑制细胞增殖:p16INK4A A20P (Ruas等人1999,Jones等人2007)p16INK4A M53I (Harland等人1997,Walker等人1999，p16INK4A P81L (Walker et al. 1999)p16INK4A P81T (Kannengiesser et al. 2009, McKenzie et al. 2010)p16INK4A D84G (Yarbrough et al. 1999)p16INK4A D84H (Ruas et al. 1999)p16INK4A D84N (Ruas et al. 1999)p16INK4A D84V (Yarbrough et al. 1999)p16INK4A D84Y (Ruas et al. 1999)p16INK4A D84Y (Ruas et al. 1999)p16INK4A R87P (Walker et al. 1999)Yarbrough et al. 1999)p16INK4A G101W (Walker et al. 1999, Kannengiesser et al. 2009, McKenzie et al. 2010, Scaini et al. 2014)p16INK4A P114L (Harland et al. 1997)p16INK4A V126D (Walker et al. 1999, Becker et al. 2001)基于受影响的氨基酸残基，以下p16INK4A错意突变体尚未检测其与CDK4或CDK6结合的能力，但已在癌症中报道并预测其具有致病性(COSMIC数据库:福布斯et al . 2017年)被注解为候选人:p16INK4A分子A20E p16INK4A分子A20T p16INK4A分子P81H p16INK4A分子P81R p16INK4A分子P81S p16INK4A分子D84A p16INK4A分子G101V p16INK4A分子P114H p16INK4A分子P114R p16INK4A分子P114S p16INK4A分子P114T p16INK4A分子V126A p16INK4A分子V126F p16INK4A分子V126I .p16INK4A P114S被证明有降低绑定到(Kannengiesser et al . 2009)和一个抑制细胞增殖的能力降低(Scaini等，2014)。 p16INK4A A20S retains the ability to bind to CDK4 and CDK6 and to inhibit cellular proliferation (Yarbrough et al. 1999). p16INK4A R87W (Walker et al. 1999) and p16INK4A R87L (Yarbrough et al. 1999) retain the ability to bind to CDK4 and CDK6, but are unable to induce cell cycle arrest. Mutants p16INK4A A20S, p16INK4A R87W and p16INK4A R87L have not been annotated.Some p16INK4A missense mutants are temperature sensitive, and their ability to bind to CDK4 and CDK6 can only be properly assessed at the physiological temperature of 37 degrees Celsius (Becker et al. 2001). Not controlling experimental temperature can be one source of inconsistencies when evaluating functionality of p16INK4A mutants, but many other variabilities in experimental systems and techniques can also influence the results of binding assays. A p16INK4A mutant with a preserved ability to bind to CDK4 and CDK6 may still not be able to inhibit their cyclin-dependent activation. However, the loss of CDK inhibitory function in p16INK4A mutants that do bind to CDK4 and CDK6 has not been tested directly.Nonsense mutations in the second exon of the CDKN2A gene that lead to premature termination of p16INK4A mRNA translation are frequent in cancer. While the mRNAs of predicted p16INK4A truncation mutants can be detected, the truncated proteins cannot:p16INK4A R58* (Castellano et al. 1997)p16INK4A R80* (Fahham et al. 2010)p16INK4A E88* (Castellano et al. 1997)p16INK4A W110* (Castellano et al. 1997)In addition, it was shown that the C-terminal half of p16INK4A is critical for binding to CDK4 and CDK6, and inhibition of cellular proliferation (Fahham et al. 2010).The following nonsense and frameshift truncation mutants have not been functionally tested and are annotated as candidates:p16INK4A E10* p16INK4A S12* p16INK4A W15* p16INK4A E26* p16INK4A E27* p16INK4A E33* p16INK4A Y44* p16INK4A Q50* p16INK4A E61* p16INK4A E69* p16INK4A C72* p16INK4A P75*p16INK4A E119* p16INK4A E120* The following recurrent frameshift truncations mutants that lack the C-terminal half of wild type p16INK4A and are therefore assumed to be unable to bind to CDK4 or CDK6 are also annotated as candidates:p16INK4A S7fs*8 p16INK4A W15fs*1 p16INK4A L16fs*9 p16INK4A T18fs*15 p16INK4A T18fs*8 p16INK4A G23fs*3 p16INK4A A36fs*17 p16INK4A L37fs*16 p16INK4A N39fs*14 p16INK4A Y44fs*1