Structural consequences of tumor-derived mutations in p16INK4a probed by limited proteolysis.
The cyclin-dependent kinase inhibitor p16(INK4a) (hereafter p16) functions as a multiple tumor suppressor. Mutations in p16, which are distributed throughout the entire protein, have been identified in a variety of human cancers and cancer-derived cell lines. It is unclear how tumor-derived mutations disrupt the structure and function of p16, especially since many of these mutations are located far away from the cyclin-dependent kinase binding site. In this study, we investigated the effect of two tumor-derived mutations, P81L and V126D, on the structure of p16 by limited proteolysis. The proteolytic products were characterized by gel electrophoresis, HPLC, and mass spectrometry. Our results show that the N-terminal half of p16 is significantly more sensitive to proteolysis in both tumor-derived mutant proteins than in the wild type, suggesting that this region is particularly unstable. Interestingly, the N-terminal half of p16 contains many residues that are important for cyclin-dependent kinase binding. Thus, our results provide a structural mechanism by which tumor-derived mutations inactivate the function of p16 and suggest that stabilization of the N-terminal region could be a useful strategy for future therapeutic development.
Zhang
Bin
B
Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06032, USA.
Peng
Zheng-yu
ZY
D017089:Ankyrin Repeat / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002942:Circular Dichroism; 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 / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007930:Leucine / Q000235:genetics; D008545:Melanoma / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D013329:Structure-Activity Relationship; D014633:Valine / Q000235:genetics
D019941:Cyclin-Dependent Kinase Inhibitor p16; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D001224:Aspartic Acid; D011392:Proline; 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; D019286:Endopeptidase K; D007930:Leucine; D014633:Valine