Chen I T, Akamatsu M, Smith M L, Lung F D, Duba D, Roller P P, Fornace A J, O'Connor P M
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Oncogene. 1996 Feb 1;12(3):595-607.
The cyclin-dependent kinase inhibitor p21Cip1/Waf1 is responsible for the p53-dependent growth arrest of cells in G1 phase following DNA damage. In the present study we investigated regions of p21 involved in inhibition of the G1/S phase cyclin-dependent kinase, cyclin E/Cdk2, as well as regions of p21 important for binding to this kinase and recombinant PCNA. To perform these studies we synthesized a series of overlapping peptides spanning the entire p21 sequence and used them in in vitro assays with cyclin E/Cdk2-immune complexes and with recombinant p21 and PCNA proteins. One amino-terminal p21 peptide spanning amino acids 15-40, antagonized p21 binding and inhibition of cyclin E/Cdk2 kinase. Antagonism of p21 binding was, however, lost in a similar peptide lacking amino acids 15-20, or in a peptide in which cysteine-18 was substituted for a serine. These results suggest that this peptide region is important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also antagonized p21-activity, but this peptide did not affect the ability of p21 to interact with cyclin E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides we tested inhibited cyclin E/Cdk2. We also found that a peptide spanning amino acids 21-45 bound recombinant p21 in ELISA assays, and additional studies revealed a requirement for amino acids 26 through 45 for this interaction. A p21 peptide spanning amino acids 139-164 was found to bind PCNA in a filter binding assay and this peptide suppressed recombinant p21-PCNA interaction. Conformational analysis revealed that peptides spanning amino acids 21-45 and 139-164 tended towards an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein. Taken together, our results provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction. Our results also suggest that a potential p21 dimerization domain may lie in the amino-terminus of p21. Continued exploration of these domains could prove useful in assessing p21-mimetic strategies for cancer treatment.
细胞周期蛋白依赖性激酶抑制剂p21Cip1/Waf1负责DNA损伤后细胞在G1期的p53依赖性生长停滞。在本研究中，我们研究了p21中参与抑制G1/S期细胞周期蛋白依赖性激酶、细胞周期蛋白E/Cdk2的区域，以及p21中对与该激酶和重组增殖细胞核抗原（PCNA）结合很重要的区域。为了进行这些研究，我们合成了一系列覆盖整个p21序列的重叠肽，并将它们用于与细胞周期蛋白E/Cdk2免疫复合物以及重组p21和PCNA蛋白的体外试验。一个覆盖第15至40位氨基酸的p21氨基末端肽拮抗了p21与细胞周期蛋白E/Cdk2激酶的结合及抑制作用。然而，在一个缺少第15至20位氨基酸的类似肽中，或者在一个将半胱氨酸-18替换为丝氨酸的肽中，p21结合的拮抗作用丧失了。这些结果表明，该肽区域对p21与细胞周期蛋白E/Cdk2的相互作用很重要。第二个肽（第58至77位氨基酸）也拮抗p21的活性，但该肽不影响p21与细胞周期蛋白E/Cdk2相互作用的能力。可能需要一个大于26个氨基酸的p21区域来抑制细胞周期蛋白依赖性激酶（Cdk），因为我们测试的肽都没有抑制细胞周期蛋白E/Cdk2。我们还发现，一个覆盖第21至45位氨基酸的肽在酶联免疫吸附测定（ELISA）中与重组p21结合，进一步的研究表明该相互作用需要第26至45位氨基酸。在滤膜结合试验中发现一个覆盖第139至164位氨基酸的p21肽与PCNA结合，并且该肽抑制了重组p21与PCNA的相互作用。构象分析表明，覆盖第21至45位氨基酸和第139至164位氨基酸的肽在三氟乙醇缓冲液中倾向于α螺旋构象，这表明这些区域在天然蛋白中可能处于卷曲构象。综上所述，我们的结果为p21中参与细胞周期蛋白E/Cdk2和PCNA相互作用的结构域提供了深入了解。我们的结果还表明，一个潜在的p21二聚化结构域可能位于p21的氨基末端。对这些结构域的持续探索可能有助于评估用于癌症治疗的p21模拟策略。
