title: Dissection of CDK4-binding and transactivation activities of p34(SEI-1) and comparison between functions of p34(SEI-1) and p16(INK4A).
abstract: Recent studies showed that p34(SEI-1), also known as TRIP-Br1 or SEI-1, plays a dual role in the regulation of cell-cycle progression. It exhibits the transactivation activity and regulates a number of genes required for G1/S transition, while it also binds and activates cyclin-dependent kinase 4 (CDK4) independent of the inhibitory activity of p16. The goals of this paper are to further dissect the two roles and to compare the functions between SEI-1 and p16. (i) Yeast one-hybrid-based random mutagenesis was first used to identify a number of SEI-1 residues important for LexA-mediated transactivation, including residues L51, K52, L53, H54, L57, and L69 located within the heptad repeat (residues 30-88), a domain required for LexA-mediated transactivation, and two residues M219 and L228 at the C-terminal segment that contributes to transactivation through modulating the heptad repeat. (ii) The functional significance of these residues was further confirmed by site-directed mutagenesis. It was also shown that the heptad repeat-involving transactivation is distinct from the well-known acidic region-involving transactivation. (iii) Yeast two-hybrid-based binding analysis was made possible with the transactivation-negative SEI-1 mutants, and the results showed that some of such mutants retain full ability to bind and activate CDK4. (iv) Site-specific mutants of CDK4 were used to show that there are notable differences among SEI-1, p16, and cyclin D2 in binding to CDK4. (v) The expression levels of SEI-1 and p16 were compared in 32 tumor specimens of human squamous cell carcinomas of the head and neck. The results indicate that SEI-1 was consistently overexpressed, while p16 was consistently underexpressed. These results provide important information on the molecular mechanism of the functions of SEI-1 and on the comparison between SEI-1 and p16 at both molecular and cellular levels.
authors.affiliation: Department of Surgery, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA. li.225@osu.edu
authors.forename: Junan
authors.initials: J
authors.lastname: Li
authors.forename: Peter
authors.initials: P
authors.lastname: Muscarella
authors.forename: Sang Hoon
authors.initials: SH
authors.lastname: Joo
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Knobloch
authors.forename: W Scott
authors.initials: WS
authors.lastname: Melvin
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Weghorst
authors.forename: Ming-Daw
authors.initials: MD
authors.lastname: Tsai
mesh_terms: D000595:Amino Acid Sequence; D002294:Carcinoma, Squamous Cell / Q000378:metabolism; D003001:Cloning, Molecular; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism / Q000502:physiology*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006258:Head and Neck Neoplasms / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D018076:DNA, Complementary; D009687:Nuclear Proteins; C402464:SERTAD1 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D012313:RNA; D051358:Cyclin-Dependent Kinase 4