Dissection of CDK4-binding and transactivation activities of p34 SEI-1 and comparison between functions of p34SEI-1 and p16INK4A

Junan Li, Peter Muscarella, Sang Hoon Joo, Thomas J. Knobloch, W. Scott Melvin, Christopher M. Weghorst, Ming Daw Tsai

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Recent studies showed that p34SEI-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.

Original languageEnglish (US)
Pages (from-to)13246-13256
Number of pages11
JournalBiochemistry
Volume44
Issue number40
DOIs
StatePublished - Oct 11 2005
Externally publishedYes

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Cyclin-Dependent Kinase 4
Dissection
Transcriptional Activation
Mutagenesis
Yeast
Cyclin D2
Yeasts
Tumors
Genes
Cells
Site-Directed Mutagenesis
Cell Cycle

ASJC Scopus subject areas

  • Biochemistry

Cite this

Dissection of CDK4-binding and transactivation activities of p34 SEI-1 and comparison between functions of p34SEI-1 and p16INK4A. / Li, Junan; Muscarella, Peter; Joo, Sang Hoon; Knobloch, Thomas J.; Melvin, W. Scott; Weghorst, Christopher M.; Tsai, Ming Daw.

In: Biochemistry, Vol. 44, No. 40, 11.10.2005, p. 13246-13256.

Research output: Contribution to journalArticle

Li, Junan ; Muscarella, Peter ; Joo, Sang Hoon ; Knobloch, Thomas J. ; Melvin, W. Scott ; Weghorst, Christopher M. ; Tsai, Ming Daw. / Dissection of CDK4-binding and transactivation activities of p34 SEI-1 and comparison between functions of p34SEI-1 and p16INK4A. In: Biochemistry. 2005 ; Vol. 44, No. 40. pp. 13246-13256.
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abstract = "Recent studies showed that p34SEI-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.",
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AU - Li, Junan

AU - Muscarella, Peter

AU - Joo, Sang Hoon

AU - Knobloch, Thomas J.

AU - Melvin, W. Scott

AU - Weghorst, Christopher M.

AU - Tsai, Ming Daw

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