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.The newly defined gene product, p34 SEI-1 , also known as TRIP-Br1 1 (hereafter abbreviated as SEI-1), has been shown to exhibit multiple biological functions (1-3): (a) SEI-1 specifically binds to multiple PHD zinc finger-and/or bromodomain-containing transcription factors, such as KRIP-1 and p300/CBP, and activates/represses a number of genes important for signal transduction and cell-cycle progression (1). (b) SEI-1 directly binds to DP1 and stimulates the transcriptional activity of the E2F1/DP1 complex in a manner that is regulated by Rb and E1A proteins, thus triggering genes required for the G1/S transition (1). (c) SEI-1 physically contacts cyclin-dependent kinase 4 (CDK4) through a domain distinct from its PHD-bromodomaininteracting region and stimulates CDK4-mdeiated phosphorylation of Rb (2, 3). In the presence of tumor suppressor p16, a quaternary complex is formed between SEI-1, CDK4, cyclin D2, and p16, and the stimulation of SEI-1 renders the kinase resistant to the inhibitory effect of p16. (d) In a yeast one-hybrid system, SEI-1 turns on the lac Z reporter † This work was supported by a research grant CA69472

CDCA4 Is an E2F Transcription Factor Family-induced Nuclear Factor That Regulates E2F-dependent Transcriptional Activation and Cell Proliferation

“…INK4a (33,66,67). If this is the case for CDCA4, CDCA4 can regulate cell proliferation by modulating the kinase activity of G 1 /S cyclin-Cdk complexes in addition to repressing the transcriptional activity of E2F1.…”

“…TRIP-Br1 is consistently overexpressed in human squamous cell carcinomas of the head and neck (67). High level amplification of TRIP-Br1 at chromosome 19q13.1 is commonly detected in ovarian cancer (70).…”

Transcriptional and subcellular regulation of the TRIP-Br family

“…SERTA domains appear in several proteins including SEI-1 (later renamed TRIP-Br1), RBT1 (RPA-binding transactivator) (Cho et al, 2000), and TARA (Marchler- Bauer and Bryant, 2004). This domain is required for the interaction between TRIP-Br1 and CDK4 (Sugimoto et al, 1999;Li et al, 2005), but the function of the SERTA domain remains unclear. The PHD-Bromo binding domain is required for proteinprotein interaction between TRIP-Br1 and KAP1 (Hsu et al, 2001).…”

“…TRIP-Br1, by interacting with CDK4, affects the kinase activity of CDK4 (Sugimoto et al, 1999;Li et al, 2005). At low serum levels, TRIP-Br1 promotes cell growth by antagonizing the function of the cdk inhibitor p16INK4a.…”

Tumor Suppressor Gene p16/INK4A/CDKN2A and Its Role in Cell Cycle Exit, Differentiation, and Determination of Cell Fate

“…p16 has also been associated with a variety of additional cell proliferation control proteins that either bind and suppress its function or compete for p16 targets. SEI-1/p34/TRIP-Br1 protein induces CDK4-mediated Rb phosphorylation through physical binding, independent of p16 (Li et al, 2005). SEI-1 facilitates CDK4 function making it resistant to p16 inhibition.…”