Identification and Functional Analysis of a Novel Cyclin E/Cdk2 Substrate Ankrd17

Abstract

Cyclin E/Cdk2 is a key regulator in G 1 -S transition. We have identified a novel cyclin E/Cdk2 substrate called Ankrd17 (ankyrin repeat protein 17) using the TAP tag purification technique. Ankrd17 protein contains two clusters of a total 25 ankyrin repeats at its N terminus, one NES (nuclear exporting signal) and one NLS (nuclear localization signal) in the middle, and one RXL motif at its C terminus. Ankrd17 is expressed in various tissues and associates with cyclin E/Cdk2 in an RXL-dependent manner. It can be phosphorylated by cyclin E/Cdk2 at 3 phosphorylation sites (Ser 1791 , Ser 1794 , and Ser 2150 ). Overexpression of Ankrd17 promotes S phase entry, whereas depletion of Ankrd17 expression by small interfering RNA inhibits DNA replication and blocks cell cycle progression as well as up-regulates the expression of p53 and p21. Ankrd17 is localized to the nucleus and interacts with DNA replication factors including MCM family members, Cdc6 and PCNA. Depletion of Ankrd17 results in decreased loading of Cdc6 and PCNA onto DNA suggesting that Ankrd17 may be directly involved in the DNA replication process. Taken together, these data indicate that Ankrd17 is an important downstream effector of cyclin E/Cdk2 and positively regulates G 1 /S transition.Progression through the cell cycle is driven by the sequential and periodic activation of cyclin/Cdk complexes. Cyclin D/Cdk4/6 4 complexes are active throughout the G 1 phase, cyclin E/Cdk2 at the G 1 /S boundary, cyclin A/Cdk2 during S phase, and cyclin A/Cdk1 and cyclin B/Cdk1 during the G 2 /M transition. Cyclin E/Cdk2 plays a central role in coordinating both the onset of S phase and centrosome duplication in cell cycle (1-4). It presumably exerts most of its biologic activities by phosphorylating its substrates, most frequently through the RXL motif in the substrate that interacts with the cyclin box (5-8). A number of RXL-containing proteins are themselves cell cycle regulatory proteins. In the case of pRb, cyclin E/Cdk2-mediated Rb phosphorylation inactivates Rb by derepressing E2F transcription factors (9, 10), whereas in the case of p27, phosphorylation of p27 by cyclin E/Cdk2 stimulates its degradation by the SCF-Skp2 ubiquitin ligase (11-15).There are a number of proteins identified as cyclin E/Cdk2 substrates that regulate cell division. NPAT is a transcription factor that controls cell cycle-dependent histone gene transcription. The phosphorylation of NPAT by cyclin E/Cdk2 promotes histone transcription (16 -19). Cells without NPAT fail to enter S phase from quiescence (20). CBP/p300 is another protein phosphorylated by cyclin E/Cdk2 at G 1 /S transition to activate its histone acetyltransferase activity (21) and may function as a cofactor for many transcription factors including E2F (22). Interestingly, cyclin E/Cdk2 phosphorylates E2F-5 and increases its transcriptional activity through CBP/p300 recruitment (23). Cyclin E/Cdk2 also phosphorylates centrosomal proteins that regulate centrosome duplication. Phosphorylation of nucleophosmin (NPM) by cyc...

Cyclin E-CDK2 Protein Phosphorylates Plant Homeodomain Finger Protein 8 (PHF8) and Regulates Its Function in the Cell Cycle

“…CREB-binding protein-p300 is phosphorylated by cyclin E-CDK2 at G 1 /S transition to activate its histone acetyltransferase activity (34). We previously identified PHF8 as a novel CDK2-interacting protein using tandem affinity purification (16). PHF8 is a H3K9me2 demethylase and can regulate rDNA transcription and cell cycle progression.…”

“…HeLa cells were synchronized at early S phase by treatment with double thymidine in the same way as described for U2OS cells in a previous report (16). Briefly, cells were synchronized at early S transition by treatment with 2 mM thymidine for 16 h in complete medium, released in fresh medium for 8 h, and then incubated with 2 mM thymidine for another 16 h. Cells were synchronized to early S phase at this point.…”

“…E-4), ␤-actin catalog no. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], and lamin B (catalog no. M-20) antibodies were purchased from Santa Cruz Biotechnology.…”

“…The truncated PHF8 (PHF8 (1-316), PHF8 (317-680), PHF8 (681-1024), and PHF8 mutants PHF8 (317-680) 381AGA, PHF8 (317-680) 641AKA, and PHF8 (681-1024) S844A and full-length CDK2 were cloned into pET41b. The mammalian expression vectors for cyclin E and CDK2 were generated as described previously (16).…”

“…However, many of these substrates remain unknown. To understand the molecular mechanisms of cyclin E-CDK2 and its associated proteins in cell cycle regulation, we employed the tandem affinity purification technique to identify 14 novel proteins interacting with CDK2, including the histone demethylase PHF8 (16). PHF8 belongs to a class of plant homeodomain-containing zinc finger proteins that play an important role in histone demethylation, gene transcription, and chromatin remodeling.…”