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.
Highlights
Cyclin/cyclindependent kinases (Cdks) are orderly activated during cell cycle progression, it is their phosphorylation substrates that carry out the ordered cellular changes that lead to cell division
Association of Ankrd17 with Cdk2 in Vitro and in Vivo—To identify potential Cdk2-associated proteins, a TAP tag purification procedure was performed. 293T cells were transduced with Cdk2 expression negative mode), the enhanced resolution scan was per- retrovirus pMSCV-c-FLAG-HA-Cdk2
The PCNA and MCM3 positive cells were scored and the results showed that the ratio of PCNA positive cells in CSK-extracted cells was significantly lower in the Ankrd17depleted cells when compared with controls (Fig. 8C, left panel), whereas the percentage of PCNA positive cells did not change in unextracted cells
Summary
TAP Tag Purification of Cdk2-associated Proteins—pMSCVc-FLAG-HA-Cdk retroviral vector was transfected into the virus packaging cell line phoenix, the viruses were harvested 48 h after transfection and subjected to infect 293T cells. Cell lysates were incubated with anti-FLAG beads (Sigma), washed with TAP buffer (20 mM Tris-HCl, pH 8.0, 10% glycerol, 5 mM MgCl2, 100 mM KCl, 0.1% Tween 20), and eluted with FLAG peptide (Sigma) (0.25 mg/ml). GST fusion proteins immobilized on glutathione beads were incubated with the lysates from 293T cells transfected with Myc-cyclin E and MycCdk in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Triton X-100, and protease inhibitor) (Roche Applied Science). Total cell lysates were incubated with antibodies at 4 °C for 2 h followed by the addition of protein A-agarose beads. After another 2 h of incubation at 4 °C, the beads were washed with lysis buffer.
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