Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells.
High-fidelity maintenance of genomic integrity in eukaryotes is ensured by cell cycle checkpoints and DNA repair. The checkpoint kinase, Chk2, has been implicated in both of these responses. In response to DNA damage, Chk2 is initially phosphorylated at Thr-68, which leads to its full activation. The fully activated Chk2 then phosphorylates downstream substrates of cell cycle control. However, the mechanism of inactivation of Chk2 is still unknown. Protein phosphatase type 2A (PP2A) plays an essential role in cell cycle regulation and induction of G2 arrest by a mechanism of phosphorylation/dephosphorylation with a variety of protein kinases. Data from our investigation provide evidence that, in response to cisplatin exposure, PP2A associates with Chk2 as a complex in cells and functions as a negative regulator of Chk2 activation by dephosphorylating p-Chk2. Results from immunostaining and coimmunoprecipitation demonstrate that Chk2 and PP2A can colocalize in cells, and the holoenzyme of PP2A (subunits A, B and C) coimmunoprecipitates with p-Chk2. Further, inhibition of PP2A by okadaic acid, an inhibitor of PP2A, and by small interfering RNA (siRNA) to PP2A results in enhanced Chk2 phosphorylation, implicating a direct enzyme-substrate relationship. An in vitro PP2A dephosphorylation assay shows that PP2A dephosphorylates p-Chk2 in a cell-free system. These findings suggest that the protein serine/threonine kinase, Chk2, is activated after cisplatin exposure and negatively regulated by a tightly associated protein serine/threonine phosphatase, PP2A.
Liang
Xiaobing
X
Department of Biochemistry and Molecular Pharmacology, Mary Babb Randolph Cancer Center, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506-9300, USA.
Reed
Eddie
E
Yu
Jing Jie
JJ
D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2; D002945:Cisplatin / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D019319:Okadaic Acid / Q000494:pharmacology; D010051:Ovarian Neoplasms / Q000378:metabolism / Q000473:pathology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D019319:Okadaic Acid; D013912:Threonine; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D002945:Cisplatin