Oncogenes and Tumor Suppressors Mitotic Arrest by Tumor Suppressor RASSF1A Is Regulated via CHK1 Phosphorylation

The tumor suppressor RAS-association domain family 1 isoform A (RASSF1A) is known to play an important role in cell-cycle regulation. However, the molecular details about RASSF1A protein regulation are unclear. In this report, checkpoint kinase 1 (CHK1) is identified as a novel RASSF1A kinase that phosphorylates RASSF1A in vitro and under cellular conditions. Using tandem mass spectrometry and biochemical analysis, it was determined that CHK1phosphorylates RASSF1Aon Serine 184, which has been shown to bemutated in a subset of human primary nasopharyngeal carcinomas. Furthermore, Serine 184 phosphorylation of RASSF1A was significantly diminished by a CHK1-specific kinase inhibitor. Similarly, a kinase-dead CHK1 mutant was unable to phosphorylate Serine 184 whereas constitutively active-CHK1 enhanced phosphorylation. Molecular substitution of Serine 184 with aspartic acid, mimicking phosphorylation, abolished the ability of RASSF1A to interact with microtubules and induceM-phase arrest. Combined, these data indicate that phosphorylation ofRASSF1AbyCHK1 is important for mitotic regulation and provide valuable new insight into the regulatory mechanisms of RASSF1A function. Implications: This study reveals that CHK1-mediated phosphorylation of RASSF1A, at Serine 184, plays an important role in cell-cycle regulation and highlights that mutation of this CHK1 phosphorylation site in nasopharyngeal carcinoma has disease relevance. Mol Cancer Res; 12(1); 119–29. 2013 AACR.