Mitotic Arrest by Tumor Suppressor RASSF1A Is Regulated via CHK1 Phosphorylation

Abstract

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 CHK1 phosphorylates RASSF1A on Serine 184, which has been shown to be mutated 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 induce M-phase arrest. Combined, these data indicate that phosphorylation of RASSF1A by CHK1 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.

Dasatinib induces DNA damage and activates DNA repair pathways leading to senescence in non-small cell lung cancer cell lines with kinase-inactivating BRAF mutations

“…RASSF1A binds to and stabilizes MST1/2 leading to activation of the HIPPO pathway [ 38 ]. ATM, Aurora A and Chk1 can phosphorylate RASSF1A leading to its activation [ 39 , 40 ]. Lats1/2 phosphorylates the transcriptional co-activators YAP and TAZ that results in their ubiquitin-mediated proteolysis.…”

Hippo pathway and protection of genome stability in response to DNA damage

“…In response to the ATR/Chk1 activation, RASSF1A is also targeted by Chk1 at Ser184. Chk1 phosphorylation decreases RASSF1A association with the microtubules . RASSF1A has been shown to associate with α‐tubulin and affect microtubule polymerization and mitotic progression .…”

RASSF tumor suppressor gene family: Biological functions and regulation

“…RASSF1A phosphorylation mimicking mutant abolishes the ability of RASSF1A to induce M-phase arrest [170] ATM (Ataxia telangiectasia mutated) S131 Phosphorylation is required for the activation of MST2 and LATS1, which leads to p73 stabilization [11] PKC (Protein kinase C) S197 and S203…”

“…Phosphorylation is required for Skp2 dependent degradation of RASSF1A [166] Chk1 (checkpoint kinase) S184 Phosphorylation causes RASSF1A to dissociate from microtubules and alters RASSF1A distribution in the cell, with ''punctate distribution pattern'', not ''network and fiber-like''. RASSF1A phosphorylation mimicking mutant abolishes the ability of RASSF1A to induce M-phase arrest [170] ATM (Ataxia telangiectasia mutated) S131 Phosphorylation is required for the activation of MST2 and LATS1, which leads to p73 stabilization [11] PKC (Protein kinase C) S197 and S203…”

“…Recently, several other kinases, including ATM (ataxia telangiectasia mutated) [11], CDK4 (cyclin-dependent kinase-4) [166], PKC (protein kinase C) [167], MST1 [168], Aurora A [165], Aurora-B [169], and Chk1 [170] have also been shown to phosphorylate RASSF1A. Each of these kinases seems to regulate different aspects of RASSF1A function (see Table 3), suggesting that regulation of RASSF1A by phosphorylation is a complex process with multiple kinases involved that modulate RASSF1A function needed for various cellular processes.…”