Role for KAP1 Serine 824 Phosphorylation and Sumoylation/Desumoylation Switch in Regulating KAP1-mediated Transcriptional Repression

Abstract

As a multifunctional protein, KRAB domain-associated protein 1 (KAP1) is reportedly subjected to multiple protein posttranslational modifications, including phosphorylation and sumoylation. However, gaps exist in our knowledge of how KAP1 phosphorylation cross-talks with KAP1 sumoylation and what the biological consequence is. Here, we show that doxorubicin (Dox) treatment induces KAP1 phosphorylation at Ser-824 via an ataxia telangiectasia mutated (ATM)-dependent manner, correlating with the transcriptional de-repression of p21 WAF1/CIP1 and Gadd45␣. A S824A substitution of KAP1, which ablates the ATM-induced phosphorylation, results in an increase of KAP1 sumoylation and repression of p21 transcription in Dox-treated cells. By contrast, a S824D mutation of KAP1, which mimics constitutive phosphorylation of KAP1, leads to a decrease of KAP1 sumoylation and stimulation of p21 transcription before the exposure of Dox. We further provide evidence that SENP1 deSUMOylase is involved in activating basal, but not Dox-induced, KAP1 Ser-824 phosphorylation, rendering a stimulation of p21 and Gadd45␣ transcription. Moreover, KAP1 and differential sumoylation of KAP1 were also demonstrated to fine-tune the transcription of three additional KAP1-targeted genes, including Bax, Puma, and Noxa. Taken together, our results suggest a novel role for ATM that selectively stimulates KAP1 Ser-824 phosphorylation to repress its sumoylation, leading to the de-repression of expression of a subset of genes involved in promoting cell cycle control and apoptosis in response to genotoxic stresses.The Krüppel-associated box zinc finger proteins (KRAB-ZFP) comprise approximately one-third of the 799 different zinc finger proteins, constituting the largest single-family transcriptional regulators in mammals (1). KRAB domainassociated protein 1 (KAP1) 3 functions as transcriptional corepressor for ZBRK1, a KRAB-ZFP member, by acting as a transcription intermediary factor to connect KRAB-ZFPs to transcriptional repression machinery. Because KAP1 itself cannot bind DNA directly, the specificity of transcriptional repression is dictated by its interaction with ZBRK1 through proteinprotein interaction. The RING finger-B box-coiled-coil domain of KAP1 associates with the KRAB domain of ZBRK1, repressing the transcription of DNA damage-responsive gene Gadd45␣ (2) and p21 WAF1/CIP1 (3). KAP1 can recruit and coordinate several components of gene silencing machinery. For example, KAP1 interacts with histone deacetylase complex NuRD and N-CoR1 and binds to histone methyltransferase SETDB1 to modify the configuration of chromatin structures (4 -6). KAP1 also recruits heterochromatin protein 1 (HP1) to histones through a PXVXL motif (7,8). In addition, KAP1 is identified as a Mdm2-binding protein that inactivates p53 (9, 10).Emerging evidence supports the idea that post-translational modifications, including phosphorylation and sumoylation, play a pivotal role in regulating transcriptional control in response to different extracellular milieu. As a multi...

Structural insights into human KAP1 PHD finger–bromodomain and its role in gene silencing

“…Unlike other SUMO E3 ligases-such as the RING/U-box and HECT domain E3s 39 , which commonly function to bring together an activated E2 SUMO and a substrate to promote SUMO conjugation-the KAP1 PHD finger-bromodomain is an intramolecular SUMO E3 ligase, thus ensuring ordered and stoichiometric site-specific SUMOylation at several lysine residues within the bromodomain and in the region N-terminal to the PHD finger, which together are required for KAP1's co-repression activity 33,41,42 .…”

Anticancer mechanisms of doxorubicin and zoledronic acid in breast cancer tumor growth in bone

“…Doxorubicin inhibits cellular proliferation via p53-dependent and p53-independent mechanisms (43)(44)(45). In p53 wild-type MCF7 breast cancer cells, doxorubicin treatment causes an accumulation of p53 (45)(46)(47)(48), p53 recruits p21 (46) leading to decreased expression of cyclin B and cdc2 resulting in a cell cycle block at G 2 -M (47, 48). However, MDA-MB-436 cells express mutated p53 and it is unlikely that increases in p53 expression seen in these tumors are capable of recruiting p21 (49).…”

Distinct phosphatases antagonize the p53 response in different phases of the cell cycle

“…The Krüppel-associated box domain-associated protein 1 (KAP1) is a transcriptional corepressor protein for the p21 gene CDKN1A and prevents transcription initiation of the poised promoter (31,32). Its activity is impeded by DNA damage through phosphorylation by ATM on S824 and by Chk1/Chk2 on S473 (31,(33)(34)(35). Dephosphorylation of these sites, in turn, is regulated by PP4 (36).…”

“…Transcription of p21 is dynamically regulated to enable rapid expression in response to stress, as well as to ensure timely shutdown of transcription when the stressor is under control (30). The Krüppel-associated box domain-associated protein 1 (KAP1) is a transcriptional corepressor protein for the p21 gene CDKN1A and prevents transcription initiation of the poised promoter (31,32). Its activity is impeded by DNA damage through phosphorylation by ATM on S824 and by Chk1/Chk2 on S473 (31,(33)(34)(35).…”