Control of HIPK2 stability by ubiquitin ligase Siah-1 and checkpoint kinases ATM and ATR

The tumour suppressor HIPK2 is an important regulator of cell death induced by DNA damage, but how its activity is regulated remains largely unclear. Here we demonstrate that HIPK2 is an unstable protein that colocalizes and interacts with the E3 ubiquitin ligase Siah-1 in unstressed cells. Siah-1 knockdown increases HIPK2 stability and steady-state levels, whereas Siah-1 expression facilitates HIPK2 polyubiquitination, degradation and thereby inactivation. During recovery from sublethal DNA damage, HIPK2, which is stabilized on DNA damage, is degraded through a Siah-1-dependent, p53-controlled pathway. Downregulation of Siah-1 inhibits HIPK2 degradation and recovery from damage, driving the cells into apoptosis. We have also demonstrated that DNA damage triggers disruption of the HIPK2-Siah-1 complex, resulting in HIPK2 stabilization and activation. Disruption of the HIPK2-Siah-1 complex is mediated by the ATM/ATR pathway and involves ATM/ATR-dependent phosphorylation of Siah-1 at Ser 19. Our results provide a molecular framework for HIPK2 regulation in unstressed and damaged cells.     

Tumor suppressor SMAR1 mediates cyclin D1 repression by recruitment of the SIN3/histone deacetylase 1 complex

Matrix attachment region binding proteins have been shown to play an important role in gene regulation by altering chromatin in a stage- and tissue-specific manner. Our previous studies report that SMAR1, a matrix-associated protein, regresses B16-F1-induced tumors in mice. Here we show SMAR1 targets the cyclin D1 promoter, a gene product whose dysregulation is attributed to breast malignancies. Our studies reveal that SMAR1 represses cyclin D1 gene expression, which can be reversed by small interfering RNA specific to SMAR1. We demonstrate that SMAR1 interacts with histone deacetylation complex 1, SIN3, and pocket retinoblastomas to form a multiprotein repressor complex. This interaction is mediated by the SMAR1(160-350) domain. Our data suggest SMAR1 recruits a repressor complex to the cyclin D1 promoter that results in deacetylation of chromatin at that locus, which spreads to a distance of at least the 5 kb studied upstream of the cyclin D1 promoter. Interestingly, we find that the high induction of cyclin D1 in breast cancer cell lines can be correlated to the decreased levels of SMAR1 in these lines. Our results establish the molecular mechanism exhibited by SMAR1 to regulate cyclin D1 by modification of chromatin.     

SMAR1-derived P44 peptide retains its tumor suppressor function through modulation of p53

The use of pharmacologically active short peptide sequences is a better option in cancer therapeutics than the full-length protein. Here we report one such 44-mer peptide sequence of SMAR1 (TAT-SMAR1 wild type, P44) that retains the tumor suppressor activity of the full-length protein. The protein transduction domain of human immunodeficiency virus, type 1, Tat protein was used here to deliver the 33-mer peptide of SMAR1 into the cells. P44 peptide could efficiently activate p53 by mediating its phosphorylation at serine 15, resulting in the activation of p21 and in effect regulating cell cycle checkpoint. In vitro phosphorylation assays with point-mutated P44-derived peptides suggested that serine 347 of SMAR1 was indispensable for its activity and represented the substrate motif for the protein kinase C family of proteins. Using xenograft nude mice models, we further demonstrate that P44 was capable of inhibiting tumor growth by preventing cellular proliferation. P44 treatment to tumor-bearing mice prevented the formation of poorly organized tumor vasculature and an increase in hypoxia-inducible factor-1alpha expression, both being signatures of tumor progression. The chimeric TAT-SMAR1-derived peptide, P44, thus has a strong therapeutic potential as an anticancer drug.     

ATMIN defines an NBS1-independent pathway of ATM signalling

The checkpoint kinase ATM (ataxia telangiectasia mutated) transduces genomic stress signals to halt cell cycle progression and promote DNA repair in response to DNA damage. Here, we report the characterisation of an essential cofactor for ATM, ATMIN (ATM INteracting protein). ATMIN interacts with ATM through a C-terminal motif, which is also present in Nijmegen breakage syndrome (NBS)1. ATMIN and ATM co-localised in response to ATM activation by chloroquine and hypotonic stress, but not after induction of double-strand breaks by ionising radiation (IR). ATM/ATMIN complex disruption by IR was attenuated in cells with impaired NBS1 function, suggesting competition of NBS1 and ATMIN for ATM binding. ATMIN protein levels were reduced in ataxia telangiectasia cells and ATM protein levels were low in primary murine fibroblasts lacking ATMIN, indicating reciprocal stabilisation. Whereas phosphorylation of Smc1, Chk2 and p53 was normal after IR in ATMIN-deficient cells, basal ATM activity and ATM activation by hypotonic stress and inhibition of DNA replication was impaired. Thus, ATMIN defines a novel NBS1-independent pathway of ATM signalling.     

Abnormal V(D)J recombination of T cell receptor beta locus in SMAR1 transgenic mice

Scaffold/matrix-associated region-1-binding protein (SMAR1) specifically interacts with the MARbeta sequence, which is located 400-bp upstream of the murine TCRbeta enhancer and is highly expressed during the DP stage of thymocyte development. To further analyze the functions of SMAR1, transgenic mice were generated that express SMAR1 in a tissue-independent manner. SMAR1-overexpressing mice exhibit severely altered frequency of the T cells expressing commonly used Vbetas (Vbeta5.1/5.2 and Vbeta8.1/8.2/8.3). The rearrangements of Vbeta5.1/5.2, Vbeta8.1/8.2/8.3 loci are also reduced in SMAR1 transgenic mice. The T cells in SMAR1 transgenic mice exhibit a mild perturbation at the early DN stage. SMAR1 transgenic mice exhibit hypercellular lymph nodes and spleen accompanied with prominent architectural defects in these organs. These results indicate that SMAR1 plays an important role in the regulation of T cell development as well as V(D)J recombination besides maintaining the architecture of the lymphoid organs.     

Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia–telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.     

Differential localization of ATM is correlated with activation of distinct downstream signaling pathways

ATM, the gene mutated in the genetic disease ataxia telangiectasia (AT), is a well-known protein involved in the DNA double-strand break response, where it plays an important role in sensing damage and signaling to DNA repair machinery and cell cycle checkpoints. However, a number of recent papers, including ours have found that ATM also plays important roles outside of the nucleus, which may explain some of the phenotypic features seen in AT patients. Our research into mechanisms of TSC2 regulation helped uncover a pathway upstream of TSC2 that is regulated by cytoplasmic ATM in response to ROS initiated by ATM activation of LKB1 and AMPK. We found that TSC2 activation results in mTORC1 repression and subsequent induction of autophagy. Elucidation of this stress response pathway provides a molecular mechanism for ATM signaling in the cytoplasm, and lays the groundwork for further studies on how ATM activity is regulated beyond DNA damage in different cellular compartments.