Tadahiro Shimazu

Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B.

Although heat-shock protein 70 (HSP70), an evolutionarily highly conserved molecular chaperone, is known to be post-translationally modified in various ways such as phosphorylation, ubiquitination and glycosylation, physiological significance of lysine methylation has never been elucidated. Here we identify dimethylation of HSP70 at Lys-561 by SETD1A. Enhanced HSP70 methylation was detected in various types of human cancer by immunohistochemical analysis, although the methylation was barely detectable in corresponding non-neoplastic tissues. Interestingly, methylated HSP70 predominantly localizes to the nucleus of cancer cells, whereas most of the HSP70 protein locates to the cytoplasm. Nuclear HSP70 directly interacts with Aurora kinase B (AURKB) in a methylation-dependent manner and promotes AURKB activity in vitro and in vivo. We also find that methylated HSP70 has a growth-promoting effect in cancer cells. Our findings demonstrate a crucial role of HSP70 methylation in human carcinogenesis.

Multiple Histone Deacetylases and the CREB-binding Protein Regulate Pre-mRNA 3-End Processing *

Trichostatin A (TSA), a specific inhibitor of histone deacetylases (HDACs), induces acetylation of various non-histone proteins such as p53 and α-tubulin. We purified several acetylated proteins by the affinity to an anti-acetylated lysine (AcLys) antibody from cells treated with TSA and identified them by mass spectrometry. Here we report on acetylation of CFIm25, a component of mammalian cleavage factor Im (CF Im), and poly(A) polymerase (PAP), a polyadenylating enzyme for the pre-mRNA 3′-end. The residues acetylated in these proteins were mapped onto the regions required for interaction with each other. Whereas CBP acetylated these proteins, HDAC1, HDAC3, HDAC10, SIRT1, and SIRT2 were involved in in vivo deacetylation. Acetylation of the CFIm25 occurred depending on the cleavage factor complex formation. Importantly, the interaction between PAP and CF Im complex was decreased by acetylation. We also demonstrated that acetylation of PAP inhibited the nuclear localization of PAP by inhibiting the binding to the importin α/β complex. These results suggest that CBP and HDACs regulate the 3′-end processing machinery and modulate the localization of PAP through the acetylation and deacetylation cycle.

Regulation of SV40 large T-antigen stability by reversible acetylation

Reversible acetylation on protein lysine residues has been shown to regulate the function of both nuclear proteins such as histones and p53 and cytoplasmic proteins such as α-tubulin. To identify novel acetylated proteins, we purified several proteins by the affinity to an anti-acetylated-lysine antibody from cells treated with trichostatin A (TSA). Among the proteins identified, here we report acetylation of the SV40 large T antigen (T-Ag). The acetylation site was determined to be lysine-697, which is located adjacent to the C-terminal Cdc4 phospho-degron (CPD). Overexpression of the CBP acetyltransferase acetylated T-Ag, whereas HDAC1, HDAC3 and SIRT1 bound and deacetylated T-Ag. The acetylation and deacetylation occurred independently of p53, a binding partner of T-Ag, but the acetylation was enhanced in the presence of p53. T-Ag in the cells treated with TSA and NA or the acetylation mimic mutant (K697Q) became unstable in COS-7 cells, suggesting that acetylation regulates stability of T-Ag. Indeed, NIH3T3 cells stably expressing K697Q showed decreased anchorage-independent growth compared with those expressing wild type or the K697R mutant. These results demonstrate that acetylation destabilizes T-Ag and regulates the transforming activity of T-Ag in NIH3T3 cells.Reversible acetylation on protein lysine residues has been shown to regulate the function of both nuclear proteins such as histones and p53 and cytoplasmic proteins such as α-tubulin. To identify novel acetylated proteins, we purified several proteins by the affinity to an anti-acetylated-lysine antibody from cells treated with trichostatin A (TSA). Among the proteins identified, here we report acetylation of the SV40 large T antigen (T-Ag). The acetylation site was determined to be lysine-697, which is located adjacent to the C-terminal Cdc4 phospho-degron (CPD). Overexpression of the CBP acetyltransferase acetylated T-Ag, whereas HDAC1, HDAC3 and SIRT1 bound and deacetylated T-Ag. The acetylation and deacetylation occurred independently of p53, a binding partner of T-Ag, but the acetylation was enhanced in the presence of p53. T-Ag in the cells treated with TSA and NA or the acetylation mimic mutant (K697Q) became unstable in COS-7 cells, suggesting that acetylation regulates stability of T-Ag. Indeed, NIH3T3 cells stably expressing K697Q showed decreased anchorage-independent growth compared with those expressing wild type or the K697R mutant. These results demonstrate that acetylation destabilizes T-Ag and regulates the transforming activity of T-Ag in NIH3T3 cells.

Selenium-based S-adenosylmethionine analog reveals the mammalian seven-beta-strand methyltransferase METTL10 to be an EF1A1 lysine methyltransferase.

Lysine methylation has been extensively studied in histones, where it has been shown to provide specific epigenetic marks for the regulation of gene expression; however, the molecular mechanism and physiological function of lysine methylation in proteins other than histones remains to be fully addressed. To better understand the substrate diversity of lysine methylation, S-adenosylmethionine (SAM) derivatives with alkyne-moieties have been synthesized. A selenium-based SAM analog, propargylic Se-adenosyl-l-selenomethionine (ProSeAM), has a wide spectrum of reactivity against various lysine methyltransferases (KMTs) with sufficient stability to support enzymatic reactions in vitro. By using ProSeAM as a chemical probe for lysine methylation, we identified substrates for two seven-beta-strand KMTs, METTL21A and METTL10, on a proteomic scale in mammalian cells. METTL21A has been characterized as a heat shock protein (HSP)-70 methyltransferase. Mammalian METTL10 remains functionally uncharacterized, although its ortholog in yeast, See1, has been shown to methylate the translation elongation factor eEF1A. By using ProSeAM-mediated alkylation followed by purification and quantitative MS analysis, we confirmed that METTL21A labels HSP70 family proteins. Furthermore, we demonstrated that METTL10 also methylates the eukaryotic elongation factor EF1A1 in mammalian cells. Subsequent biochemical characterization revealed that METTL10 specifically trimethylates EF1A1 at lysine 318 and that siRNA-mediated knockdown of METTL10 decreases EF1A1 methylation levels in vivo. Thus, our study emphasizes the utility of the synthetic cofactor ProSeAM as a chemical probe for the identification of non-histone substrates of KMTs.

Effects of downregulated HDAC6 expression on the proliferation of lung cancer cells

Histone deacetylase 6 (HDAC6) is a multifunctional, cytosolic protein deacetylase that primarily acts on alpha-tubulin. Here we report that stable knockdown of HDAC6 expression causes a decrease in the steady-state level of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor alpha, in A549 lung cancer cells. The decreased levels of in EGFR in HDAC6-knockdown cells, which correlated with increased acetylation of microtubules, were due to increased turnover of EGFR protein. Despite the decrease in EGFR levels, A549 cells lacking functional HDAC6 appeared to grow normally, probably due to increased expression of extracellular signal-regulated kinases 1 and 2. Indeed, HDAC6-knockdown cells were more sensitive than control cells to the MEK inhibitor U0126. These results suggest that HDAC6 inhibitors combined with inhibitors of growth factor signaling may be useful as cancer therapy.

Nucleolin is involved in interferon regulatory factor-2-dependent transcriptional activation

We have previously shown that interferon regulatory factor-2 (IRF-2) is acetylated in a cell growth-dependent manner, which enables it to contribute to the transcription of cell growth-regulated promoters. To clarify the function of acetylation of IRF-2, we investigated the proteins that associate with acetylated IRF-2. In 293T cells, the transfection of p300/CBP-associated factor (PCAF) enhanced the acetylation of IRF-2. In cells transfected with both IRF-2 and PCAF, IRF-2 associated with endogenous nucleolin, while in contrast, minimal association was observed when IRF-2 was transfected with a PCAF histone acetyl transferase (HAT) deletion mutant. In a pull-down experiment using stable transfectants, acetylation-defective mutant IRF-2 (IRF-2K75R) recruited nucleolin to a much lesser extent than wild-type IRF-2, suggesting that nucleolin preferentially associates with acetylated IRF-2. Nucleolin in the presence of PCAF enhanced IRF-2-dependent H4 promoter activity in NIH3T3 cells. Nucleolin knock-down using siRNA reduced the IRF-2/PCAF-mediated promoter activity. Chromatin immunoprecipitation analysis indicated that PCAF transfection increased nucleolin binding to IRF-2 bound to the H4 promoter. We conclude that nucleolin is recruited to acetylated IRF-2, thereby contributing to gene regulation crucial for the control of cell growth.

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

The subcellular localization and activity of cortactin is regulated by acetylation and interaction with Keap1

Promoting acetylation and thus nuclear localization of a cytoskeletal remodeling protein may limit cancer metastasis. Acetylation against cell migration The actin-binding protein cortactin promotes cell migration through cytoskeletal remodeling in the cell cortex and is abundant in certain types of aggressive cancers. Ito et al. found that the cytosolic protein Keap1 promoted the localization of cortactin to the cell cortex and thus cell migration. Cortactin shuttled between the cytoplasm and the nucleus; however, upon acetylation, cortactin no longer bound to Keap1 and became predominantly localized in the nucleus. Thus, increasing the acetylation of cortactin or preventing it from binding to Keap1 may suppress the metastasis of cancer cells. Cortactin is an F-actin–binding protein that localizes to the cell cortex, where the actin remodeling that is required for cell migration occurs. We found that cortactin shuttled between the cytoplasm and the nucleus under basal conditions. We identified Kelch-like ECH-associated protein 1 (Keap1), a cytosolic protein that is involved in oxidant stress responses, as a binding partner of cortactin that promoted the cortical localization of cortactin and cell migration. The ability of cortactin to promote cell migration is regulated by various posttranslational modifications, including acetylation. We showed that the acetylated form of cortactin was mainly localized to the nucleus and that acetylation of cortactin decreased cell migration by inhibiting the binding of cortactin to Keap1. Our findings reveal that Keap1 regulates cell migration by affecting the subcellular localization and activity of cortactin independently of its role in oxidant stress responses.

Self-activation of Serine/Threonine Kinase AfsK on Autophosphorylation at Threonine-168

A Hanks-type protein kinase AfsK autophosphorylates on threonine residue(s) and phosphorylates AfsR, a global regulator for secondary metabolism in Streptomyces coelicolor A3(2). Mass spectrometry of a tryptic digest of the autophosphorylated form of AfsKΔC corresponding to the kinase catalytic domain (Met-1 to Arg-311) of AfsK, together with subsequent site-directed mutagenesis of the candidate amino acids, identified threonine-168 as a single autophosphorylation site. Threonine-168 is located in the activation loop that is known for some Ser/Thr kinases to modulate kinase activity on phosphorylation of one or more threonine residues within the loop. Consistent with this, mutant T168D, in which Thr-168 was replaced by Asp, became a constitutively active kinase; it phosphorylated AfsR to the same extent as AfsKΔC produced in and purified from Escherichia coli cells during which a considerable population of it had been already phosphorylated intermolecularly. All these findings show that autophosphorylation or intermolecular phosphorylation of threonine-168 in AfsK accounts for the self-activation of its kinase activity.

Role of METTL20 in regulating β-oxidation and heat production in mice under fasting or ketogenic conditions

METTL20 is a seven-β-strand methyltransferase that is localised to the mitochondria and tri-methylates the electron transfer flavoprotein (ETF) β subunit (ETFB) at lysines 200 and 203. It has been shown that METTL20 decreases the ability of ETF to extract electrons from medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) and glutaryl-CoA dehydrogenase in vitro. METTL20-mediated methylation of ETFB influences the oxygen consumption rate in permeabilised mitochondria, suggesting that METTL20-mediated ETFB methylation may also play a regulatory role in mitochondrial metabolism. In this study, we generated Mettl20 knockout (KO) mice to uncover the in vivo functions of METTL20. The KO mice were viable, and a loss of ETFB methylation was confirmed. In vitro enzymatic assays revealed that mitochondrial ETF activity was higher in the KO mice than in wild-type mice, suggesting that the KO mice had higher β-oxidation capacity. Calorimetric analysis showed that the KO mice fed a ketogenic diet had higher oxygen consumption and heat production. A subsequent cold tolerance test conducted after 24 h of fasting indicated that the KO mice had a better ability to maintain their body temperature in cold environments. Thus, METTL20 regulates ETF activity and heat production through lysine methylation when β-oxidation is highly activated.