Incorporation Of 32P Research Articles

A new method for quantifying the enzyme activity of DGKs

Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the ATP-dependent conversion of diacylglycerol (DAG) to phosphatidic acid (PtdOH). A common approach to examine the activity of these enzymes relys on a radiometric assay (Epand and Topham, 2007; Tu-Sekine and Raben, 2017). This assay quantifies the DGK-catalyzed incorporation of 32P into DAG from AT32P to generate 32PtdOH and is perhaps been the most widely used assay. While sensitive, its drawbacks are the expense and the potential negative impacts on health and the environment. In this report, we describe a new assay which utilizes fluorescent labeled NBD-DAG (1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl]-sn-Glycero-3-diacylglycerol) to quantify the DGK-θ-catalyzed conversion of NBD-DAG to NBD-PtdOH. Furthermore, we show the assay is sufficiently sensitive as the measured specific activity was similar to that previously determined with AT32P (Tu-Sekine and Raben, 2012) and was able to detect the activation of DGK-θ by synaptotagmin-1 (Barber et al., 2022). Overall, this assay is inexpensive, sensitive, and reproducible making it an attractive alternative to currently established assays.

R.M.C. (Rex) Dawson, FRS, 1924–2021

R.M.C. (Rex) Dawson, FRS, 1924–2021

Raymond L. Erikson, PhD: In Memoriam (1936–2020)

![][1] > I hoed and trenched and weeded, And took the flowers to fair. > > The Shropshire Lad by A. E. Houseman In 2016, Ray started a personal reflection on his career in science titled “Four Decades of Protein Phosphorylation and Cancer Research.” Nothing more aptly fit his

Direct effect of protein kinase A on four putative phosphorylation sites of aquaporin 2 in vitro

The water channel aquaporin 2 (AQP2) has four phosphorylation sites at Ser256, Ser261, Ser264, and Ser269 in the C-terminus and these sites are important for AQP2 bioactivity. However, the exact role of each phosphorylation site still remains unclear. In this study, we generated unique AQP2 mutants in which we eliminated three phosphorylation sites but maintained only one site at the C-terminal end. The AQP2 phosphorylation of each single site by protein kinase A (PKA) was examined by in vitro translation and 32P incorporation. The ability of AQP2 trafficking to the cell membrane was evaluated by cell surface biotinylation. Among the four phosphorylation sites, AQP2 mutant with only S256 preserved the most ability of AQP2 to cell membrane expression. The AQP2 water permeability was measured in oocyte. Ser256 is the most important site for AQP2 function. Interestingly, Ser261 and Ser264 significantly inhibit AQP2 activity. Ser269 slightly but not statistically reduced AQP2 activity. Our data suggest that the four phosphorylation sites execute differential roles in concert in AQP2 functional regulation. AQP2 activity regulated by phosphorylation at Ser256 can be counterbalanced by phosphorylation at Ser261 and Ser264.

In Vitro Detection of Long Noncoding RNA Generated from DNA Double-Strand Breaks.

DNA damage response (DDR) is essential for the maintenance of genomic integrity. We have recently discovered the generation of noncoding RNA from a DNA double-strand break (DSB) in an MRE11-RAD50-NBS1 complex-dependent manner, which are necessary for full DDR activation. The low abundance of these noncoding RNA makes them difficult to identify and study. In this chapter, we describe an in vitro biochemical assay to study the generation of damage-induced long noncoding RNA (dilncRNA) from a DNA DSB. In this assay, transcriptionally competent cell-free extracts upon incubation with a linear DNA support RNA synthesis from DNA ends, as monitored by incorporation of 32P[UTP] in discrete products resolved on a denaturing polyacrylamide gel. This approach can be used to identify the role of different DDR proteins in generating dilncRNA.

Phosphorylation of phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) in the flesh of fruits

This study determined whether phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) are phosphorylated in the flesh of a range of fruits. This was done by incubating fruit flesh with 32P[P] (where 32P[P] = 32PO43−), then PEPCK and PEPC were immunoprecipitated from extracts using specific antisera. The incorporation of 32P[P] into these enzymes was then determined by autoradiography of SDS-PAGE gels. Both enzymes were subject to phosphorylation in vivo in the flesh of grape, tomato, cherry and plum. PEPCK was also subject to phosphorylation in vivo in developing grape seeds. Proteolytic cleavage of PEPCK showed that it was phosphorylated at a site(s) located on its N-terminal extension. Potentially phosphorylation of these enzymes could contribute to the coordinate regulation of their activities in the flesh of fruits and in developing seeds.

Abstract A184: Listeria-32P, a new approach to treat pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (pancreatic cancer), is the 4th leading cause of cancer deaths. This “silent killer” is characterized by metastases occurring often before the primary tumor can be detected, resulting in a five-year survival rate of only 4%, underscoring the need for new alternative therapies. Listeria monocytogenes-based cancer therapy could be such an alternative. Our laboratory discovered that attenuated Listeria exhibits novel pathways that are particularly useful against metastatic cancer. We found that Listeria infects metastases and primary tumors, and kills tumor cells through high levels of reactive oxygen species (ROS), and that myeloid-derived suppressor cells (MDSC) deliver Listeria selectively to the tumor site(s) through chemo/cytokines produced by the tumor cells, and by protecting them, after infection, from immune clearance through their immune suppressive character. Based on these results we now use Listeria as a platform for the delivery of anticancer agents to the tumor microenvironment (TME). One novel application is the delivery of radioisotopes, which emit cytocidal radiation such as beta-particles coupled to Listeria, selectively into the tumor cells. This leads to the synergistic destruction of tumor cells by ionizing radiation, through beta-particles and through ROS generation by Listeria. We were the first to demonstrate that in a highly aggressive model of pancreatic cancer (Panc-02), therapeutic treatment with Listeria reduced the number of metastases by 50%, and when coupled to radioisotope 188-Rhenium (188Re) by 90%. This correlated with a selective accumulation of radioactivity in the metastases without significant side effects. Experimental procedures: Most recently, we explored a completely new method to RL generation using 32Phosphorus (32P), by incorporating 32P into the cell wall of Listeria (Listeria-32P) during culturing of Listeria. Panc-02 mice received 12 doses of Listeria-32P over a period of 28 days. Results: Treatment with Listeria-32P completely eliminated the metastatic cancer in 90% of the mice (visible by eye), while survival studies demonstrated that 43% of the mice were still alive and free of cancer (confirmed by histological examination) 3 months after the last treatment. Untreated mice die within 30-35 days. Most importantly, the incorporation of 32P delivered through Listeria into normal tissues including bone marrow (BM) was hardly detectable, in contrast to 32P alone, which strongly incorporated into the BM. In conclusion: We have developed a novel non-toxic strategy that is highly effective against pancreatic cancer in a preclinical model Panc-02, with a goal to treat patients with pancreatic cancer in the near future. Currently, we are testing Listeria-32P in KPC transgenic mice (conditionally express endogenous Kras-G12D and p53-R172H mutant alleles), which is a humanized model of spontaneous pancreatic cancer. Citation Format: Dinesh Chandra, Ziqiang Yuan, Steven K. Libutti, Ekaterina Dadachova, Claudia Gravekamp. Listeria-32P, a new approach to treat pancreatic cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A184.

PKA tightly bound to human placental mitochondria participates in steroidogenesis and is not modified by cAMP

IntroductionProtein phosphorylation plays an important role in the modulation of steroidogenesis and it depends on the activation of different signaling cascades. Previous data showed that PKA activity is related to steroidogenesis in mitochondria from syncytiotrophoblast of human placenta (HPM). PKA localization and contribution in progesterone synthesis and protein phosphorylation of HPM was assessed in this work. MethodsPlacental mitochondria and submitochondrial fractions were used. Catalytic and regulatory PKA subunits were identified by Western blot. PKA activity was determined by the incorporation of 32P into proteins in the presence or absence of specific inhibitors. The effect of PKA activators and inhibitors on steroidogenesis and protein phosphorylation in HPM was tested by radioimmunoassay and autoradiography. ResultsThe PKAα catalytic subunit was distributed in all the submitochondrial fractions whereas βII regulatory subunit was the main isoform observed in both the outer and inner membranes of HPM. PKA located in the inner membrane showed the highest activity. Progesterone synthesis and mitochondrial protein phosphorylation are modified by inhibitors of PKA catalytic subunit but are neither sensitive to inhibitors of the regulatory subunit nor to activators of the holoenzyme. DiscussionThe lack of response in the presence of PKA activators and inhibitors of the regulatory subunit suggests that the activation of intramitochondrial PKA cannot be prevented or further activated. ConclusionsThe phosphorylating activity of PKA inside HPM could be an important component of the steroidogenesis transduction cascade, probably exerting its effects by direct phosphorylation of its substrates or by modulating other kinases and phosphatases.

Mechanisms Underlying CaMKII Regulation of Astrocytic Glutamate Transporters

Excitotoxic calcium signaling, a hallmark of aberrant neuronal activity during cerebral ischemia and brain trauma, induces a loss of CaMKII activity that appears to sensitize neurons to glutamate-induced toxicity. Because CaMKII is present in astrocytes and other glia, we postulate that aberrant CaMKII signaling could compromise neuronal signaling and survival to excitotoxicity by disrupting astrocyte function. In support of this model, inhibition of CaMKII in cortical astrocytic cultures with a peptide (tat-CN21) as well as a small molecule (KN-93) inhibitor significantly reduces glutamate uptake compared to inactive controls, suggesting that glutamate transporters in astrocytes require CaMKII for their basal activity. The two primary excitatory amino acid transporters (EAAT) in astrocytes are EAAT 1 and EAAT2. Co-immunoprecipitation from rodent brain and immunocytochemical studies in cortical astrocytes reveal an association and co-localization respectively between EAAT2 and CaMKII. Although additional studies are required to determine if these interactions exist for EAAT1 and CaMKII in cells, in vitro studies using GST-fusion proteins and peptide tiling arrays indicate that purified δCaMKII binds to the N terminal and second intracellular loop of EAAT1, whereas, CaMKII preferentially binds to the N terminus and third intracellular loop of EAAT2. Using P32 incorporation as an index of CaMKII phosphorylation, phosphorylation of immunoprecipitated transporters as well as peptide tiling assays for EAAT2 reveal that astrocytic glutamate transporters are indeed CaMKII substrates. In total, our data support the hypothesis that astrocytic glutamate transporters are regulated by CaMKII binding and/or phosphorylation. Further studies are required to determine if a loss of CaMKII activity that is associated with pathological states, including epilepsy and ischemic stroke, compromises neuronal survival due to reduced glutamate transporter function previously associated with excitotoxicity.

Thermodynamic Effects of the Alteration of the Axial Ligand on the Unfolding of Thermostable Cytochrome c

The role the axial methionine plays in the conformational properties and thermostability of the heme active site has been investigated with the help of site-specific mutations at the axial Met69 position with His (M69H) and Ala (M69A) in thermostable cytochrome c(552) from Thermus thermophilus. Detailed circular dichroism, direct electrochemistry, and other spectroscopic studies have been employed to investigate the thermally induced and GdnHCl-induced unfolding properties of the heme active site of the wild type and the mutants of cytochrome c(552). We observed an unusually high thermodynamic and thermal stability of the M69A mutant compared to that of wild-type cytochrome c(552). However, the M69H mutant exhibited a slightly lower unfolding free energy compared to that of the wild-type protein. The high conformational stability of the M69A mutant was attributed to the presence of residual structure in the unfolded state as well as to the altered conformation in the folded state of this mutant of cytochrome c(552). This study thus supports the view that apart from the folded state, the unfolded state of a protein may also make a significant contribution to the stability of a protein.

Nafion–Zirconium Phosphate Composite Membrane: A New Approach to Prepare 32P Patches for Superficial Brachytherapy Applications

This article describes a method for the preparation of (32)P patch for the treatment of skin cancer. It is based on the surface modification of a Nafion film by treatment with ZrOCl(2) solution, impregnation of a predicted quantity of (32)P into the film, and its subsequent immobilization into a nonleachable matrix by lamination. The effect of variations of critical parameters on the incorporation of (32)P into the membrane, such as solution pH, contact time, reaction volume, inactive carrier concentration of the feed, reaction temperature, and so on, was investigated to arrive at the conditions resulting in optimum retention of (32)P activity. The morphology of the membrane was evaluated by scanning electron microscope and energy dispersive spectral analyses. Quality control tests were carried out to ensure nonleachability, uniform distribution of activity, and stability of the patches.

Abstract 2048: Phosphorylation and regulation of PTEN by Polo-like kinase 1

Abstract The tumor suppressor PTEN plays a critical role in the regulation of cell survival, proliferation, and angiogenisis. Deregulated expression and/or activities frequently result in tumor development. Several recent studies have revealed that PTEN also localizes to the nucleus and that nuclear PTEN may regulate the level and activity of mitotic checkpoint proteins, thus suppressing chromosomal instability during cell division. However, the mechanism by which PTEN is translocated to the nucleus remains unclear. Given that PTEN is heavily phosphorylated and that Plk1 is an important mitotic kinase, we have asked whether Plk1 directly phosphorylates PTEN and whether Plk1-mediated phosphorylation of PTEN functions as an important regulatory mechanism during cell division. Here we report that a significant fraction of PTEN localized to nuclear chromatin. The chromatin association of PTEN was enhanced during mitosis, which was correlated with an increase in PTEN phosphorylation at threonine 366 (T366) and serine 370 (S370). Using purified recombinant proteins, we demonsatrated that Plk1 was at least partly responsible for this phosphorylation. Plk1 strongly catalyzed the incorporation of 32P into PTEN, resulting in increased signals detected by a phospho-specific antibody that recognized T366/S370 phosphorylation. Furthermore, BI-2536, a potent Plk1 inhibitor, significantly blocked PTEN phosphorylation by Plk1 in vitro, as well as its chromatin association in vivo. Combined, these results strongly suggest that Plk1 phosphorylates PTEN at T366 and/or S370 and that this phosphorylation promotes its association with chromatin during the cell cycle. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2048. doi:1538-7445.AM2012-2048

The Multifunctional Calcium/Calmodulin-Dependent Protein Kinase II Delta (CaMKIIδ) Phosphorylates Titin N2B and PEVK Spring Elements

Titin is phosphorylated by several kinases that significantly affect titin mechanical properties. PKA and PKG phosphorylates titin N2B element and decreases titin's stiffness, while PKCα phosphorylates titin PEVK spring element and increases titin's stiffness. CaMKII is a Ca2+ and calmodulin dependent serine/threonine kinase that is activated by increases in cellular Ca2+. Four isoforms have been described (α, β, γ, and δ). CaMKII delta (CaMKIIδ) is the predominant isoform in the heart. It has been found that increases in CaMKIIδ activity causes severe left ventricular dysfunction. The aim of the present study is to determine whether CaMKIIδ phosphorylates titin and, if so, identify its target domains. To determine whether CaMKIIδ phosphorylates titin in mouse left ventricular muscle, LV skinned fibers were incubated with recombinant human CaMKIIδ expressed in insect cells and [γ-32P]ATP. We found that CaMKIIδ phosphorylates titin in mouse LV skinned fibers. Further, pre-incubation of the LV skinned fiber with protein phosphatase 1 (PP1) significantly increases 32P incorporation on titin. To identify the CaMKIIδ titin target domains, human titin N2B, PEVK, Ig8-15, I27-34, and murine N2B recombinant proteins expressed in E. coli were incubated with CaMKIIδ and [γ-32P]ATP. We found that titin N2B and PEVK spring elements, but not Ig domains, were phosphorylated by CaMKIIδ. Prediction of the specific phosphorylation sites suggest overlap with the PKC and PKA sites (including S170 of the PEVK element a PKC substrate) and work to indentify the specific substrate sites and the possible effect on mechanics is in progress. In summary, the present study showed that CaMKIIδ phosphorylates titin spring elements.

Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.

Liver-specific Inducible Nitric-oxide Synthase Expression Is Sufficient to Cause Hepatic Insulin Resistance and Mild Hyperglycemia in Mice

Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, plays an important role in obesity-induced insulin resistance. Inhibition of iNOS by gene disruption or pharmacological inhibitors reverses or ameliorates obesity-induced insulin resistance in skeletal muscle and liver in mice. It is unknown, however, whether increased expression of iNOS is sufficient to cause insulin resistance in vivo. To address this issue, we generated liver-specific iNOS transgenic (L-iNOS-Tg) mice, where expression of the transgene, iNOS, is regulated under mouse albumin promoter. L-iNOS-Tg mice exhibited mild hyperglycemia, hyperinsulinemia, insulin resistance, and impaired insulin-induced suppression of hepatic glucose output, as compared with wild type (WT) littermates. Insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and -2, and Akt was significantly attenuated in liver, but not in skeletal muscle, of L-iNOS-Tg mice relative to WT mice without changes in insulin receptor phosphorylation. Moreover, liver-specific iNOS expression abrogated insulin-stimulated phosphorylation of glycogen synthase kinase-3β, forkhead box O1, and mTOR (mammalian target of rapamycin), endogenous substrates of Akt, along with increased S-nitrosylation of Akt relative to WT mice. However, the expression of insulin receptor, IRS-1, IRS-2, Akt, glycogen synthase kinase-3β, forkhead box O1, protein-tyrosine phosphatase-1B, PTEN (phosphatase and tensin homolog), and p85 phosphatidylinositol 3-kinase was not altered by iNOS transgene. Hyperglycemia was associated with elevated glycogen phosphorylase activity and decreased glycogen synthase activity in the liver of L-iNOS-Tg mice, whereas phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and proliferator-activated receptor γ coactivator-1α expression were not altered. These results clearly indicate that selective expression of iNOS in liver causes hepatic insulin resistance along with deranged insulin signaling, leading to hyperglycemia and hyperinsulinemia. Our data highlight a critical role for iNOS in the development of hepatic insulin resistance and hyperglycemia.

Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2).

Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK) is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of 32P [counts per minute (CPM)] for each peptide substrate was determined by the radiolabel assay using [γ-32P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135) were phosphorylated by other enzymes (PKA, PKCα, and ERK1), R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2.

Letter: The effect of local anaesthetics on the incorporation of 32P into the phosphoinositides of rabbit vagus nerve.

Letter: The effect of local anaesthetics on the incorporation of 32P into the phosphoinositides of rabbit vagus nerve.

Splice Variants of the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 4 (DYRK4) Differ in Their Subcellular Localization and Catalytic Activity

Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.

The Hippo Tumor Pathway Promotes TAZ Degradation by Phosphorylating a Phosphodegron and Recruiting the SCFβ-TrCP E3 Ligase

The TAZ transcription co-activator promotes cell proliferation and epithelial-mesenchymal transition. TAZ is inhibited by the Hippo tumor suppressor pathway, which promotes TAZ cytoplasmic localization by phosphorylation. We report here that TAZ protein stability is controlled by a phosphodegron recognized by the F-box protein β-TrCP and ubiquitylated by the SCF/CRL1(β-TrCP) E3 ligase. The interaction between TAZ and β-TrCP is regulated by the Hippo pathway. Phosphorylation of a phosphodegron in TAZ by LATS primes it for further phosphorylation by CK1ε and subsequent binding by β-TrCP. Therefore, the Hippo pathway negatively regulates TAZ function by both limiting its nuclear accumulation and promoting its degradation. The phosphodegron-mediated TAZ degradation plays an important role in negatively regulating TAZ biological functions.

Identification of a Coiled Coil in Werner Syndrome Protein That Facilitates Multimerization and Promotes Exonuclease Processivity

Werner syndrome (WS) is a rare progeroid disorder characterized by genomic instability, increased cancer incidence, and early onset of a variety of aging pathologies. WS is unique among early aging syndromes in that affected individuals are developmentally normal, and phenotypic onset is in early adulthood. The protein defective in WS (WRN) is a member of the large RecQ family of helicases but is unique among this family in having an exonuclease. RecQ helicases form multimers, but the mechanism and consequence of multimerization remain incompletely defined. Here, we identify a novel heptad repeat coiled coil region between the WRN nuclease and helicase domains that facilitates multimerization of WRN. We mapped a novel and unique DNA-dependent protein kinase phosphorylation site proximal to the WRN multimerization region. However, phosphorylation at this site affected neither exonuclease activity nor multimeric state. We found that WRN nuclease is stimulated by DNA-dependent protein kinase independently of kinase activity or WRN nuclease multimeric status. In addition, WRN nuclease multimerization significantly increased nuclease processivity. We found that the novel WRN coiled coil domain is necessary for multimerization of the nuclease domain and sufficient to multimerize with full-length WRN in human cells. Importantly, correct homomultimerization is required for WRN function in vivo as overexpression of this multimerization domain caused increased sensitivity to camptothecin and 4-nitroquinoline 1-oxide similar to that in cells lacking functional WRN protein.