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Intramural Papers of the Month

By Erin D. Hopper and Tara Cartwright
July 2010

A Magnesium-Coordinating Threonine Plays a Critical Role in GTPase Catalysis

NIEHS scientists from the Laboratory of Neurobiology have demonstrated the critical role of the Mg2+-coordinating Thr residue in the catalytic rate of regulatory GTPases. Mutation of Thr 204 to Gln or Ala did not affect the ability of the G-protein alpha subunit (Gsα) to be activated by GTP, but it significantly reduced the rate of GTPase activity.

Gsα contains two switch domains, SWI and SWII, that undergo a conformational change upon Mg2+ and GTP binding, and this conformational change leads to protein activation. The bound Mg2+ is coordinated to six oxygens - two from GTP, two from water, one from a Thr residue in SWI, and one from a Ser residue.

Mutation of the Ser residue results in a protein with reduced capacity for binding Mg2+, but a dominant action that reduces and even prevents wild type molecules from acting. In contrast, mutation of the Thr residue results in the activation of the SWII domain and a reduction in the rate of GTPase activity.

This study revealed the mechanistic basis for the differences in the effects of mutating the coordinating Ser and Thr residues. Unlike the Ser, the Thr plays a critical role in the catalysis of GTP by Gsα, which it does by helping to move the hydrolytic water molecule into the proper position for catalysis. This feature of catalysis is expected to be common to all regulatory GTPases.

Citation: Zurita A, Zhang Y, Pedersen L, Darden T, Birnbaumer L( Exit NIEHS. 2010. Obligatory role in GTP hydrolysis for the amide carbonyl oxygen of the Mg2+-coordinating Thr of regulatory GTPases. Proc Natl Acad Sci USA 107(21): 9596-9601.

Cell Survival is Modulated by Phosphorylation of SIRT1

A study from the NIEHS Laboratory of Signal Transduction and Laboratory of Structural Biology has revealed a new mechanism of SIRT1 regulation that demonstrates the important role of the protein in cell fate.

Two anti-apoptotic members of the DYRK (dual specificity tyrosine-phosphorylated and regulated kinase) family bind and phosphorylate SIRT1. Using mass spectrometry, the authors showed that these two DYRK members, DYRK1A and DYRK3, phosphorylated the Thr(522) residue, resulting in SIRT1 activation.

Activated SIRT1 deacetylates p53, and this deacetylation promotes cell survival. However, the knockdown of DYRK1A and DYRK3 results in the hyperacetylation of p53, resulting in p53 activation. This activation makes cells more sensitive to genotoxic stress and leads to the promotion of stress-induced cell death.

Although SIRT1 has been considered as a possible therapeutic target for cancer, the regulatory network governing SIRT1 activity is not well understood. Whether SIRT1 acts as a tumor suppressor or a tumor promoter may depend on whether the cancer cells express wild type or mutant p53. Further studies may provide insight into the role of p53 in the anti- and pro-apoptotic effects of SIRT1.

Citation: Guo X, Williams JG, Schug TT, Li X( Exit NIEHS. 2010. DYRK1A and DYRK3 promote cell survival through phosphorylation and activation of SIRT1. J Biol Chem 285(17): 13223-13232.

Nuclear Receptor CAR Represses the Death of Mouse Primary Hepatocytes

NIEHS scientists in the Laboratory of Reproductive and Developmental Toxicology have identified the anti-apoptotic factor Growth Arrest and DNA Damage-Inducible 45beta (GADD45B) as a Constitutive Active/Adrostane Receptor (CAR)-regulated signaling molecule, through which CAR represses Tumor Necrosis Factor alpha (TNFα)-induced cell death.

To examine the role of the nuclear receptor CAR in cell death, a team of investigators treated primary hepatocytes from Car+/+, Car-/-, Gadd45b+/+ and Gadd45b-/- mice with TNFα and Actinomycin D in the presence or absence of TCPOBOP, the CAR activating ligand. Team members also used GST-pull down and co-immunoprecipitation assays to investigate the protein-protein interactions of CAR with GADD45B and MKK7. Kinase assays determined whether the formation of a CAR protein complex alters the ability of MKK7 to phosphorylate JNK1.

These studies demonstrated that CAR required the presence of an AF2 domain to bind to GADD45B, and that this binding forms a protein complex that inhibits MKK7-dependent phosphorylation of JNK1, repressing the death of TNFα-induced mouse primary hepatocytes. Taken together, these findings provide further insight into the molecular mechanism by which CAR regulates the promotion of HCC development in mice.  

Citation: Yamamoto Y, Moore R, Flavell RA, Lu B, Negishi M. ( Exit NIEHS 2010. Nuclear receptor CAR represses TNFalpha-induced cell death by interacting with the anti-apoptotic GADD45B. PLoS One 5(4):e10121.

Stem Cell Survival Advantage Toward Arsenic Drives Malignant Transformation

Researchers from the National Cancer Institute (NCI) at NIEHS, now with NTP Laboratories, reported that the carcinogen arsenic targets stem cells for transformation, eventually producing cancers enriched in cancer stem cells (CSCs). This is facilitated by a stem cell survival advantage toward arsenic during malignant transformation.

The investigators compared both innate and acquired resistance of the human prostate stem cell line (WPE-stem) with the mature parental nontumorigenic cell line (RWPE-1) following acute (24-72 hours) and chronic (6 weeks) arsenite exposure. Analysis of CSCs included the utilization of holoclone and sphere formation assays, growth in soft agar, and expression of stem cell biomarkers.

WPE-stem exhibited an innate resistance and hyperadaptability to arsenite, including apoptosis, compared to the parental RWPE-1. WPE-stem demonstrated a higher expression of antiapoptotic genes, lower expression of proapoptotic genes, and increased arsenic-induced stress response and arsenic efflux-related genes during transformation.

These observations further strengthen the argument that arsenic most likely targets cells that have either a stem or progenitor phenotype and undergo survival selection during arsenic-induced malignant transformation.

Citation: Tokar EJ, Qu W, Liu J, Liu W, Webber MM, Phang JM, Waalkes MP( Exit NIEHS. 2010. Arsenic-specific stem cell selection during malignant transformation. J Natl Cancer Inst. 102(9):638-649.

(Erin Hopper, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Structural Biology Mass Spectrometry Group. Tara Ann Cartwright, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Neurobiology Membrane Signaling Group.) 

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