Mol Life Sci. 2005 Mar;62(5):606-18. J
Pharmacol Exp Ther.
2005 Nov;315(2):872-8. Epub
2005 Jul 22. Nucleic
Acids Res. 2006 Mar 20;34(5):1620-32. Print
A second protein kinase CK1-mediated step negatively regulates Wnt signalling by disrupting the lymphocyte enhancer factor-1/beta-catenin complex.
Hammerlein A, Weiske J, Huber O.
ABDA - Federal
Deregulated activation of the canonical Wnt signalling pathway leads to stabilization of beta-catenin and is critically involved in carcinogenesis by an inappropriate induction of lymphocyte enhancer factor (LEF-1)/beta-catenin-dependent transcription of Wnt target genes. Phosphorylation of the pathway components beta-catenin, Dishevelled, Axin and APC (adenomatous polyposis coli) by glycogen synthase kinase-3beta, CK1 and CK2 is of central importance in the regulation of the beta-catenin destruction complex. Here, we identify CK1 and CK2 as major kinases that directly bind to and phosphorylate LEF-1 inducing distinct, kinase-specific changes in the LEF-1/DNA complex. Moreover, CK1-dependent phosphorylation in contrast to CK2 disrupts the association of beta-catenin and LEF-1 but does not impair DNA binding of LEF-1. Sequential phosphorylation assays revealed that for efficient disruption of the LEF-1/beta-catenin complex, beta-catenin also has to be phosphorylated. Consistent with these observations, CK1-dependent phosphorylation inhibits, whereas CK2 activates LEF-1/beta-catenin transcriptional activity in reporter gene assays. These data are in line with a negative regulatory function of CK1 in the Wnt signalling pathway, where CK1 in addition to the beta-catenin destruction complex at a second level acts as a negative regulator of the LEF-1/beta-catenin transcription complex, thereby protecting cells from development of cancer.
PMID: 15747065 [PubMed - indexed for MEDLINE]
Nicotine regulates DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) phosphorylation at multiple sites in neostriatal neurons.
Hamada M, Hendrick JP, Ryan GR, Kuroiwa M, Higashi H, Tanaka M, Nairn AC, Greengard P, Nishi A.
Department of Pharmacology,
Nicotinic acetylcholine receptors (nAChRs) regulate dopaminergic signaling in the striatum by modulating the release of neurotransmitters. We have recently reported that nicotine stimulates the release of dopamine via alpha4beta2(*) nAChRs and/or alpha7 nAChRs, leading to the regulation of DARPP-32 at Thr34, the site involved in regulation of protein phosphatase-1 (PP-1). In this study, we investigated the regulation of DARPP-32 phosphorylation at its other sites, Thr75 [cyclin-dependent kinase-5 (Cdk5) site], Ser97 (CK2 site), and Ser130 (CK1 site), that serve to modulate Thr34 phosphorylation and dephosphorylation. In neostriatal slices, nicotine (100 microM) increased phosphorylation of DARPP-32 at Ser97 and Ser130 at an early time point (30 s) and decreased phosphorylation of DARPP-32 at Thr75 at a late time point (3 min). The increase in Ser97 and Ser130 phosphorylation was mediated through the release of dopamine via activation of alpha4beta2(*) nAChRs and alpha7 nAChRs and the subsequent activation of dopamine D1 and D2 receptors. The decrease in Thr75 phosphorylation was mediated through the release of dopamine via activation of alpha4beta2(*) nAChRs and the subsequent activation of dopamine D1 receptors. These various actions of nicotine on modulatory sites of phosphorylation would be predicted to result in a synergistic increase in the state of phosphorylation of DARPP-32 at Thr34 and thus would contribute to increased dopamine D1 receptor/DARPP-32 Thr34/PP-1 signaling.
PMID: 16040813 [PubMed - indexed for MEDLINE]
Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase.
Hill JW, Evans MK.
Laboratory of Cellular and Molecular Biology, National Institute on Aging, National Institutes of Health,
Human 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme for repairing 8-oxoguanine (8-oxoG), a mutagenic guanine base lesion produced by reactive oxygen species (ROS). A frequently occurring OGG1 polymorphism in human populations results in the substitution of serine 326 for cysteine (S326C). The 326 C/C genotype is linked to numerous cancers, although the mechanism of carcinogenesis associated with the variant is unclear. We performed detailed enzymatic studies of polymorphic OGG1 and found functional defects in the enzyme. S326C OGG1 excised 8-oxoG from duplex DNA and cleaved abasic sites at rates 2- to 6-fold lower than the wild-type enzyme, depending upon the base opposite the lesion. Binding experiments showed that the polymorphic OGG1 binds DNA damage with significantly less affinity than the wild-type enzyme. Remarkably, gel shift, chemical cross-linking and gel filtration experiments showed that S326C both exists in solution and binds damaged DNA as a dimer. S326C OGG1 enzyme expressed in human cells was also found to have reduced activity and a dimeric conformation. The glycosylase activity of S326C OGG1 was not significantly stimulated by the presence of AP-endonuclease. The altered substrate specificity, lack of stimulation by AP-endonuclease 1 (APE1) and anomalous DNA binding conformation of S326C OGG1 may contribute to its linkage to cancer incidence.
PMID: 16549874 [PubMed - indexed for MEDLINE]
Protection of INS-1 cells from free fatty acid-induced apoptosis by targeting hOGG1 to mitochondria.
Rachek LI, Thornley NP, Grishko VI, LeDoux SP, Wilson GL.
Department of Cell Biology and Neuroscience, College of Medicine, University of South Alabama, Mobile, AL 36688, USA. email@example.com
Chronic exposure to elevated levels of free fatty acids (FFAs) impairs pancreatic beta-cell function and contributes to the decline of insulin secretion in type 2 diabetes. Previously, we reported that FFAs caused increased nitric oxide (NO) production, which damaged mitochondrial DNA (mtDNA) and ultimately led to apoptosis in INS-1 cells. To firmly establish the link between FFA-generated mtDNA damage and apoptosis, we stably transfected INS-1 cells with an expression vector containing the gene for the DNA repair enzyme human 8-oxoguanine DNA glycosylase/apurinic lyase (hOGG1) downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase. Successful integration of MTS-OGG1 into the INS-1 cellular genome was confirmed by Southern blot analysis. Western blots and enzyme activity assays revealed that hOGG1 was targeted to mitochondria and the recombinant enzyme was active. MTS-OGG1 cells showed a significant decrease in FFA-induced mtDNA damage compared with vector-only transfectants. Additionally, hOGG1 overexpression in mitochondria decreased FFA-induced inhibition of ATP production and protected INS-1 cells from apoptosis. These results indicate that mtDNA damage plays a pivotal role in FFA-induced beta-cell dysfunction and apoptosis. Therefore, targeting DNA repair enzymes into beta-cell mitochondria could be a potential therapeutic strategy for preventing or delaying the onset of type 2 diabetes symptoms.
PMID: 16567524 [PubMed - in process]
Cell Mol Life Sci. 2005 Mar;62(5):606-18.
J Pharmacol Exp Ther. 2005 Nov;315(2):872-8. Epub 2005 Jul 22.
Nucleic Acids Res. 2006 Mar 20;34(5):1620-32. Print 2006.
Diabetes. 2006 Apr;55(4):1022-8.