Defective human MutY phosphorylation exists in colorectal cancer cell lines with wild-type MutY alleles.
Oxidative DNA damage can generate a variety of cytotoxic DNA lesions such as 8-oxoguanine (8-oxoG), which is one of the most mutagenic bases formed from oxidation of genomic DNA because 8-oxoG can readily mispair with either cytosine or adenine. If unrepaired, further replication of A.8-oxoG mispairs results in C:G to A:T transversions, a form of genomic instability. We reported previously that repair of A.8-oxoG mispairs was defective and that 8-oxoG levels were elevated in several microsatellite stable human colorectal cancer cell lines lacking MutY mutations (human MutY homolog gene, hmyh, MYH MutY homolog protein). In this report, we provide biochemical evidence that the defective repair of A.8-oxoG may be due, at least in part, to defective phosphorylation of the MutY protein in these cell lines. In MutY-defective cell extracts, but not extracts with functional MutY, A.8-oxoG repair was increased by incubation with protein kinases A and C (PKA and PKC) and caesin kinase II. Treatment of these defective cells, but not cells with functional MutY, with phorbol-12-myristate-13-acetate also increased the cellular A.8-oxoG repair activity and decreased the elevated 8-oxoG levels. We show that MutY is serine-phosphorylated in vitro by the action of PKC and in the MutY-defective cells by phorbol-12-myristate-13-acetate but that MutY is already phosphorylated at baseline in proficient cell lines. Finally, using antibody-isolated MutY protein, we show that MutY can be directly phosphorylated by PKC that directly increases the level of MutY catalyzed A.8-oxoG repair.
Parker
Antony R
AR
Department of Pathology, The Johns Hopkins University, Baltimore, Maryland 21205, USA.
O'Meally
Robert N
RN
Sahin
Fikret
F
Su
Gloria H
GH
Racke
Frederick K
FK
Nelson
William G
WG
DeWeese
Theodore L
TL
Eshleman
James R
JR
D000225:Adenine / Q000737:chemistry; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000483:Alleles; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D002273:Carcinogens; D047390:Casein Kinase II; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D015179:Colorectal Neoplasms / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003596:Cytosine / Q000737:chemistry; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D006151:Guanosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D018895:Microsatellite Repeats; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D012984:Software; D013755:Tetradecanoylphorbol Acetate; D015854:Up-Regulation
D000276:Adjuvants, Immunologic; D002273:Carcinogens; D007211:Indoles; D008301:Maleimides; D020033:Protein Isoforms; C467001:bisindolylmaleimide IX; D006151:Guanosine; C046215:8-hydroxyguanosine; D012694:Serine; D003596:Cytosine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D000225:Adenine; D013755:Tetradecanoylphorbol Acetate