| Co-Investigator(Kenkyū-buntansha) |
ASANO Shigetaka Institute of Medical Science, University of Tokyo Department of Medicine, Professor, 医科学研究所・病態薬理学, 教授 (50134614)
NAKAHATA Tatsutoshi Institute of Medical Science, University of Tokyo Department of Pediatric Hematology/Oncology, Professor, 医科学研究所・癌病態学, 教授 (20110744)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Fanconi anemia (FA) is an autosomal recessive genomic instability syndrome characterized by progressive bone marrow failure, congenital anomalies, and cancer susceptibility. Cells derived from FA patients show hypersensitivity to DNA cross-linking agents, abnormal cell cycle progression and reduced cell survival. FA patients constitute at least eight different complementation groups (FA-A to FA-H) as defined by cell fusion analysis. The genes for group C (FANCC), group A (FANCA), group G (FANCG) have been cloned. We recently proposed that the FANCA phosphorylation, formation of FANCA/FANCC complex and nuclear accumulation of the protein complex define the FA pathway, based on the observation that these biochemical events are disturbed in FA cells derived from groups A, B, C, E, F, G and H.In support of this notion, physical interaction of FANCG and FANCA was documented. In order to analyze molecular mechanisms of FANCA phosphorylation, we have developed an in vitro kinase system. When
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anti-FANCA immune complexes from wole cell lysates were incubated with 32P-ATP, FANCA was phosphorylated in vitro. V8 protease-digested phosphopeptides from this protein was identical to those from 32P-labeled FANCA in vivo. Phosphoamino acid analyses showed that serine residues were phosphorylated. Cell fractionation studies showed that phosphorylated FANCA and the kinase activities localize to the cytoplasm. These results indicate that a serine protein kinase (FANCA-PK) binds to and phosphorylates FANCA in the cytoplasm. Consistently, a mutant FANCA protein, which has no NLS and fails to translocate into the nuclei, is still phosphorylated in vivo and in vitro. When wt-FANCA was overexpressed in non-AFA cells, FANCA phosphorylation was restored in vitro as well as in vivo but defective FANCA/FANCC binding or MMC hypersensitivity, a marker phenotype of FA, was not corrected. Patient-derived mutants FANCA (H1110P) and FANCA (R1117G), overexpressed in FANCA-null cells, was phosphorylated in vitro similarly to wt-FANCA, whereas its cellular phosphorylation remained markedly reduced. Taken together, these results indicate that FANCA-PK constitutievly binds to FANCA and phosphorylates the substrate without the other FA proteins but that FANCA phosphorylation alone is insufficient for function of the FA pathway. A protein domain of FANCA containing His at 1110 and Arg at 1117 appears to have a critical role for FANCA phosphorylation in vivo. Less
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