RADIOBIOLOGY DIVISION

13.RADIOBIOLOGY DIVISION

    Research in the Radiobiology Division is focused on the two basic pathways in cancer: the RB pathway and the p53 pathway. In particular, the physiological meaning of phosphorylation of these proteins has been studied using many phospho-specific antibodies to recognize the phosphorylation sites on p53 and the RB proteins.
Phosphorylation of p53

    Upon DNA damage, p53 protein accumulates rapidly through a post-transcriptional mechanism(s) and is also activated as a transcription factor, which then leads to growth arrest or apoptosis. An important question is how these two different pathways are selected by p53. Studies in the Radiobiology Division showed last year that the switch between promoters of a G1-arrest gene, p21Waf1, and an apoptosis gene, p53AIP1, is regulated by phosphorylation of Ser46 on p53. Thus, purification and identification of this Ser46-kinase have been tried. This was neither p38 MAP kinase nor JNK. It was found that Ser46-kinase is composed of several proteins and contains p53DINP1, a newly found protein which is induced by p53.⁽¹⁸⁹⁾
    The stress-responsive p38 MAP kinase, when activated by genotoxic stresses such as UV radiation, enhances p53 activity by phosphorylation and leads to cell cycle arrest or apoptosis. It was found that p53-inducible Wip1 phosphatase mediates a negative feedback regulation of p38-p53 signaling in response to UV regulation.⁽¹⁹⁰⁾ P38 MAP kinase was also shown to phosphorylates Ser33 but not Ser46 of p53 upon DNA damage by chemotherapeutic agents.⁽¹⁹¹⁾
    In contrast to other stresses, it was found that p53 accumulates but is functionally impaired when DNA synthesis is blocked.⁽¹⁹²⁾
    The relationship between phosphorylation of p53 and other stresses, cisplatin, taxol, hypoxia and nitric oxide was also studied and found that these stresses induce phosphorylation of a variety of sites on p53.^(193-¹⁹⁵⁾
A Novel ATM Family

    Nonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates imperfect mRNAs that contain premature translation termination codons (PTCs) and code for nonfunctional or potentially harmful polypeptides. It was shown that a novel PI3-kinase-related protein kinase, hSMG-1, is a human ortholog of a product of C. elegans smg-1, one of seven smg genes involved in NMD. HSMG-1 phosphorylates hUPF1/SMG-2 in vivo and in vitro at specific serine residues in SQ motifs. hSMG-1 can associate with hUPF1/SMG-2 and other components of the surveillance complex. In particular, overexpression of a kinase-deficient point mutant of hSMG-1 results in a marked suppression of the PTC-dependent beta-globin mRNA degradation, whereas that of wild type hSMG-1 enhances it. It was also shown that inhibitors of hSMG-1 induce the accumulation of truncated p53 proteins in human cancer cell lines with p53 PTC mutation. Therefore, it was concluded that hSMG-1 plays a critical role in NMD through the direct phosphorylation of hUPF1/SMG-2 in the evolutionally conserved mRNA surveillance complex.⁽¹⁹⁶⁾
RB Proteins and Cyclin-dependent Kinases

    The hyper-phosphorylated RB protein generated by the action of cdk2/cyclin E strongly stimulated the activity of DNA polymerase alpha, interacting with the catalytic subunit of that polymerase.⁽¹⁹⁷⁾
    PKC-eta was found to associate with the cyclinE/cdk2/p21 complex, phosphorylating p21 and inhibiting cdk2 kinase in keratinocytes.⁽¹⁹⁸⁾
    The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl) retinamide can inhibit the growth and induce apoptosis of tumor cells. It was suggested that its antiproliferative activity arises from its capacity to maintain the RB protein in a de-phosphorylated growth-suppressive status in S-G2/M, possibly through cyclin D1 downregulation.^(199,²⁰⁰⁾




13.RADIOBIOLOGY DIVISION



	Research in the Radiobiology Division is focused on the two basic pathways in cancer: the RB pathway and the p53 pathway. In particular, the physiological meaning of phosphorylation of these proteins has been studied using many phospho-specific antibodies to recognize the phosphorylation sites on p53 and the RB proteins. Phosphorylation of p53 Upon DNA damage, p53 protein accumulates rapidly through a post-transcriptional mechanism(s) and is also activated as a transcription factor, which then leads to growth arrest or apoptosis. An important question is how these two different pathways are selected by p53. Studies in the Radiobiology Division showed last year that the switch between promoters of a G1-arrest gene, p21Waf1, and an apoptosis gene, p53AIP1, is regulated by phosphorylation of Ser46 on p53. Thus, purification and identification of this Ser46-kinase have been tried. This was neither p38 MAP kinase nor JNK. It was found that Ser46-kinase is composed of several proteins and contains p53DINP1, a newly found protein which is induced by p53.⁽¹⁸⁹⁾ The stress-responsive p38 MAP kinase, when activated by genotoxic stresses such as UV radiation, enhances p53 activity by phosphorylation and leads to cell cycle arrest or apoptosis. It was found that p53-inducible Wip1 phosphatase mediates a negative feedback regulation of p38-p53 signaling in response to UV regulation.⁽¹⁹⁰⁾ P38 MAP kinase was also shown to phosphorylates Ser33 but not Ser46 of p53 upon DNA damage by chemotherapeutic agents.⁽¹⁹¹⁾ In contrast to other stresses, it was found that p53 accumulates but is functionally impaired when DNA synthesis is blocked.⁽¹⁹²⁾ The relationship between phosphorylation of p53 and other stresses, cisplatin, taxol, hypoxia and nitric oxide was also studied and found that these stresses induce phosphorylation of a variety of sites on p53.^(193-¹⁹⁵⁾ A Novel ATM Family Nonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates imperfect mRNAs that contain premature translation termination codons (PTCs) and code for nonfunctional or potentially harmful polypeptides. It was shown that a novel PI3-kinase-related protein kinase, hSMG-1, is a human ortholog of a product of C. elegans smg-1, one of seven smg genes involved in NMD. HSMG-1 phosphorylates hUPF1/SMG-2 in vivo and in vitro at specific serine residues in SQ motifs. hSMG-1 can associate with hUPF1/SMG-2 and other components of the surveillance complex. In particular, overexpression of a kinase-deficient point mutant of hSMG-1 results in a marked suppression of the PTC-dependent beta-globin mRNA degradation, whereas that of wild type hSMG-1 enhances it. It was also shown that inhibitors of hSMG-1 induce the accumulation of truncated p53 proteins in human cancer cell lines with p53 PTC mutation. Therefore, it was concluded that hSMG-1 plays a critical role in NMD through the direct phosphorylation of hUPF1/SMG-2 in the evolutionally conserved mRNA surveillance complex.⁽¹⁹⁶⁾ RB Proteins and Cyclin-dependent Kinases The hyper-phosphorylated RB protein generated by the action of cdk2/cyclin E strongly stimulated the activity of DNA polymerase alpha, interacting with the catalytic subunit of that polymerase.⁽¹⁹⁷⁾ PKC-eta was found to associate with the cyclinE/cdk2/p21 complex, phosphorylating p21 and inhibiting cdk2 kinase in keratinocytes.⁽¹⁹⁸⁾ The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl) retinamide can inhibit the growth and induce apoptosis of tumor cells. It was suggested that its antiproliferative activity arises from its capacity to maintain the RB protein in a de-phosphorylated growth-suppressive status in S-G2/M, possibly through cyclin D1 downregulation.^(199,²⁰⁰⁾