Studies on the Relationship between the RB Pathway and the p53 Pathway

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Proteins working at the RB Pathway and the p53 Pathway have been studied.1. we have generated almost all antibodies to distinguish about 13 in vivo phosphorylation sites on the RB proteins. Using these antibodies, we have shown that Cdk4-cyclin D-specific phosphorylation on RB proteins is used for relaease of E2F1 while Cdk2-cyclin E-specific phosphorylation is used for release of LXCXE-motif-containing proteins RBP1, which recruits histone deacetylase, and Brg1, chromatin remodeling protein. In addition, we have found that only E2F1 among E2F family members binds to RB proteins upon DNA damge of cells. Moreover, phosphorylation of Ser612 on RB perotein stimulated this complex formation. This phosphorylation is likely to be performed by a kinase other than Cdk because it is not inhibited by Cdk inhibitors.2. Mdmx collaborates with Mdm2 to negatively regulates p53 activity, but the mechanism is not known. We have found that 14-3-3 binds to Mdmx when Ser367 of Mdmx is phosphorylated, leading to ubiquitination of Mdmx by Mdm2 and activation of p53.3. Clathrin plays an important role in endocytosis. We have found, unexpectedly, that clathrin heavy chain is also localized in nucleas and plays a necessary role for transactivation activity of p53. Moreover, we have found that clathrin light chain and p53 compete for binding to the C-terminal region of clathrin heavy chain.