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

已经研究了在RB途径和p53途径工作的蛋白质。我们已经产生了几乎所有的抗体来区分RB蛋白上的约13个体内磷酸化位点。使用这些抗体，我们已经表明，Cdk 4-细胞周期蛋白D特异性磷酸化RB蛋白用于释放E2 F1，而Cdk 2-细胞周期蛋白E特异性磷酸化用于释放LXCXE基序的蛋白RBP 1，它招募组蛋白去乙酰化酶，和Brg 1，染色质重塑蛋白。此外，我们还发现E2 F家族成员中只有E2 F1在细胞DNA损伤后与RB蛋白结合。此外，RB perotein上的Ser 612的磷酸化刺激了这种复合物的形成。这种磷酸化很可能是由激酶而不是Cdk进行的，因为它不受Cdk底物的抑制。Mdmx与Mdm 2协同负调节p53活性，但机制尚不清楚。我们已经发现，当Mdmx的Ser 367被磷酸化时，14-3-3与Mdmx结合，导致Mdm 2对Mdmx的泛素化和p53.3的激活。网格蛋白在胞吞作用中起重要作用。我们意外地发现，网格蛋白重链也定位于细胞核中，并对p53的反式激活活性起着必要的作用。此外，我们发现网格蛋白轻链和p53竞争结合网格蛋白重链的C-末端区域。

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.