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

已经研究了RB通路和p53通路上的蛋白。我们已经产生了几乎所有的抗体来区分RB蛋白上大约13个体内磷酸化位点。利用这些抗体，我们发现RB蛋白上的Cdk4-cyclin d特异性磷酸化用于E2F1的释放，而Cdk2-cyclin e特异性磷酸化用于LXCXE-motif-containing蛋白RBP1的释放，RBP1募集组蛋白去乙酰化酶和Brg1，染色质重塑蛋白。此外，我们发现在细胞DNA损伤时，E2F家族成员中只有E2F1与RB蛋白结合。此外，RB蛋白上Ser612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Cdk以外的激酶进行的，因为它不受Cdk抑制剂的抑制。Mdmx与Mdm2协同负调控p53活性，但机制尚不清楚。我们发现14-3-3与Mdmx结合时，Mdmx的Ser367被磷酸化，导致Mdmx被Mdm2泛素化，p53.3被激活。网格蛋白在胞吞作用中起重要作用。出乎意料的是，我们发现网格蛋白重链也定位于细胞核中，并对p53的转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在网格蛋白重链的c端区结合上存在竞争。

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.