Dunaief Joshua L, King Ayala, Esumi Noriko, Eagen Matthew, Dentchev Tzvete, Sung Ching-Hwa, Chen Shiming, Zack Donald J
FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.
Curr Eye Res. 2002 May;24(5):392-6. doi: 10.1076/ceyr.24.5.392.8524.
To identify and characterize retinoblastoma protein (pRb) binding proteins that may influence retinoblast proliferation and retinal pigment epithelial cell survival.
The yeast two-hybrid system was used to screen a bovine retinal cDNA library and to characterize positive clones. DNA sequencing and site-directed mutagenesis were used for further analysis. Co-immunoprecipitation experiments were used to confirm the results of the two-hybrid system in vivo.
In the two-hybrid system, Protein Phosphatase 1alpha1 (PP1alpha1) binds the retinoblastoma protein. Unlike several other pRb binding proteins, PP1alpha1 binds only weakly to the Rb family member p107, and does not demonstrate detectable binding to p130. Confirming the two-hybrid results, endogenous PP1 in a human retinal pigment epithelial (RPE) cell line co-immunoprecipitates with endogenous pRb but not p107 or p130. Site directed mutagenesis of two pRb binding motifs in PP1alpha1 from LXSXE to LXCXE leads to slight increases in its two-hybrid interaction with pRb but does not alter its binding preference for pRb over the other family members. The complete sequence of bovine PP1alpha1 is reported.
The strong two-hybrid interaction between PP1alpha1 and pRb, but not p107 or p130, suggests that the phosphorylation status of members of the pRb family may be regulated by different phosphatases, contributing to fine control of cell cycle progression. Conversely, PP1 activity may be specifically regulated by pRb and not p107 or p130. Mutagenesis studies suggest that PP1alpha1's LXSXE motif is not responsible for its binding preference for pRb over p107 and p130. Disruption of the PP1-pRb interaction may influence retinoblastoma tumorigenesis as well as RPE cell proliferation and survival.
鉴定并表征可能影响视网膜母细胞瘤细胞增殖和视网膜色素上皮细胞存活的视网膜母细胞瘤蛋白（pRb）结合蛋白。
采用酵母双杂交系统筛选牛视网膜cDNA文库并鉴定阳性克隆。利用DNA测序和定点诱变进行进一步分析。通过免疫共沉淀实验在体内证实双杂交系统的结果。
在双杂交系统中，蛋白磷酸酶1α1（PP1α1）与视网膜母细胞瘤蛋白结合。与其他几种pRb结合蛋白不同，PP1α1仅与Rb家族成员p107弱结合，且未显示出与p130的可检测结合。在人视网膜色素上皮（RPE）细胞系中，内源性PP1与内源性pRb进行免疫共沉淀，而不与p107或p130共沉淀，从而证实了双杂交结果。将PP1α1中两个pRb结合基序从LXSXE定点突变为LXCXE，导致其与pRb的双杂交相互作用略有增加，但并未改变其对pRb相对于其他家族成员的结合偏好。报道了牛PP1α1的完整序列。
PP1α1与pRb之间存在强烈的双杂交相互作用，而与p107或p130无此相互作用，这表明pRb家族成员的磷酸化状态可能受不同磷酸酶调控，有助于对细胞周期进程进行精细控制。相反，PP1活性可能受pRb而非p107或p130的特异性调控。诱变研究表明，PP1α1的LXSXE基序并非其对pRb相对于p107和p130具有结合偏好的原因。PP1与pRb相互作用的破坏可能会影响视网膜母细胞瘤的肿瘤发生以及RPE细胞的增殖和存活。
