Bianchi M, Villa-Moruzzi E
Department of Experimental Pathology, University of Pisa, via Roma 55, 56126 Pisa, Italy.
Biochem Biophys Res Commun. 2001 Jan 12;280(1):1-3. doi: 10.1006/bbrc.2000.4067.
Protein Ser/Thr phosphatase-1 (PP1) controls the retinoblastoma protein (pRb) function, including its dephosphorylation at mitotic exit. Since PP1delta was found to coimmunoprecipitate with pRb from mitotic and early G1 cells, we further investigated the PP1delta-pRb association using GST-full length and GST-deletion mutants of delta. GST-delta pulled-down pRb from G2, mitotic and G1 HeLa cells, thus confirming the coimmunoprecipitation results. Among the delta deletion mutants tested, pRb was pulled down by mutant 159-295, which reproduces the C-terminal domain of delta without the C-terminus, whereas the C-terminus alone did not pull-down pRb. Further fragmentation of the 159-295 mutant indicated that pRb was pulled down by fragment 195-260, which includes several residues involved in substrate binding, and by fragment 159-212, which contains the putative pRb-binding motif LxSxE. Altogether the results supported the hypothesis that PP1delta may contribute to the dephosphorylation of pRb at mitotic exit and that the PP1delta-pRb interaction may be at multiple sites.
蛋白丝氨酸/苏氨酸磷酸酶-1（PP1）调控视网膜母细胞瘤蛋白（pRb）的功能，包括其在有丝分裂退出时的去磷酸化。由于发现PP1δ能与有丝分裂期和G1早期细胞中的pRb进行共免疫沉淀，我们使用δ的GST全长及GST缺失突变体进一步研究了PP1δ与pRb的相互作用。GST-δ能从G2期、有丝分裂期和G1期的HeLa细胞中拉下pRb，从而证实了共免疫沉淀结果。在所测试的δ缺失突变体中，突变体159-295能拉下pRb，该突变体重现了δ的C末端结构域但不含C末端，而单独的C末端不能拉下pRb。159-295突变体的进一步片段化表明，片段195-260（包含几个参与底物结合的残基）和片段159-212（包含假定的pRb结合基序LxSxE）能拉下pRb。总体而言，这些结果支持了以下假说：PP1δ可能有助于有丝分裂退出时pRb的去磷酸化，且PP1δ与pRb的相互作用可能发生在多个位点。
