DIFFERENTIAL SPECIFICITY FOR BINDING OF RETINOBLASTOMA BINDING-PROTEIN-2 TO RB, P107, AND TATA-BINDING PROTEIN
DIFFERENTIAL SPECIFICITY FOR BINDING OF RETINOBLASTOMA BINDING-PROTEIN-2 TO RB, P107, AND TATA-BINDING PROTEIN
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DOI：
10.1128/mcb.14.11.7256
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发表时间：
1994-11-01
影响因子：
5.3
通讯作者：
KAYE, FJ
中科院分区：
文献类型：
--
作者：
KIM, YW;OTTERSON, GA;KAYE, FJ
RB和p107产物的生长抑制活性被认为是由细胞蛋白质的异质家族与两种蛋白质中存在的保守T/E1 A口袋结构域的可逆结合介导的。为了研究这些相互作用的功能作用，我们研究了细胞视网膜母细胞瘤结合蛋白2（RBP 2）与RB，p107和相关的TATA结合蛋白（TBP）产物的结合特性。我们观察到，尽管RBP 2仅与p107的T/E1 A口袋结合，但它可以通过独立的T/E1 A和非T/E1 A结构域与RB相互作用，并且仅通过非T/E1 A结构域与TBP相互作用。与此观察结果一致，我们发现RBP 2的Leu-X-Cys-X-Glu基序内的突变导致沉淀p107的能力丧失，而RB和TBP结合活性保留。我们将RBP 2的非T/E1 A结合位点定位在一个15 kDa的片段上，该片段独立于Leu-X-Cys-X-Glu基序，编码RB和TBP的结合活性，但不与p107相互作用。尽管存在非T/E1 A结合位点，但是，重组RBP 2保留了从全细胞裂解物中优先沉淀活性低磷酸化RB的能力。此外，我们发现共转染RBP 2可以逆转体内RB介导的E2 F活性抑制。这些发现证实了相关RB，p107和TBP蛋白的差异结合特异性，并支持核RBP 2产物上存在多功能结构域，这可能允许与细胞转录机制的复杂相互作用。
The growth suppressor activities of the RB and p107 products are believed to be mediated by the reversible binding of a heterogeneous family of cellular proteins to a conserved T/E1A pocket domain that is present within both proteins. To study the functional role of these interactions, we examined the properties of cellular retinoblastoma binding protein 2 (RBP2) binding to RB, p107, and the related TATA-binding protein (TBP) product. We observed that although RBP2 bound exclusively to the T/E1A pocket of p107, it could interact with RB through independent T/E1A and non-T/E1A domains and with TBP only through the non-T/E1A domain. Consistent with this observation, we found that a mutation within the Leu-X-Cys-X-Glu motif of RBP2 resulted in loss of ability to precipitate p107, while RB- and TBP-binding activities were retained. We located the non-T/E1A binding site of RBP2 on a 15-kDa fragment that is independent from the Leu-X-Cys-X-Glu motif and encodes binding activity for RB and TBP but does not interact with p107. Despite the presence of a non-T/E1A binding site, however, recombinant RBP2 retained the ability to preferentially precipitate active hypophosphorylated RB from whole-cell lysates. In addition, we found that cotransfection of RBP2, can reverse in vivo RB-mediated suppression of E2F activity. These findings confirm the differential binding specificities of the related RB, p107, and TBP proteins and support the presence of multifunctional domains on the nuclear RBP2 product which may allow complex interactions with the cellular transcription machinery.