Inactivation of pRB-Related Proteins p130 and p107 Mediated by the J Domain of Simian Virus 40 Large T Antigen

Abstract

Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.The retinoblastoma tumor suppressor gene (Rb-1) is mutated in all cases of retinoblastoma and is also frequently mutated in a variety of other cancers (55,91,92). Loss of the antiproliferative activity of the RB protein (pRB) is thought to play a critical role in oncogenic transformation. There are two other members of the RB family of proteins, p107 and p130. These proteins have a high degree of sequence homology to pRB (31,38,56,62). pRB, p107, and p130 share a number of functional properties, including the ability to associate with and negatively regulate members of the E2F family of transcription factors (3,6,8,36,42,78,89) and the ability to induce a G 1 arrest in certain sensitive cell types (16,43,103). The presence of a p130-E2F complex has been proposed to define a quiescent, or G 0 , state of a cell (80). pRB, p107, and p130 are phosphorylated in a cell cycle-dependent manner (2,4,10,21,61,96). Phosphorylation in the mid/late G 1 phase inactivates the growth-suppressive properties of pRB and possibly also of p107 and p130 (15,43,103). pRB, p107, and p130 associate with cyclins (28,30,32,38,47,54,56,78) and are likely to be phosphorylated by cyclin-dependent kinases (cdks). Despite all these similarities, no mutations in p107 or p130 have yet been described in human tumors, and the role of these two proteins in oncogenic transformation remains unclear. pRB appears to be an essential protein in mouse embryonic development, as mice with homozygous deletions of the Rb-1 gene die before birth (14, 46, 52). Moreover, mice heterozygous for Rb-1 develop pituitary tumors with a penetrance of almost 100% (40,45,46). In contrast, mice lacking either p107 or p130 apparently develop norm...

Different functions are required for initiation and maintenance of immortalization of rat embryo fibroblasts by SV40 large T antigen

“…One interpretation of our results is that a functional J domain is required for initiation of immortalization but is dispensable once the immortal state is established. Recent work has suggested that the J domain is required to release the pocket proteins from E2F complexes, resulting in transcriptionally active`free' E2F (Sheng et al, 1997;Stubdal et al, 1997;Zalvide et al, 1998). Our data potentially contradict this hypothesis.…”

“…When T antigen binds these pocket proteins, they are unable to interact with, and repress the activity of, the transcription factor E2F, so E2F-dependent transcription proceeds and the cell enters the cell cycle. It has been suggested that an intact J domain is required to disrupt a pocket protein-E2F complex (Srinivasan et al, 1997;Stubdal et al, 1997;Zalvide et al, 1998), and that this function can only be supplied in cis (Stubdal et al, 1997). Others have found that the J domain can be supplied in trans (Montano et al, 1990;Porras et al, 1996), either by another T molecule, or by the homologus region from small t antigen.…”

Loss of LIN9, a member of the DREAM complex, cooperates with SV40 large T antigen to induce genomic instability and anchorage-independent growth

“…To confirm that the retinoblastoma pathway has been targeted efficiently by the D434-444 mutant, we analyzed levels of p130. It has been shown that SV40 LT species that bind to the pocket proteins alter the phosphorylation state and steady-state levels of p130 (Stubdal et al, 1996(Stubdal et al, , 1997Chao et al, 2000). We found low levels of p130 in cells expressing wild-type LT and D434-444, and high levels of p130 in cells expressing the LT K1 mutant.…”

Interaction of retinoblastoma protein family members with large T-antigen of primate polyomaviruses

“…Reintroducing a LXCXE domain to an N-terminally truncated T-antigen confers on cells the ability to grow to high density (Tevethia et al, 1997). Mutation studies demonstrated that T-antigen-mediated degradation of pRb2/p130 requires the T-antigen J domain as does the effect of T-antigen on p107 and pRb2/p130 phosphorylation (Stubdal et al, 1997). Furthermore, the J domain is required to override the repression of E2F activity mediated by pRb and pRb2/ p130 implying that whereas the LXCXE domain serves as a binding site for the pocket proteins, the J domain is involved in disrupting their function (Zalvide et al, 1998).…”