We isolated a novel protein, EID-1, that interacts biochemically and functionally with pRB and p300 or CBP. An accompanying manuscript describes the isolation of this protein by a second group using a strategy similar to our own (45
). We found that EID-1 blocks differentiation in several models. This activity correlates with its ability to inhibit transcriptional activation by proteins such as MyoD that utilize p300 and CBP as coactivators. Furthermore, we showed that EID-1 directly blocks p300 and CBP HAT activity. EID-1 is ubiquitinated and rapidly degraded as cells exit the cell cycle, provided its C-terminal pRB- and p300-binding domain is intact. Thus, EID-1 is poised to couple cell cycle exit to the execution of a differentiation program.
We have thus far been unable to detect complexes of pRB and EID-1 in untransfected cells. One possibility is that the isolation of EID-1 in our pRB two-hybrid screen was fortuitous. This seems unlikely, however, for several reasons. First, eight of eight clones that we recovered in our screen encoded fragments of EID-1. When tested with the two-hybrid screen, EID-1 bound to pRB with higher affinity than known pRB interactors such as E2F1 and E7 (data not shown). Biochemical and structural studies show that E7 possesses a core 9mer motif with the sequence DL
N that is necessary and sufficient for high-affinity pRB-binding (underlined are critical residues that directly contact pRB) (35
). This sequence is well conserved in EID-1.
There are several reasons why detection of endogenous pRB–EID-1 complexes in cells may be inherently difficult. First, EID-1, like other pocket-binding LXCXE proteins, binds exclusively to the un(der)phosphorylated form of pRB. Secondly, EID-1 is a short-lived protein of low abundance. More importantly, EID-1 is rapidly degraded as cells exit the cell cycle. Thus, under the conditions where pRB becomes un(der)phosphorylated, EID-1 disappears. Indeed, as described below, these two phenomena may be linked. In this regard, we find that overproduced EID-1 is more stable than endogenous EID-1, perhaps because one or more cellular proteins required for its degradation become limiting. Our attempts to stabilize pRB–EID-1 complexes with proteasomal inhibitors have thus far been unsuccessful (data not shown). A caveat, however, is that these agents block degradation but not ubiquitination. It is possible that EID-1 cannot bind to pRB once it has been ubiquitinated.
In functional assays, pRB and EID-1 antagonize one another. pRB, but not tumor-derived mutants, prevents EID-1 from inhibiting transcription. Conceivably, this activity relates to the earlier observation that pRB cooperates with certain transcription factors and promotes differentiation. Conversely, forced production of EID-1 inhibits pRB-dependent differentiation. Importantly, an EID-1 mutant that cannot form stable complexes with pRB [EID-1(1-157)] retains this property. This would place EID-1 downstream of pRB in a differentiation control pathway.
Several lines of evidence suggest that inhibition of p300- and CBP-dependent transcriptional activation by EID-1 does not merely reflect nonspecific transcriptional squelching. Firstly, EID-1 had no significant effect on the CMV promoter used as an internal control in our experiments. Secondly, EID-1(1-157) more potently inhibited p300- and CBP-dependent transactivation than did wild-type EID-1 and yet did not, itself, act as a transcriptional activator at any concentration tested when fused to Tetr. Finally, we easily obtained stable SAOS-2 and C2C12 clones producing high levels of EID-1 (data not shown). This last observation argues against a nonspecific “toxic” effect by EID-1.
Instead, our biochemical studies suggest that EID-1 inhibits p300 and CBP HAT activity. Importantly, both wild-type EID-1 and EID-1(1-157) inhibited HAT activity in vitro and blocked differentiation. In contrast, EID-1(1-157Δ53Δ92) was defective for both of these activities, suggesting that these two properties are linked. It is noteworthy that EID-1(1-157) retained the ability to inhibit p300 and CBP HAT despite a diminished (but not absent) ability to bind to these two proteins. In this way, EID-1 resembles E1A and TWIST (5
Several lines of evidence lead us to hypothesize that pRB and/or p300 play a role in targeting EID-1 for ubiquitin-dependent proteolysis upon cell cycle exit. First, only those EID-1 mutants that measurably bound to p300 or pRB were ubiquitinated in cells. Second, both p300 and pRB can bind to MDM2 (23
), which is a known E3 ubiquitin ligase (30
). The results of our coimmunoprecipitation assays are consistent with the hypothesis that p300 and/or pRB serve as adapters that recruit MDM2 to EID-1. Finally, degradation of EID-1 was temporally related to dephosphorylation of pRB during leukemic cell differentiation. Conversely, overproduction of a pRB mutant that can bind to EID-1, but cannot bind to MDM2, stabilized EID1.
EID-1 bears some functional similarity to HBP1 (61
). HBP1 inhibits myogenic differentiation and can inhibit pRB-dependent differentiation but cannot override a pRB-induced cell cycle block (68
). In contrast to EID-1, however, HBP1 requires an intact LXCXE motif for these activities (68
). Indeed, our data raise the possibility that the above-noted effects of HBP1 were due to displacement of EID-1 from pRB.
pRB is a more potent inducer of differentiation than p107 and p130 (47
). In an adipocyte model, pRB promoted differentiation whereas p107 and p130 inhibited differentiation and antagonized pRB (9
; M. Classon and E. Harlow, personal communication). It will be important to determine whether p107 and p130 physically interact with EID-1 and, if so, whether the functional consequences of such interactions differ from that described here for pRB.
Both the pRB family and p300 and CBP are targeted for inactivation during viral transformation. The majority of human tumors harbor mutations which, directly or indirectly, perturb pRB function (58
). Deletions and translocations of p300 and CBP have recently been identified in some solid tumors such as gastric and colon carcinomas as well as in leukemias (20
). EID-1 may act at a nodal point that couples the activity of the pRB pathway to the p300-CBP pathway. Deregulation of EID-1 may contribute to the failure of cancer cells to differentiate in vivo. This raises the interesting possibility that EID-1 may, itself, function as an oncogene and be a target of mutations in human cancer.