[Frontiers in Bioscience 3, d532-547, June 8, 1998]|
NEW PERSPECTIVES ON RETINOBLASTOMA FAMILY FUNCTIONS IN DIFFERENTIATION
Amy S. Yee, Heather H. Shih, and Sergei G. Tevosian
The Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111
Received 5/10/98 Accepted 5/29/98
2.1 General Background of the RB Family
Cell differentiation is a fundamental process that imparts unique identity through a coordinated tissue-specific gene expression program. For example, the muscle and adipocyte differentiation programs consist of genes that specify contractile proteins and fat mobilization, respectively. Despite the unique differences in tissue-specific gene expression, a general feature of many tissues is a notable lack of proliferation and the maintenance of an irreversible cell cycle exit. During differentiation, the expression of tissue-specific genes is tightly coordinated with cell cycle exit. Many studies have provided knowledge on cell cycle arrest and on key regulatory transcription factors that give tissue specific gene expression. However, little is known on the mechanisms that insure tight coordination of cell cycle exit and tissue-specific gene expression. Yet, these are necessary features in which lapses in coordination have severe consequences. For example, the abrogation of the cell cycle exit allows re-entry of otherwise differentiated tissues into the cell cycle. In disease, this aberrant proliferation is a characteristic of early changes that may lead to cancer in some tissues. In normal functions, the proliferation of differentiated tissues allows regeneration. A notable case is the regeneration of liver in response to injury. Thus, the regulation of cell cycle exit in the context of differentiation has important implications for both cancer and normal tissue biogenesis. Recent studies with the Retinoblastoma (RB, p107, p130) family have provided new insights into the coordination of cell cycle and tissue-specific events during differentiation.
Studies from cancer biology, cell biology, and virology have converged to establish RB and E2F in one paradigm for G1 regulation. RB was first discovered as a tumor suppresser gene with frequent mutation in human cancer. The expression of RB led to suppression of the G1 phase of the cell cycle. As summarized in figure 1A, a key discovery is the identification of the E2F transcription factor as the first nuclear target for RB. E2F regulates the expression of numerous genes necessary for S-phase of the cell cycle, such as thymidine kinase, DHFR, DNA Pol a, cdc6. For example, the direct link of cdc6 to DNA replication provides a satisfying example of E2F coordination of S-phase (1). RB inhibits E2F-dependent transcription of these essential genes and provides one mechanism for the observed G1 arrest by RB. The under-phosphorylated form of RB is associated with inhibition of E2F. In G1, sequential RB phosphorylations by Cyclin D/CDK4 and by Cyclin E/CDK2 lead to dissociation of RB and E2F. The net result is free E2F and the activation of genes for S-phase entry and progression. In S-phase, E2F is inactivated through phosphorylation by Cyclin A/CDK2 and thereby allowing exit from S-phase (2-4). Thus, the phosphorylation of RB and E2F by CDKs contribute to regulation of cell cycle progression in normal cells. This fundamental pathway has been subverted by the viral oncoproteins of the DNA tumor virus (E1A, Large T, E7) through the sequestration of RB and release of E2F to give S-phase (reviewed in (5, 6) and references within).
Figure 1. Summary of the E2F and RB family regulation in the cell cycle. The E2F activity is composed of a heterodimer of E2F and DP proteins to achieve the maximal DNA binding activity. The paradigm of E2F and RB regulation is diagrammed in simple (A) and reaslistic (B) view. The simplified view highlights the basic features of E2F and RB family members. A detailed discussion can be found in the text and within (5,7, 8). Figure 1C is a schematic diagram of the RB family. The RB family consists of RB, p107 and p130. A common feature is the pocket, which is the region of tumorigenic mutations, viral oncoprotein binding and E2F interactions. The A and B pocket domains are denoted in yellow. The C domain is unique to RB and is denoted in green. The spacer regions between the A and B are similar in p107 and p130, denoted in a grid pattern. The spacer region is different in RB, denoted in a plain pattern. All other regions are largely divergent, denoted in varying shades of blue. Please see text for more details (reviewed in (17).
While the inhibition of E2F by RB was a satisfying explanation, the rapidly emerging knowledge suggests that this paradigm is far too simple (see figure 1B and reviewed in (7, 8)). There are three RB family members (RB, p107, and p130), 6 E2Fs (E2F1-6) and 3 DPs(1,2,3). A summary of the various E2F complexes is provided in figure 1B. The challenge remains the functional delineation of each RB and E2F family member. The overall E2F levels are further regulated on the protein level through ubiquitin mediated degradation (9-11). While the studies of RB have been dominated by E2F, there is growing appreciation that RB has a diverse role in cellular regulation and that functionally important non-E2F targets of RB must exist. A simple argument is that the concentration of RB family members is much greater than that of E2F. Thus, the beautiful work with E2F may be just a glimpse of regulation by RB family members.
Figure 1C depicts a schematic diagram of RB, p107, and p130. RB is the first and best characterized member in which most studies have focused on the pocket domain. This region is the site for interactions with E2F and other proteins. In the case of E2F, disruption of the pocket:E2F interaction leads to the release of inhibition and to subsequent activation of E2F. Disruption could be achieved by mutation, viral oncoprotein binding, or CDK phosphorylation. The observation that numerous cancers are correlated with RB pocket domain mutations remains a seminal discovery in tumor biology. Despite extensive efforts, there has been no correlation of pocket mutations in p107 and p130 with cancer. Loss of p130 expression has been observed in small cell lung carcinoma (12). A possibility is the inactivation of p130 and p107 functions may still lead to cancer.Unlike RB, this neutralization may not result from genetic mutation, but could arise through disruptive protein interactions or phosphorylation. However, this remains to be proven.
Numerous viral and cellular proteins use a conserved LXCXE motif for pocket interaction with RB, p107, and p130. Notably, E2Fs use a different motif (13). RB also contains a C-pocket domain that is necessary for interaction with E2F1-3 and with c-abl (for review, see(14)). Notably, The RB C-pocket is not found in p107 or p130. Additionally, the p107 and p130 proteins contain a spacer region between the A and B pockets that directs the interaction and apparent inhibition of Cyclin A/CDK2 or Cyclin E/CDK2 ((15, 16)). The p107 and p130 spacer regions differ from RB, which does not interact with CDK complexes. The N-terminal regions of RB, p107, and p130 are divergent and may provide the specificity within the family (reviewed in (17)).
This review will highlight the RB family in three distinct aspects of cell differentiation: cell cycle exit, tissue-specific gene expression, and apoptosis protection. The multiple functions of RB suggests a role in global coordination to insure fidelity during differentiation. The primary focus will be on the muscle and adipocyte differentiation models. The elegant and extensive work on MyoD and c/EBP families of transcriptional regulators has provided a backdrop for elucidating how cell cycle regulation coordinates with tissue specific gene expression. Recent observations in several labs suggest the existence of both positive and negative regulation to control the progression of differentiation. We hypothesize the existence of a differentiation checkpoint that coordinates cell cycle and tissue-specific events in a full differentiation pathway.