Figure presents a comprehensive molecular interaction map of regulators of cell cycle and DNA repair processes. The current map, however, is limited to events in the mammalian cell nucleus. Because of limits on what can legibly be formatted onto a journal page, the map is divided into two parts. Figure A maps the interactions involving E2F, pRb, Cyclin, and Cdk family members, their activators and inhibitors, as well as some important interactions with other components. Figure B focuses on the p53-Mdm2 subsystem and on subsystems related to DNA repair. Molecular components are grouped in putative subsystems according to mutual interactions or functional coherence. It remains to be determined whether subsystems can be identified on the basis of objective criteria.
The monomolecular species included in Figure are listed alphabetically in an index (Table ), which gives grid coordinates to help locate each species.
Index of monomolecular species
Some of the interaction patterns shown in the map deserve special comment. References are cited in the annotation list and can be found using the identifiers marked on the interaction lines in the map. These identifiers are italicized letter–number combinations, which are used also in the following text to specify particular interactions on the map.
We are concerned here primarily with molecular interactions rather than biological effects. The latter may eventually be understood as emerging from the former.
The E2F-pRb Box
The possible occupancy states of an E2F recognition element in a promoter comprise a multiplicity of complex patterns. The method of representing these patterns was shown by the simplified example in Figure . The simplest arrangement would be an E2F:DP heterodimer bound to an E2 element (E5). (The italicized letter–number combinations identify particular interactions in Figure and refer to entries in the annotation list where references are cited.) Because promoters generally have two or more E2F recognition elements, however, the actual situation even in this case may be far from simple. Most E2F family proteins can activate transcription (E6) by way of a transactivation domain. E2F-6, however, has instead a transcription repressor domain (E9). E2F:DP heterodimers (other than those of E2F-6) can tether pRb family proteins onto promoters, thereby converting a potential gene activator into a repressor (E7). E2F in fact more often functions to repress rather than activate.
E2F proteins (as heterodimers with a DP protein [E1]) have individual binding preferences for pRb family members. The E2F-pRb box shows that pRb prefers E2F-1-, -2-, or -3-containing heterodimers (E2) (with lesser interaction with E2F-4), p107 binds only E2F-4 heterodimers, and p130 can bind heterodimers of E2F-4 or 5 (E3,4). All of these complexes can bind and repress E2F recognition elements (E6). E2F-6, however, does not bind any pRb family protein. E2F-1, -2, and -3 are marked together as a unit on the map, because of their mutual preference for pRb. Although there are significant functional distinctions between them, the molecular bases for these differences is not known. No major functional differences among different DP family members have been defined.
A further level of complexity arises from the ability of some E2F complexes to bind other transcription factors, such as Sp1, and to synergize the transcriptional activation.
Another mode of regulation arises from the ability of histone deacetylase (HDAC1) to bind to pRb:E2F:DP-type complexes (E13). HDAC1 could deactivate transcription that has been enhanced by histone acetylation (H1) and thus could contribute to gene down-regulation by pRb family proteins.
pRb sometimes functions as a gene activator rather than repressor. For example, it can activate the Jun family and CCAAT/enhancer-binding protein (C/EBP) transcription factors (E16,17). The detailed mechanism by which this occurs is not known.
Taken together, the interactions noted on the map add up to perhaps 20 different possible states of an individual E2F recognition element.
The interaction capabilities of the constituent proteins are subject to modulation by phosphorylation, protein binding, and regulated degradation. In addition, regulated nuclear–cytoplasmic transport is emerging as an important process. Translocations can be represented as indicted in Figure but, because of space limitations, have been omitted from the current version of the map. pRb is subject to different sets of multiple phosphorylation by CycD:Cdk4/6 (C31) and cycE:Cdk2 (C32). The manner of depiction of these phosphorylations and their effects was explained in Figure . Only the fully phosphorylated pRb is impaired with respect to E2F binding (C33). The E2F binding of p107 and p130 is also inhibited by phosphorylation (E11). Phosphorylation of E2F and/or DP by CycA:Cdk2 (which forms stable complexes with E2F-1) (E20) inhibits the E2F-DP interaction and could serve to turn off E2F function when cycA accumulates late in S phase.
pRb family proteins may also be inhibited by binding to other proteins, such as Raf-1 (E22). This may be one of the logical connections, suggested by current work, which may communicate signals from the cell surface to the cell cycle control circuitry.
The Cyclin-Cdk Box
The activity of Cdks is intricately regulated. To begin with, Cdk activity requires binding to a Cyclin. The map shows Cdk4 or Cdk6 (which have the same molecular interactions) binding to CycD (C3), Cdk2 binding to Cyclins E or A (C4), and Cdk1 (also known as Cdc2) binding to Cyclins A or B (C5). (The subtypes of CycB and CycD are not differentiated here.)
A second class of controls on Cdks are stimulatory and inhibitory phosphorylations, which are controlled by several kinases and phosphatases noted on the map. All Cdks are activated by phosphorylation of Thr160 (or 161), carried out by CycH:Cdk7 (C14), which functions also as a constituent of the transcription factor IIH (TFIIH) complex. Cdk1, and to some extent other Cdks, can be inhibited by phosphorylations corresponding to Thr14 and/or Tyr15 (C17,19,20). These sites are phosphorylated by Wee1 or Myt1 (C16). In the case of Cdk1, these inhibitory phosphorylations are removed by dual-action phosphatase Cdc25C, which is in turn activated by phosphorylations (C18) introduced by mammalian polo-like kinase 1 (Plk1) (C37) and/or Cdk1 (C36). Cdc25C can be phosphorylated at Ser216 by Chk1 (C38) or C-TAK1 (C39). Ser216 phosphorylation generates a binding site for 14-3-3, and this binding inhibits the phosphatase (C40).
A positive feedback loop that can be traced on the map consists of just two components: Cdc25C and CycB:Cdk1. Cdc25C is activated by hyperphosphorylation (C18); activated Cdc25C dephosphorylates Thr14 and Tyr15 of Cdk1, thereby removing the inhibitory effect of these phosphates on the kinase (C17) and increasing the activating phosphorylation of Cdc25C (C36). This positive feedback could help produce switch-like behavior and may operate in the G2 to M cell cycle phase transition.
A third class of controls acts through the binding of specific Cyclin:Cdk inhibitors, including p16ink4a, p21cip1, p27kip1, and p57kip2. p16ink4a inhibits by binding Cdk4/6 in competition with cycD (C8). p21cip1, p27kip1, and p57kip2 can bind Cyclin complexes of Cdk4/6 and Cdk2 (C7,23). There may be an additional complication, however, because p21 can stabilize and enhance the activity of cycD:Cdk4 when a single p21 molecule is bound but can inhibit the same activity when a second p21 molecule binds to the complex (C22). p27 can be phosphorylated by the kinase it inhibits, CycE:Cdk2 (C21). This seemingly paradoxical relationship might be due to intermolecular action of an active CycE:Cdk2 on an inactive CycE:Cdk2:p27 complex.
The Cyclin:Cdk system can interact with elements of the DNA replication and repair systems through binding of p21 (R6) or Cyclin D (R11) to proliferating cell nuclear antigen (PCNA). This action may also involve Gadd45, which can bind simultaneously to p21 (C34) and PCNA (R10). p21, PCNA, and Gadd45 are all transciptionally activated by p53 (P43,44).
The p53-Mdm2 Box
The map shows the remarkable richness of p53 interconnections and the diversity of functionally determinative p53 modifications. Eleven phosphorylation or acetylation sites (or groups of sites) for which functionality has been surmised are shown. If all of these could occur independently, there would be ~2000 possible modification states of p53 monomers. Some interdependent modifications have been noted: phosphorylation of Thr18 requires previous phosphorylation of Ser15 (P3) (Appella, personal communication); acetylation of Lys320 requires tetramer structure of p53 and is inhibited by phosphorylation of Ser378 (P23) (Sakaguchi and Appella, personal communication). Other dependencies certainly exist, some perhaps having major functional impact, whereas many could have subtle quantitative effects, which may or may not convey a selective evolutionary advantage. Nevertheless, p53-expressing cells may contain hundreds of different modification states of p53 monomers.
Some p53 modifications and interactions are especially notable. Ser15 appears to be the site of phosphorylation responses to DNA damage signals communicated by way of the kinases ataxia telangiectasia mutated gene/protein (ATM) (P2) and DNA-dependent protein kinase (DNA-PK) (P6). Phosphorylation of Ser18 or Ser20 prevents stable binding to Mdm2 (P5), thus abrogating the Mdm2-mediated inhibition (P29) and degradation (P31) of p53. Because these sites are located within the region required for Mdm2 binding, it is plausible that their phosphorylation could inhibit this interaction. p53 forms a stable complex with p300 (P25), as a result of which p300 acetylates p53 Lys382 (P21). This acetylation, as well as the acetylation of Lys320 by PCAF (P23), enhances the sequence-specific binding of p53 to promoters, probably indirectly by inhibiting nonspecific DNA binding (P22). A similar mechanism of enhanced promoter binding (P16) may occur as a result of binding of the p53 C-terminal region to 14-3-3 (P13), which requires 14-3-3 to be dimerized (P14).
p53 can bind to a number of proteins that are involved in DNA repair functions, cell cycle control, or general control functions. In approximate order of binding location from N to C termini of p53, these include the following: Mdm2 (which has a pocket that binds a p53 N-terminal peptide) (P28); p300 C-terminal region (P25); DP1 (P26); poly(ADP-ribose) polymerase (PARP) (P46); c-Abl (A4); replication protein A (RPA) (S6); high-mobility group protein (HMG) (P52); TFIIH constituent helicases xeroderma pigmentosum complementation group B (XPB) and XPD and DNA repair protein CSB (P27); p19ARF (P40); p300 N-terminal region (P33); BRCA1 (P47); and 14-3-3 (P13). Some of these interactions (p300, BRCA1, and 14-3-3) stimulate and some (Mdm2, PARP, and RPA) inhibit the transcriptional activity of p53. (Stimulations may be indirect: 14-3-3 may block nonspecific binding of p53 to DNA [P16]; p300 may do the same consequential to acetylation of K382 [P21,22].)
p53 can form homotetramers and must be in tetramer form for sequence-specific DNA binding and transcriptional activation. The map shows the dependencies relating to tetramers; tetramerization is stimulated by phosphorylation of Ser392, and this enhancement can be inhibited by phosphorylation of Ser315 (P17). The ability to form tetramers is further modulated by other modifications and protein interactions. Influence on tetramerization may be how p53 transcriptional activity is stimulated by binding BRCA1 and inhibited by binding Mdm2, PARP, or RPA. This could be the major mechanism of regulation of p53 transcriptional activity. The activation of p53 seems to be exquisitely controlled by a large number of determinative inputs. The transition to active tetramers could be very sharp because of a possible fourth-power dependence on the concentration of tetramerization-competent monomers.
Mdm2 is an intimate part of the p53 control system. Mdm2 contains a pocket that binds a p53 N-terminal peptide (P28). Mdm2 binding blocks the transcriptional activation domain of p53 (P29) and is instrumental in p53 degradation (P31). p53, in turn, transcriptionally up-regulates Mdm2, probably forming a negative feedback loop. Mdm2 can itself activate some genes, such as Cyclin A (P37). In addition to binding p53, Mdm2 reportedly binds E2F1:DP1 (P35), pRb (P35), TATA-binding protein (TBP) (P36), TBP-associated factor II250 (TAFII250) (P37), p19ARF(P34), and p300 (P33). Some of these interactions may compete for the same Mdm2 site, as may be the case for p19ARF and p300. Binding to p53 is abrogated by phosphorylation of Mdm2 on Ser17, perhaps through the kinase activity of DNA-PK (P49).
p19ARF, an alternate reading frame (ARF) product from the ink4a locus that also codes for p16, has recently emerged as an additional player in the p53-Mdm2 system. p19ARF binds to and inhibits the actions of Mdm2 (P34,41). It also can bind to p53 (P40). Moreover, p19ARF is transcriptionally up-regulated by E2F1:DP1 (P42). This link between p53 and E2F1 may be crucial to the control of S-phase and apoptosis.
The map includes three phases of nucleotide excision repair (NER). The first phase, lesion recognition and local opening of the DNA helix, is carried out by a molecular assembly, which includes the XPC:HR23B heterodimer (N1,2), XPA (N3), and TFIIH (N13). This phase opens the DNA helix in the vicinity of the lesion and allows access to other DNA repair proteins. If the DNA is opened by another process, such as transcription, XPC is dispensable, and repair can begin with the second phase.
The second phase, excision of a short DNA strand segment containing the lesion, is carried out by an assembly of the XPG and XPF:excision repair cross-complementing 1 (ERCC1) endonucleases (N6,8,9
), together with XPA, RPA, TFIIH, and PCNA (which can bind XPG) (R9
). This assembly appears to be held together in part through RPA, which binds to single-stranded DNA (ssDNA) regions in the vicinity of lesion (N10
), and at the same time may be able to bind XPG and XPF:ERCC1 (N7
), as well as XPA (N4
). In going from the recognition to the excision phase, the molecular assembly rearranges as XPC:HR23B is replaced by XPG (N13
) (Wakasugi and Sancar, 1998
In the third phase, gap filling, x-ray repair cross-complementing gene/protein 1 (XRCC1) appears to function as a platform for the assembly of DNA polymerase β (DPase β) (N20
), DNA ligase III (N19
), and PARP (N18
). This assembly is held together, in part, via breast cancer protein 1 C-terminal module (BRCT) modules in the constituent proteins (Masson et al., 1998
). The binding of PARP by XRCC1 may function to block the further action of PARP during this phase at a repair site. The assembly of XRCC1 with DPase β and DNA ligase III may also function in the single-nucleotide replacement pathway of base excision repair (Cappelli et al., 1997
Through its binding to DNA single-stranded regions, RPA may also recruit Rad52 and Rad51 to sites of DNA damage (S14,15). Rad51 also binds to ssDNA and, together with RPA, may function in recombinational repair (N11).
Rad51 may also provide links to a network of mutually interacting components via its binding to c-Abl (N12). c-Abl may bind ATM (A1), DNA-PK (B7), pRb (E18), and p53 (A4), although it is not fully determined which of these interactions can occur simultaneously and which are mutually exclusive. Rad51, ATM, and DNA-PK bind to a Src homology 3 (SH3) domain in the c-Abl N-terminal region, whereas pRb and p53 may bind to the c-Abl C-terminal region. c-Abl may regulate Rad51 function by phosphorylating Rad51 on Tyr54, thereby abrogating the direct binding of Rad51 to ssDNA (N12).
Another set of interactions is implicated in the processing of DNA double-strand breaks. DNA double-strand ends are recognized and bound by the Ku70:Ku80 heterodimer (Ku) (B1,2
), which can recruit DNA-PK to the site (B3
), thereby activating the kinase (B4
). DNA-PK, however, can also bind to RPA (S16
). RPA is a heterotrimer (S1,2
) that binds ssDNA regions (S3
). DNA-PK can thus be recruited to ssDNA regions formed transiently at replication forks. DNA-PK may then be available for interaction with a double-stranded DNA (dsDNA) end, which could appear in the vicinity as a consequence of replication fork encounters with open topoisomerase I DNA complexes trapped by drugs such as camptothecin (Shao et al., 1999
). It is noteworthy that DNA-PK does not always require Ku for activation, because it can be activated by tethering to DNA via other molecules, such as chromatin constituents of the HMG family (B10
A further capability of RPA could arise from its ability to bind p53 (S6) and from the abrogation of this binding by phosphorylation of the RPA2 subunit by DNA-PK (S11), ATM (S12), or CycA:Cdk2 (S10).
We thus begin to see some of the intricate mechanism of the DNA repair machinery. This example shows how a molecular interaction map can represent DNA-targeted processes and the transitions between multimolecular assemblies.