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Crossing borders in search of functions: The role of Cdk2 in meiosis

As is the case for most stress response factors, the activity of Chk2 kinase, a central effector of the DNA damage response, is homeostatically regulated by a fine balance of positive and negative regulation. Prives and colleagues investigate the negative regulation of Chk2 by ubiquitin/ proteasome degradation in the February 1, 2009 issue of Cell Cycle,1 and in so doing, expose a significant gap in our somewhat dogmatic knowledge of MDM2 biology and function. Kass et al. show that both MDM2, a well-studied E3 that targets p53, and the recently identified E3, P/CAF2 (better known for its HAT function and participation in transcription regulation), target Chk2 for ubiquitination and degradation. Targeting of Chk2 by MDM2 was blocked by DNA damage stimulated phosphorylation of Chk2 S456, allowing for stress induced stabilization of Chk2 during DNA damage responses. Unexpectedly, the ability of MDM2 to ubiquitinate Chk2 did not require its RING E3 domain, which is required for ubiquitination of all of its other known targets—most notably, p53. Given that P/CAF binds and ubiquitinates MDM2, the authors also looked for an impact of P/CAF on Chk2, and found that it independently ubiquitinated and destabilized Chk2. MDM2 and P/CAF together synergistically ubiquitinated Chk2, and all three molecules could be found in one complex. As seen with MDM2, mutation of the putative P/CAF E3 domain had no effect on its Chk2 destabilizing activity. The authors propose that P/CAF and MDM2 may act within a larger multi-subunit E3 (a la SCF or APC) to target Chk2, and serve mainly as recognition factors/scaffolds rather than actual E3 enzymes. In support of this idea, MDM2 interacted more strongly with Chk2-S456A, a mutant which is preferentially hyperubiquitinated in the presence of MDM2. Presuming the effects of P/CAF and MDM2 on Chk2 ubiquitination are direct—this work leaves the obvious mystery of the identity of the Chk2 E3. What known MDM2-P/CAF binding partners might be viable candidates? Most obvious is MDM-X, the heterodimerization partner of MDM2.3 MDM-X, though structurally similar to MDM2, with a conserved C-terminal RING E3 domain, does not target p53 for degradation by itself. It can participate with MDM2 in targeting p53 when heterodimerized (or perhaps in larger oligomeric complexes) with MDM2. Thus, if the MDM2 E3 defective mutant and the E3-defective P/CAF mutant can both interact with MDM-X, perhaps its RING contributes the missing E3. Another viable candidate would be CUL4 and its associated SCF complex, as two publications have identified CUL4 as a direct binding partner of MDM2, and also a participant with MDM2 in p53 ubiquitination.4,5 Lastly, p300 and/or CBP may provide the missing E3 activity. p300 interacts with both MDM2 and P/CAF through separate domains (C/H1-TAZ1 and C/H3-TAZ2, respectively), while p300 and P/CAF both interact with MDM2 through its central domain.2,6,7 Thus, there is a distinct possibility given all the possible contact surfaces, that p300 (and/or CBP), P/CAF and MDM2 participate in a tripartite complex that could then interact with Chk2. p300/CBP are better known as HATs (as is the distantly related P/CAF) that globally regulate histone acetylation, but also act as non-histone acetylases that regulate dozens (at least) of transcription factors, most notably, p53. The ubiquitin ligase activity of p300 was recently described as specifically targeting p53 for polyubiquitination, but only when p53 had been oligoubiquitinated by MDM2 first.8 CBP shares a similar “E4” activity for p53.9 This activity is centered within the N-terminal 595 amino acids of p300 where a substrate independent generic E3 activity is also located. Initially perplexing was the lack of a canonical E3 domain in p300 (RING, Hect, U-box, etc.), but since 2003 there has been a growing list of such non-canonical E3’s, which includes A20,10 P/CAF and Rabex-5.11 Though no evolutionarily conserved domains are shared by these proteins, at least p300 and A20 share non-RING (and non-homologous) Zn2+-binding domains, which are required for E3 activity, or are at least present within the putative E3 domain. P/CAF has a potential Zn2+ binding sequence within its E3 domain, and this sequence aligns with a portion of p300 TAZ1 sequence.12 Given that p300 and P/CAF do share evolutionary origins, their E3 domains may very well share structural similarity, perhaps centering on the known (for p300) and putative (for P/CAF) Zn2+-binding domains of each protein. Setting aside the details of exactly how MDM2 and P/CAF accomplish the ubiquitination of Chk2 without their own E3 domains, one wonders— why this way? Given evolutionary pressures, the regulation of Chk2 may have evolved in this manner due to the need for Chk2 ubiquitination in contexts where the MDM2 RING or P/CAF E3 domains are not available. Two possibilities arise. Perhaps the Chk2 regulatory function was encoded in a more evolutionarily ancient form of MDM2 or P/CAF, prior to E3 activities being joined to the molecules. More likely, especially for MDM2, there are prevalent forms of the molecule that lack the RING domain, yet should still bind to Chk2 and P/CAF. A substantial fraction of MDM2 protein exists in a caspase-cleaved form, where cleavage after residue 361 by distinct caspases in resting13 and apoptotic14 cells, leaves intact a C-terminally truncated RING-less form that maintains an intact central domain, and presumably, p300 and P/CAF binding activities. As the exact functional relevance of caspase-cleaved MDM2 has remained mysterious, perhaps examination of its effects on Chk2 in resting, DNA damaged and apoptotic cells would be worthwhile, and also shed light on why nature seems to have chosen an arcane mechanism more reminiscent of Rube Goldberg than Occam, when it comes to MDM2-P/CAF regulation of Chk2.