Cell cycle suppression by Cdk5 is achieved through its participation in a multi-protein complex that includes E2F1. The involvement of E2F1 in the mechanics of Cdk5 cell cycle suppression is significant. The E2Fs are a family of transcription factor proteins best known for their role in regulating cell proliferation, differentiation, and apoptosis through transcriptional regulation (Ishida et al., 2001
; Muller et al.,2001
; Ren et al., 2002
; Trimarchi and Lees, 2002
). Our EMSA data provide evidence that Cdk5 has the capacity to reduce the transcription of genes with E2F1 response elements. The basis for this inhibition can be found in our studies of the impact of Cdk5 on the association of E2F1 with its cofactor, DP1. It has been shown that, in the absence of DP1, E2F1-dependent transcriptional activation decreases dramatically (Bandara et al., 1993
). By binding E2F1 and displacing DP1, wild type Cdk5 and KDCdk5 both decrease the occupancy of the E2F1 promoter element and hence can function as effective cell cycle suppressor proteins. This is a p35-dependent process as can be seen from the fact that the Cdk5(S159T) mutant does not affect promoter occupancy and, as would be predicted, does not function as a cell cycle suppressor.
The requirement for p35 binding in the formation of the complex is intriguing. While its binding to Cdk5 is well known, its association with E2F1 () is unexpected. We note that p35 is most similar in size and structure to cyclin A and there is a known cyclin A binding site on E2F1. Further, the binding of cyclin A blocks the ability of E2F1 to support the transcription needed for full cell cycle progression (Krek et al., 1994
; Mudryj et al., 1991
). Thus, we would predict that the interaction of p35 with E2F1 is through this cyclin A site. Cyclin A and D also can bind with Cdk5 (Guidato et al., 1998
; Lee et al., 1996; Xiong et al., 1992
; Zhang et al.,1993
), but surprisingly the S159T mutation has no effect on this interaction (Supplemental Figure 4A
). Similarly, the T286A mutation of cyclin D1, which is important for its subcellular localization, binds to Cdk5 as well as its wild type counterpart. Finally, Cdk5 binding to cyclins D2 and A is very weak (Figure S4B and Figure S4C
). Taken together, these data argue that Cdk5 is not ‘buffering’ other cyclins to block their access to Cdk4 and Cdk6. Rather it is p35 that is the factor most directly involved in Cdk5 cell cycle suppression.
As the main activator of Cdk5, p35 contains a myristolation signal motif, which allows it to be anchored to the plasma membrane. Since it is the p10 fragment that contains the myristolation anchor, cleavage of membrane-bound p35 liberates p25 to the cytoplasm (Nikolic et al1998; Patrick et al., 1999
). Although this would predict that most p35 would be membrane bound, in cultured neurons and other cells p35 has been also found in the cell nucleus (Nikolic et al., 1996
; Qu et al., 2002
; Gong et al., 2003
; Xinrong Fu, et al 2006
). Our BiFC data strongly support this nuclear location (). In this context, it is significant that of the three known activators of Cdk5 only nuclear p35 demonstrates cell cycle suppression ability. The closely related p39 protein is capable of compensating for developmental p35 deficiency in vivo (Ko et al., 2001), but this redundancy does not appear to apply to the cell cycle activity. The failure to observe cell cycle suppression with p25 is also significant. Kim et al (2008)
recently have reported that p25 induces neuronal cell cycle reentry thorough stimulating Cdk5 phosphorylation of HDAC1 and the induction of DNA damage. Many of the effects reported in this study occurred over a period of weeks, suggesting that chronic Cdk5 activity in the nucleus might have a very different outcome than shorter, acute episodes such as those we have studied here.
It is noteworthy that in certain cell types E2F1 itself appears to function as a cell cycle suppressor (Field et al., 1996
). Indeed, our laboratory has explicitly argued that E2F1 acts as a cell cycle suppressor in neurons (Wang et al., 2007
). This would appear to be at odds with the model presented in . We have begun to resolve the apparent inconsistency by monitoring the levels of other E2F family members. We find E2F3 levels are dramatically up-regulated in E2f1−/−
brains compared with other E2Fs (Figure S5
). shows that the nuclear E2F3 level in E2f1−/−
were increased about 15 times compared with wild type. This suggests that elevated levels of E2F3 are the motive force behind the observed neuronal cell cycle events in the E2f1−/−
brain. Thus E2F1 acts as a cell cycle suppressor by a feedback mechanism that normally keeps the levels of E2F3 low. The implication is that the failure of cell cycle suppression is indirect and mediated by E2F3.
Figure 9 A model of the regulation of the neuronal cell cycle by Cdk5. For a normal cell cycle to precede, E2F1 must bind its co-factor, DP1, to fully activate a variety of cell cycle related genes. When Cdk5 is located in the cytoplasm, Cdk5 cannot make a complex (more ...)
It is telling that the complexes we describe differ dramatically depending on whether the players assemble in the nucleus or the cytoplasm. Our data show that Cdk5, p35, DP1 and E2F1 can bind each other as heterodimers in all combinations and in all locations. This means that each protein has a potential binding site for each of the other three proteins. Yet, when all four are present at the same time, Cdk5 and DP1 apparently compete to bind a p35-E2F1 complex. In the cytoplasm, DP1 keeps Cdk5 out of the complex resulting in a DP1-p35-E2F1 trimer. In the nucleus, the situation reverses; Cdk5 keeps DP1 out of the complex to make a Cdk5-p35-E2F1 trimer ().
Our findings have implications for neurodegenerative diseases in general and Alzheimer’s disease in particular. Cdk5 has been demonstrated to be an effective tau kinase. Since hyperphosphorylated tau is a major constituent of the neurofibrillary tangles found in Alzheimer’s and other dementias, inhibition of Cdk5 activity has been proposed as a therapeutic approach to slowing the advance of the disease (Gong and Iqbal, 2008
; Camins et al., 2006
; Kaminosono et al., 2008
). Kinase inhibition may prove effective in blocking the formation of tangles, but our results suggest caution in this approach. Cell cycle re-entry is a common prelude to neurodegeneration in a variety of conditions. As nuclear Cdk5 acts as a cell cycle suppressor both in vivo and in vitro, it would follow that this characteristic of Cdk5 may be worth promoting, rather than inhibiting. Thus, any strategy aimed at blocking Cdk5 activity as a means of combating neurodegenerative disease should be carefully monitored for either direct or indirect effects on the levels or location of the Cdk5 protein. Treatments that lower overall protein levels or encourage nuclear emigration may produce unexpected and possibly unwanted consequences for the CNS.