Attempts to identify the cyclin-cdk complexes responsible for the phosphorylation and functional inactivation of pRb have been frustrated by conflicting evidence generated by different types of experiments. Resolution of this issue is essential for understanding the control of cell cycle progression, as pRb phosphorylation appears to be critical for the advance of most cell types through the G1
phase into S phase. Some experiments have gauged the activities of G1
cyclin-cdk complexes through their incubation in vitro with pRb substrate (6
). Yet others have studied the phosphorylation of pRb in cells in which high, superphysiologic levels of various cyclins and cdk has been achieved through introduction of different expression vectors (10
) Each of these approaches would appear to be vulnerable to artifact. The specificity of G1
cyclin kinases for different substrates is relatively weak (27
), and a recent study has shown that by increasing the amount of a cyclin-cdk above a certain threshold level, one can cause the phosphorylation of additional sites beyond those modified at lower levels of these complexes (6
). With these observations in mind, we avoided the ectopic expression of active cyclins and cdks and instead attempted to inhibit the functions of endogenous cyclin-cdk complexes expressed at physiologic levels by the cell.
Our analysis of phosphopeptides of pRb from cells arrested by cdk2DN, in which cdk2 activity has been effectively blocked, indicates that D-type cyclin-directed phosphorylation in vivo is restricted to a subset of sites on pRb. Tryptic phosphopeptides obtained from pRb that has been phosphorylated both in vitro and in vivo have been extensively characterized (6
). However, in the present experiments, the lack of precision in phosphopeptide analysis of manipulated, sorted cells makes it difficult to draw further conclusions from our data. For example, the population of cdk2DN-transfected cells may contain a small amount of contaminating untransfected cells, so that we cannot conclusively determine that all the pRb phosphopeptides from these cells represent phosphorylation by D-type cyclin kinases alone. More significantly, however, the relative absence of specific pRb phosphopeptides from cdk2DN-transfected cells indicates that these sites are less favored targets for phosphorylation by D-type cyclin-associated kinases in vivo and indeed unlikely to be modified at all by these cyclin-cdk complexes. We suspect but cannot prove directly that these particular sites are targeted specifically by cyclin E-cdk2 complexes following initial phosphorylation of pRb on other sites by cyclin D-cdk4/6 complexes.
The present experiments suggest that cyclin D-cdk4/6 complexes alone were unable to inactivate pRb in vivo. We were not able to directly measure the growth-suppressive properties of pRb which has been modified by cyclin D-cdk4/6 complexes, since the concurrent inhibition of cdk2 activity might also affect other essential steps in G1
. Instead, we analyzed other characteristics associated with active pRb. pRb mediates growth suppression in part by binding to and apparently sequestering a number of other cellular growth-promoting proteins. Prominent among these are members of the E2F family of transcription factors. Only hypophosphorylated pRb binds E2F, and pRb inactivated by deletion, mutation, or phosphorylation is incapable of binding to or repressing E2F (2
). As shown here, E2F-mediated transcription is repressed in cdk2DN-transfected cells, despite phosphorylation of pRb by D-type cyclin kinase. These results provided an indication that the partial modification of pRb effected by cyclin D-cdk4/6 complexes does not succeed in causing release of E2Fs from the inhibitory effects of pRb.
While we suppose here that E2F activity is controlled by the state of phosphorylation of pRb, an alternative scenario might be suggested by recent work demonstrating that E2F activity can in certain circumstances be modulated directly by cyclin E-cdk2, independent of any involvement of pRb (9
). This mechanism, if operative, would force us to reinterpret the experiments here in which we analyzed E2F activity in cells expressing the cdk2DN plasmid which interferes directly with cdk2 activity. However, using the same cells that we have studied here, others have shown that the effects of the cdk2DN allele can be reversed by wild-type simian virus 40 large T antigen but not by a mutant thereof that is incapable of binding pRb (22
). Such results strongly support the notion that the effects on E2F activity observed by us here derive from the functioning of pRb, which in the present case are regulated by its state of phosphorylation.
Further evidence supporting the notion that partial phosphorylation of pRb, obtained by blocking cdk2 activity, does not abrogate the binding of pRb to E2F comes from our analysis of both an osteosarcoma and a keratinocyte cell line. Specifically, the form of pRb that migrates at a rate intermediate between those of fully phosphorylated and unphosphorylated pRb retains its ability to bind E2Fs and is enriched in a population of cdk2DN-transfected cells.
In contrast to our observations, others have recently reported that pRb that is phosphorylated in vitro by cyclin D1-cdk4 complexes, cyclin E-cdk2 complexes, or cyclin A-cdk2 complexes can no longer bind E2F (6
). However, we would argue that the substrate specificities of protein kinases are often abrogated in vitro and, as mentioned above, that the excessive amounts of cyclin-cdk complexes can phosphorylate additional sites that are not modified by lower amounts of these complexes (6
). This notion is borne out by our own earlier results showing that ectopic expression of cyclin E or cyclin D in the living cell can drive pRb phosphorylation to completion, which would not seem to reflect the normal activities of the endogenous proteins in cells.
As demonstrated some years ago, phosphorylated forms of pRb lose their binding affinity to nuclear structures, as manifested by the ability to leach them from the nuclei of detergent-permeabilized cells with low-salt buffers (43
). As shown here, in cdk2DN-transfected cells, pRb undergoes partial phosphorylation by cyclin D-cdk4/6, retains its ability to bind and repress E2Fs, but loses its tight tethering to the nucleus. These data indicate that pRb undergoes a succession of functional alterations as the cell traverses G1
; in this case, the loss of nuclear tethering of pRb precedes the loss of ability to bind E2F. Since E2F binding appears to be tightly connected with the growth-controlling functions of pRb, we believe that loss of nuclear binding is not equivalent, as previously assumed, to loss of the ability to inhibit cell proliferation (43
Previous experiments have suggested that pRb phosphorylation occurs in a sequential manner in other types of cells. In particular, full phosphorylation of human pRb ectopically expressed in yeast requires both D-type and E cyclin kinase activity or the activity of analogous yeast cyclins. In this yeast expression system, the presence of cyclin D1 alone also leads to partial pRb phosphorylation (19
). Further correlative evidence for sequential phosphorylation of pRb comes from analysis of phytohemagglutinin-stimulated T cells. In these cells, the initial forms of phosphorylated pRb appear at a point in time when only cyclin D-associated (and not cyclin E-associated) kinase can be detected, while the slowest-migrating fully phosphorylated form or pRb does not appear until after cyclin E is active (7
). Together, these observations provide correlative evidence supporting the mechanistic model that has been tested here directly by the specific inhibition of the responsible cyclin-cdk complexes.
Although our observations further elucidate pRb phosphorylation in G1
, several critical issues remain. While we can conclude that cyclin D-cdk4/6 complexes are able to phosphorylate pRb only partially in vivo, we lack direct evidence demonstrating that cyclin E-cdk2 completes this process. Cell physiologic evidence suggests that pRb undergoes functional inactivation at a time in the cell cycle when cyclin E-cdk2 becomes active in mid/late G1
, a period when cyclin A-cdk2 complexes are not yet active (29
). Nonetheless, other, still undefined kinases in addition to cyclin E-cdk2 may contribute to the complete phosphorylation of pRb that occurs in mid/late G1
. Furthermore, during S phase, the activities of cyclin A-associated complexes may extend the work of the cyclin D- and cyclin E-associated complexes that is initiated in G1
A second issue is provoked by the observation that cyclin E-cdk2 appears unable to phosphorylate pRb that has not been previously modified by the actions of cyclin D-cdk4/6. The molecular mechanism by which this occurs is unclear. We do not believe that this is merely an artifact of p16INK4A
expression, as others have also recently observed that cyclin E kinase is unable to phosphorylate pRb in the absence of cyclin D kinase activity (34
). Cyclin E kinase complexes may not be able to recognize or gain access to native unphosphorylated pRb that is bound to certain tethering sites in the nucleus; our work indicates that cyclin D-cdk4/6-mediated phosphorylation releases pRb from these tethers. Alternatively, cyclin D-cdk4/6-mediated phosphorylation may evoke a conformational change in pRb that makes it into a better substrate for cyclin E-cdk2. Such a model of progressive phosphorylation of a protein by distinct kinases is not without precedent. For example, the phosphorylation of glycogen synthase kinase on certain sites by glycogen synthase kinase 3 requires its prior phosphorylation on other sites by casein kinase II (50
A third issue is suggested by the observation that the entire pool of cellular pRb appears to be held in a functionally inactive state after the cell has passed the restriction point in late G1. This would seem to require that pRb molecules that are synthesized de novo in S and G2 phases undergo phosphorylation shortly after their synthesis at a time when cyclin E is no longer active. More importantly, this phosphorylation would appear to occur during periods in S, G2, and M phase when D-type cyclins may no longer be active. For example, cells in S phase that are then deprived of serum mitogens will continue their cell cycle advance and apparently continue to successfully phosphorylate pRb in the presence of only cyclin A-cdc2 complexes. We speculate therefore that B-like cyclin–cdk complexes (i.e., involving cyclin A or B) may be able to completely phosphorylate pRb even in the absence of preparatory phosphorylation by D-type cyclin-associated cdk and may thus, with respect to pRb, be more wide-ranging in their substrate specificities than are the cyclin D- and cyclin E-associated kinase complexes.
Finally, the purpose of sequential, cooperative phosphorylation is unclear. It may facilitate an additional dimension of control on pRb inactivation. Alternatively, it may create a gradation of gene activations in which certain transcription factors are liberated by the actions of cyclin D-cdk4/6 on pRb while yet others, such as E2Fs, are released only following the actions of cyclin E-cdk2. It may also provide the cell cycle apparatus with additional specificity in regulating alternative cell fates such as proliferation and differentiation.