Analysis of cyclin E-containing complexes in the cell demonstrated that cyclin E associated with several cellular proteins. These include its kinase partner, cdk2, and molecules that affect the activity of cyclin E-cdk2 complexes both positively (cdc25A) and negatively (p27). In addition, this approach has previously yielded the identification of novel substrates, including both p107 and p130, suggesting that cdks can form stable enzyme-substrate complexes in vivo. This stable interaction may provide a mechanism by which increased specificity and selectivity can be achieved.
In this study, we have characterized two novel cyclin E-associated proteins as components of the mammalian SWI-SNF complex. Both BAF155 and BRG1 contain cdk consensus phosphorylation sites, and both could be phosphorylated by cyclin E-cdk2-associated kinase activity in vitro. Furthermore, BRG1 and BAF155 are in a phosphorylated form in the cyclin E complex. Another component of the SWI-SNF apparatus, the Ini1-hSNF5 protein, is also present in cyclin E immunoprecipitations, which suggests that the entire SWI-SNF complex may be recognized by cyclin E. Our experiments further demonstrate an intriguing requirement for the presence of BRG1 in the SWI-SNF complex to promote the recruitment of cyclin E. This observation suggests that either BRG1 recruits some essential factor to the complex, or the SWI-SNF apparatus is somehow modified in the presence of BRG1 so that it is recognized by cyclin E. The presence of the BRG1-related molecule hBRM in cyclin E immunoprecipitations implies that at least two different SWI-SNF complexes may be targeted by cyclin E, since hBRM and BRG1 are believed to be mutually exclusive components of the SWI-SNF apparatus (56
). Immunodepletion experiments suggest that the cyclin E-BAF155-BRG1 complex is distinct from those cyclin E complexes containing p107 and p130 and that neither pRb nor its related family members p107 and p130 are needed for cyclin E-BAF155-BRG1 complex assembly.
To demonstrate that the interaction of the SWI-SNF apparatus with cyclin E was functionally significant, we took advantage of a flat-cell assay established previously for BRG1 (11
). BRG1 is capable of inducing a flattened-cell morphology in SW13 cells. These cells do not divide and express markers indicative of replicative senescence, namely, SA β-galactosidase activity. Both cyclin E and cyclin D1 could abrogate this property of BRG1, reducing flat-cell induction by as much as 50%, suggesting that these cyclins can modulate the activity of BRG1. The flat-cell phenotype described in this study is similar to the one observed with the introduction of pRb into SAOS-2 cells (18
). In SAOS-2 cells, pRb similarly causes growth arrest and induction of SA β-galactosidase activity, which can be rescued by overexpression of G1
). While a dependence on BRG1 for pRb-mediated growth suppression has not yet been shown, a mutant form of BRG1 that is defective in the ability to bind pRb can no longer induce growth arrest in SW13 cells (11
). Hence, it has been proposed that pRb and BRG1 may function together to induce cell cycle arrest.
It will be important to establish the mechanism by which cyclin E suppresses flat-cell development and whether it is similar in both the SW13 and SAOS-2 assays. The ability of cyclins to rescue growth in the SAOS-2 cotransfection experiments was originally interpreted to be through pRb phosphorylation (18
). However, more recent experiments show that cyclins can also rescue the growth arrest induced by a nonphosphorylatable form of pRb (30
). The ability of cyclins to rescue growth in the SW13 cotransfection experiments may also be independent of the pRb family of proteins, since even in their absence, cyclin E can associate with SWI-SNF components. Furthermore, complexes between BRG1 and pRb appear unaffected by overexpression of cyclin E (data not shown). Our experiments thus raise the possibility that cyclin E may impinge on the SWI-SNF apparatus directly to revert the flat-cell phenotype. We are currently examining the phosphorylation status of various components of the SWI-SNF complex to see if they are altered with cyclin E overexpression.
A recent report by Sif et al. has demonstrated that the mitotic inactivation of the human SWI-SNF complex is caused by phosphorylation of various SWI-SNF subunits, including BRG1 (48
). The identity of the kinases responsible for these regulatory phosphorylations remains unknown. These observations, taken together with our results, suggest that the SWI-SNF apparatus may be modulated both positively and negatively through the cell cycle. While mitotic phosphorylation of BRG1 may be required for chromatin-mediated transcriptional repression during mitosis, phosphorylation on different sites of BRG1 or of other SWI-SNF subunits may be required for chromatin remodeling during G1
as cells prepare for DNA synthesis. Targets for modulation may include Ini1-hSNF5, because the yeast homolog Sfh1p is phosphorylated in a cell cycle-dependent manner during G1
). Since Ini1-hSNF5 is present in cyclin E immunoprecipitations, it will be important to address whether it also serves as a substrate for cyclin E-cdk2. A detailed analysis of the phosphorylation status of each of the SWI-SNF components as a function of cell cycle progression, and how this affects the chromatin remodeling activity of the complex, will help to elucidate how the SWI-SNF apparatus is coordinately regulated.
The demonstration that BRG1 may induce senescence is significant, because it implies that chromatin reorganization may be important during cell cycle exit and perhaps in the maintenance of a postmitotic state. Recent data suggest that inhibition of cdk activity can induce a senescent state (2
). Our data are consistent with a role for cyclin E in preventing cells from exiting from the cell cycle permanently through modulation of the SWI-SNF complex. It may be significant that Ini1-hSNF5 (23
) has recently been shown to be mutated in aggressive cancers (54
). Such observations provide further evidence that critical components of the chromatin remodeling machinery may act as growth suppressors that can be regulated by the cell cycle machinery. Overexpression of cyclin E in many human tumors may thus be a mechanism by which malignant cells escape cell cycle exit and senescence.