Several forkhead transcription factors have been associated with controlling periodic gene expression during the mammalian cell cycle, including FOXM1 and FOXO family members (reviewed in Refs. 10
). Moreover, both FOXM1 and FOXO transcription factors act as direct targets for CDK·cyclin complexes, the key drivers of the cell cycle. Here we have demonstrated that an additional forkhead transcription factor, FOXK2, is also subject to regulatory phosphorylation by CDK·cyclin complexes.
FOXK2 phosphorylation was revealed by the appearance of a slower mobility species on denaturing gels. Phosphorylation peaks in mitotic cells, where all of the FOXK2 migrates as a lower mobility form. In agreement with this, the mitotically active CDK1·CLNB appears to be the major kinase complex acting on FOXK2 in vivo
. Importantly, siRNA knockdown experiments suggest that CDK2 and CLNA also influence FOXK2 phosphorylation, indicating that phosphorylation might be initiated prior to mitosis. In fact, low levels of serine 368 phosphorylation can be seen throughout the cell cycle but with a distinct peak in mitotic cells. This behavior closely resembles the sequential integration of CDK1·CLB5 (an S phase cyclin) and CDK1·CLB2 (a G2
-M phase cyclin) on the activity of the forkhead transcription factor Fkh2p in S. cerevisiae
The large mobility shift in FOXK2 upon phosphorylation suggests that the slower mobility species probably represents a hyperphosphorylated form containing multiple phosphorylation events. Indeed, we have identified two sites, serines 368 and 423, that are targets for CDK·cyclin complexes in vitro
and contribute to phosphorylation and the generation of the slower mobility species in vivo
. However, there are clearly more CDK-dependent sites phosphorylated in vivo
. Whereas CDK inhibitors and CDK·cyclin knockdown efficiently reduce FOXK2 phosphorylation (), in comparison, FOXK2 phosphorylation is much less affected in the FOXK2(S368A/S423A) mutant (A
). Importantly, this latter experiment reveals additional intermediary bands that are indicative of partial phosphorylation. Thus, serines 368 and 423 are probably contributory rather than the sole determinants of CDK-mediated phosphorylation of FOXK2. The two CDK-dependent serines identified in this study were obtained by a combination of in vitro
kinase assay and mass spectrometry, which may not reflect the total phosphorylation events on FOXK2 in vivo
. Indeed, by using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, Ser393
was identified as an additional phosphorylation site of FOXK2 in HeLa cells arrested in the mitotic phase of the cell cycle (37
). However, it remains unknown whether these sites are directly phosphorylated by CDKs or other kinases activated during mitosis. Furthermore, although the results in this paper focus on a C-terminal fragment of FOXK2, we have also detected phosphorylation of the N-terminal region of FOXK2 by CDK·cyclin complexes in vitro
(data not shown). This further suggests a multisite phosphorylation mechanism whereby phosphorylation of different CDK sites would occur over time, potentially through the activity of different CDK·cyclin complexes, but ultimately result in complete multisite phosphorylation at a precise point of the cell cycle (i.e.
in mitosis). Such switchlike mechanisms are operative at other points in the cell cycle (reviewed in Ref. 38
). It is also possible that phosphorylation of FOXK2 might be important at other times during the cell cycle because phosphorylation increases in cells blocked during early S phase (B
) albeit to a lesser extent than in mitotic cells. Thus, although we clearly show the functional importance of the C-terminal phosphorylation events, additional regulatory events triggered through alternative phosphorylation sites will require further investigation.
FOXK2 seems to function as a transcriptional repressor protein, and one target appears to be the cell cycle regulator p21. By using a phosphomimetic version of FOXK2, our data indicate that one role of phosphorylation is likely to be a reduction in p21 repression. Because maximal FOXK2 phosphorylation occurs during mitosis, this loss of repression might contribute to increasing p21 levels and thereby enhance the activity of CDK4·CLND complexes during G1 phase. We also find that phosphorylation of FOXK2 appears to be important in regulating its stability, with the phosphorylated form being less stable. FOXK2 instability is also apparent in cell release from nocodazole block (C), but the timing of this is variable, and we have been unable to demonstrate this effect with transiently expressed proteins, most likely due to overexpression (data not shown). This precludes a comparative analysis of the FOXK2(S368A/S423A) mutant protein, so we are unable to make a definitive connection between the changes caused by CDK·cyclin-mediated phosphorylation and the effects seen on FOXK2 levels in cells released from nocodazole block. The changes in FOXK2 levels could, however, contribute to the loss of repression of key target genes as cells exit mitosis. Although we have focused on serines 368 and 423, it is likely that other CDK sites will contribute to both of these regulatory activities and hence produce more profound effects on FOXK2 activity. However, it is clear that phosphorylation at serines 368 and 423 is vitally important because loss of phosphorylation at these sites cannot be tolerated, because the mutant FOXK2(S368A/S423A) promotes apoptosis, and stable cell lines containing this mutant cannot be generated. It is unclear how this is manifested at the transcriptional level and whether p21 might be a critical target of FOXK2 in this context or whether other targets might be more relevant. Further studies will be directed to uncovering the function of FOXK2 during the cell cycle and its repertoire of target genes in this context.