We have previously shown that reduction in the level of Ikaros and its family members in lymphocytes facilitates lymphocyte activation and causes the rapid development of leukemias and lymphomas, thus classifying these factors as potent tumor suppressors (2
). In the present investigation, we provide evidence of new G1
-S-dependent Ikaros phosphorylation events, which downmodulate its ability to control entry into the S phase, possibly by altering its affinity for DNA. Importantly, CKII activity that has been implicated in lymphomagenesis plays a major role in these Ikaros phosphorylation events.
In search of modifications that alter Ikaros activity during the cell cycle, we have identified two major areas of phosphorylation in the Ikaros proteins (p1 and p2). The first (p2) contains a single serine phosphorylation site located in exon 4 at amino acid 63 and is present in a subset of the Ikaros DNA binding isoforms. The second (p1) contains several phosphorylation sites located between amino acids 383 to 404 in exon 8 and is shared by all Ikaros isoforms. These Ikaros phosphorylation events can take place in both lymphoid and nonlymphoid cells, indicating that the kinases involved are not cell type specific, although Ikaros protein expression is tissue restricted. The level of phosphorylation at these sites does appear to vary among different cell types (data not shown), indicating that the signaling pathways involved may be more active in some cells. For example, levels of Ikaros phosphorylation are low in NIH 3T3 cells, which may explain why ectopic expression of Ikaros in these cells exerts a block in the G1-S transition.
Ikaros phosphorylation undergoes dynamic changes during the cell cycle observed in both cell lines and primary lymphocytes. In the late G1 phase, the protein is not phosphorylated. Upon transition into the replicative (S) phase, Ikaros becomes phosphorylated. The Ikaros p1 and p2 phosphopeptides and the molecular regulation they may provide are maintained from the replicative through the mitotic phases. Their phosphorylation is lost in the G1 phase of the next cycle and becomes reestablished during the S phase.
We have previously shown that a reduction in Ikaros levels or deletion of its DNA binding domain in T cells causes their hyperproliferation in response to TCR engagement and accelerates the G1
-S transition (2
). Ectopic expression of Ikaros DNA binding isoforms in NIH 3T3 fibroblasts as presented in this study provides further insight into Ikaros's role in cell cycle regulation. Here we have shown that Ikaros blocks the G1
-S transition and that the extent of the block is dependent on Ikaros protein levels. We have also demonstrated that a combination of mutations in the shared Ikaros p1 region that prevents its hyperphosphorylation increases the protein's ability to block the cell cycle in late G1
. Thus, hyperphosphorylation of the Ikaros p1 region is a critical regulatory event that downmodulates the protein's negative effect on the G1
Mutations of S/T residues to negatively charged amino acids are frequently used to provide a phosphomimetic effect (32
). Such mutations in the Ikaros major phosphorylation area (residues 383 to 404) failed to reveal such an effect (data not shown). There have been other reports describing S or T substitutions for negatively charged amino acids that were unable to mimic a constitutive phosphorylation state (8
). Interestingly, in both Ikaros and these reported proteins, phosphorylation of several residues was involved, and the introduction of negative charges could not substitute for the actual phosphorylation events (8
Given the previously established hypothesis that Ikaros DNA binding is crucial for its role in development and proliferation, we examined whether the Ikaros p1 and p2 phosphorylation events affected Ikaros's ability to bind DNA in vitro. Mutations in S63 (p2) had no effect on either DNA binding or the stability of the protein. However, mutations in the S/T residues in the region 383 to 404 (p1) increased the ability of the protein to bind DNA by threefold but had no effect on its stability. Since the S/T phosphorylation area lies outside the DNA binding domain, its effect on inhibiting DNA binding must be indirect. Phosphorylation of the Ikaros protein in the p1 region may induce a conformational change that affects the accessibility of the DNA binding domain. Alternatively, p1 phosphorylation may promote interactions with other factors, including components of the previously described Ikaros-associated chromatin remodeling complexes, whose presence may reduce Ikaros's DNA binding.
In search of the kinases responsible for Ikaros phosphorylation, we identified the serine/threonine kinase CKII as a major player. Previous studies have implicated CKII in the regulation of the G1
-S transition (22
). Although CKII is constitutively active, its expression is upregulated upon mitogenic stimulation (33
). Transgenic mice that express CKII in the thymus and Ikaros-deficient mice both develop T-cell thymomas (29
), supporting the hypothesis that functional interactions between these two factors are responsible for T-cell homeostasis. Under this model, CKII phosphorylates Ikaros in the p1 region during the G1
-S transition and reduces Ikaros activity as a negative regulator of this transition. Other kinases, such as cdk2 and GSK3, may also play a role in regulating Ikaros phosphorylation upon entry into the replicative phase of the cell cycle.
Taking into consideration past and current studies, we propose the following models for regulation of Ikaros activity and cell cycle control (Fig. ). Ikaros impedes the G1-S transition by modulating expression of genes that function as positive or negative effectors of the cell cycle (i.e., have a negative effect on cyclins and/or a positive effect on cell cycle inhibitors p27, p21, etc.). Ikaros may affect expression of some of these genes directly by binding to their transcriptional regulatory regions (Fig. ). In support of this model, mutations in the Ikaros DNA binding domain alleviate its ability to control the cell cycle (Fig. ). Under physiological conditions, Ikaros DNA binding activity is temporarily downmodulated through CKII-dependent phosphorylation of its C-terminal region that facilitates the G1-S transition (Fig. ). Mutations in Ikaros that abolish p1 phosphorylation eliminate this type of regulation on Ikaros DNA binding and block cells in G1 (Fig. ).
FIG. 7. Models for Ikaros regulation of the G1-S transition. (A) Ikaros is present in a dephosphorylated state during the late G1 phase. In this state, Ikaros is active in DNA binding and restricts the G1-S transition, possibly by regulating transcription of (more ...)
The Ikaros exon 8 phosphorylation events described here first occur during the G1
-S transition but are maintained through the M phase. In a recent study, Dovat et al. (7
) have shown that during M phase, Ikaros becomes phosphorylated in the linker region of the DNA binding zinc fingers. These M-phase phosphorylation events also reduce DNA binding of the Ikaros protein and may exclude this family of proteins from binding to mitotic chromosomes (7
). Thus, there appears to be a gradual decrease in Ikaros activity during the cell cycle that is affected by phosphorylation. During the G1
-S transition, Ikaros becomes phosphorylated in exon 8, an event that reduces its activity as a negative regulator of the G1
-S transition and its DNA binding. In the M phase, Ikaros becomes phosphorylated at the zinc finger linker regions, which further reduces its DNA binding to possibly exclude it from mitotic chromosomes.
-S transition is a critical checkpoint in cell cycle regulation. The expression and activity of proteins involved in this transition are tightly regulated, and a failure to do so frequently results in cellular apoptosis or neoplastic transformation (5
). In fact, many human cancers present genetic lesions in key regulators of the G1
-phase progression and G1
-S transition (30
). Lymphocytes are terminally differentiated cells with the unique property of undergoing proliferative expansions upon encountering antigen. In addition to ubiquitously expressed cell cycle regulators, tissue-specific negative regulators such as Ikaros provide for controlled proliferation of lymphocytes and prevent the development of leukemias. Ikaros modifications may allow only lymphocytes that have received appropriate levels of signaling to enter the replicative phase of the cell cycle. It is tempting to propose that Ikaros's phosphorylation at multiple residues within the p1 region may be responsible for translating different levels of receptor signaling into different levels of Ikaros activity and lymphocyte activation.
The interplay between positive and negative signals that underlies the Ikaros cell cycle-regulated phosphorylation events remains to be elucidated. It will be important to determine whether phosphatases function in concert with CKII and other kinases to provide for the changes in Ikaros phosphorylation detected during the cell cycle. Deregulation of such signaling events in primary lymphocytes may affect the phosphorylation state of Ikaros and its ability to control the cell cycle and thus be responsible for transition to a neoplastic state.
Finally, there are interesting parallels between Ikaros and previously described cell cycle regulators. One of the best-studied tumor suppressors is the nuclear factor Rb, which is involved in the regulation of the G1
-S transition. Rb is inactivated during G1
by concerted phosphorylation events initiated by different cyclin-cdk complexes, which induce a progressive conformational change in this negative regulator (12
). Ikaros also negatively regulates the G1
-S transition, and phosphorylation relieves its ability to do so. However, Ikaros phosphorylation appears to occur later than that of Rb during the G1
-S transition. In addition, Rb phosphorylation affects its interactions with other proteins, whereas Ikaros phosphorylation reduces its ability to bind DNA. It is possible, however, that Ikaros phosphorylation may also affect its interactions with other proteins.