Nuclear transport of the Forkhead transcription factor, AFX, plays a critical role in the regulation of its transcriptional activity. Phosphorylation of AFX by PKB results in a rapid change in the steady-state distribution of the protein from the nucleus to the cytoplasm. However, the mechanism by which this relocalization occurs has not been determined, although it has been proposed by others that export may be regulated by PKB and that 14-3-3 may function as a chaperone for export, as has also been suggested for the regulation of cdc25 export (19
). Based on the data presented in this paper, we propose a different model, in which nuclear import, not export, of AFX is the principal target of regulation by PKB (Fig. ).
FIG. 8 Nuclear import, not export, of AFX is regulated by PKB. (A) Unphosphorylated AFX appears nuclear at steady state but is actually shuttling rapidly between the nucleus and the cytoplasm. Therefore, the rate of import of unphosphorylated AFX exceeds its (more ...)
Unphosphorylated AFX is predominantly nuclear at steady state. However, heterokaryon fusion assays (Fig. C) demonstrated that the protein is, in fact, continually shuttling between the nucleus and the cytoplasm. The rate constant for unphosphorylated AFX import, therefore, must exceed the rate constant for its export (Fig. A). Importantly, the nuclear export of AFX appears to be unaltered by PKB phosphorylation. Nuclear export of both phosphorylated and unphosphorylated AFXs is likely mediated by the export receptor, Crm1. Both wild-type HA-AFX and the triple mutant, HA-A3, which cannot be phosphorylated by PKB, bind Crm1 in the presence of RanGTP (Fig. A). In support of the identification of Crm1 as the export receptor, we show that AFX export from the nucleus in response to insulin is blocked by the addition of LMB (Fig. A), a potent and specific inhibitor of Crm1 function.
The addition of insulin to cells results in the activation of PKB and its translocation into the nucleus, where it phosphorylates Forkhead family members (Fig. B). Treatment of cells with LMB traps AFX and FKHRL1 in the nucleus. However, both are phosphorylated in response to insulin to the same extent and at the same residues as in cells not treated with LMB (Fig. B). This result, therefore, strongly indicates that PKB enters the nucleus when activated by insulin to phosphorylate Forkhead transcription factors and other target proteins. Phosphorylation alone, however, is not sufficient to inhibit the transcriptional activity of AFX. We have shown, using transcriptional activation assays, that blockade of AFX nuclear export by the addition of LMB results in a loss of insulin-induced transcriptional control (Fig. B). Therefore, both phosphorylation by PKB and nuclear export mediated by Crm1 are essential for complete inhibition of AFX transcriptional activity.
We have found that AFX enters the nucleus by an active, Ran-dependent mechanism (Fig. A) and that import requires a basic region in AFX encompassing PKB phosphorylation site S193 (Fig. ). Importantly, the addition of a negative charge at S193 substantially attenuates nuclear import (Fig. ), most likely by reducing the affinity of AFX for its nuclear import receptor. We propose, therefore, that phosphorylation by PKB at S193 reduces the rate of AFX import. Since the steady-state localization of a protein is determined by its relative rate constants for import and export, the localization of AFX would shift from the nucleus to the cytoplasm (Fig. B). This alteration in the transport rate constants in response to phosphorylation by PKB, in conjunction with proposed cytoplasmic retention by binding 14-3-3 proteins (14
), would result in both the efficient nuclear exclusion of AFX and the inhibition of its transcriptional activity. In conclusion, we propose that this nuclear exclusion mechanism is required to regulate the activity of AFX.
These data are consistent with the results of transcriptional activation assays reported previously for FKHR (30
). The PKB phosphorylation site S256 in FKHR is analogous to S193 in AFX. Mutation of S256 to an alanine in the context of full-length FKHR abolished the ability of insulin and PKB to inhibit FKHR activity. In contrast, mutation of S256 to an aspartate resulted in a substantial inhibition of its transcriptional activity. These effects were not observed when the other two PKB sites were mutated independently. Although the localization of the S256D mutant was not assayed, our data suggest that the inhibition of FKHR activity may be due, in part, to a deficit in nuclear import.
“Classical” nuclear localization sequences are characterized by short amino acid stretches that are enriched in basic amino acids. The NLS of the large T antigen of simian virus 40 was identified by deletion analysis that resulted in mislocalization of the protein to the cytoplasm (45
). It was later defined as a seven-amino-acid sequence (PKKKRKV) sufficient to confer nuclear localization when conjugated to a carrier protein (46
). Some proteins contain similar sequences that are referred to as monopartite NLSs. Other proteins, such as nucleoplasmin, contain bipartite NLSs that consist of two patches of basic residues separated by a 10-amino-acid spacer (23
). Generally, proteins carrying classical or bipartite NLSs bind a cytoplasmic receptor, importin α (28
). Importin α associates with importin β, a protein that docks import complexes at the NPC and translocates import cargo into the nucleus. There are, however, many exceptions to this type of import, and there is a large family of importins and exportins that can recognize distinct NLSs and mediate transport of different subsets of cargo. For example, ribosomal proteins (33
) and histones (32
) have been shown to bind directly to several different importin β family members and dock at the NPC independently of importin α. In addition, other proteins, such as hnRNP K (51
) and β-catenin (75
), can translocate through the NPC in the absence of any soluble factors.
We have delineated residues 180 to 221 of AFX as a novel type of nuclear import signal. This region contains a small portion of the DNA binding domain. Therefore, like other Forkhead family members, the DNA binding domain of AFX contributes to DNA binding and nuclear localization (59
). This region of AFX contains 12 lysine and arginine residues. Although several groups of these basic residues could act as monopartite or bipartite NLSs, the information provided by mutational analysis suggests that AFX contains a nonclassical NLS. Mutation of arginines 188 to 190 in the NLS of AFX has the greatest inhibitory effect on nuclear import, followed by the mutation of lysines 209 to 211. These groups of basic residues are separated by 18 amino acids, and both are contained within similar sequence repeats (KAPRRR and KAPKKK). These repeats may be important for import receptor binding.
The identity of the nuclear import receptor of AFX remains to be established. Since we do not observe binding of AFX to several classical import receptors, including importins α1, α3, and β, it may bind to a novel member of the importin family (which comprises >20 members in mammalian cells). Alternatively, AFX may enter the nucleus in other ways, for instance, by piggybacking on another protein that contains a classical NLS. The role of PKB-mediated phosphorylation and 14-3-3 binding in altering AFX import can be more thoroughly studied once the receptor is identified.
Binding of Forkhead family members to 14-3-3 proteins has been proposed to play a role in the retention of phosphorylated Forkheads in the cytoplasm (14
). Phosphorylated FKHRL1 (14
) and AFX (Fig. A) both bind 14-3-3 proteins. However, we have never observed any specificity of AFX binding to a particular 14-3-3 isoform. In fact, we have observed binding to 14-3-3β, 14-3-3
, and 14-3-3ζ (data not shown). Recently, it has been proposed that 14-3-3 protein binding may impart no specific information about subcellular targeting (53
). Instead, a 14-3-3 protein may obscure the NLS or NES of the protein to which it binds. In this way, 14-3-3 proteins would affect the subcellular localization of their target proteins by interfering with the binding of transport receptors. 14-3-3 binding to Forkhead family members may sterically inhibit import receptor binding and, thereby, act to prevent import.
The proposal has also been made that 14-3-3 possesses an NES that is recognized by Crm1 and participates directly in the export of binding partners through a Crm1 interaction (63
). In our hands, however, 14-3-3 does not bind Crm1 directly, nor is its association with AFX necessary for the interaction of AFX with Crm1. Therefore, in this case at least, 14-3-3 isoforms appear to play no part in regulating AFX nuclear export.
What is the AFX NES? Leucine-rich regions found in several Forkhead family members conform to the consensus sequence for a Crm1-dependent NES (9
). This region in AFX corresponds to residues 300 to 308 (LELLDGLNL). Deletion of residues 298 to 308 results in a protein that is unable to relocalize from the nucleus to the cytoplasm on stimulation with insulin (unpublished data). This region, therefore, likely represents the Crm1-dependent NES.
Distinct chromosomal translocations in pediatric alveolar rhabdomyosarcoma and acute lymphoblastic leukemia (ALL) involve two human Forkhead genes. Alveolar rhabdomyosarcomas are associated with unique chromosomal translocations that arise from fusion of PAX3 or PAX7 to the FKHR gene (27
). The PAX3-FKHR fusion protein is a more potent transcriptional activator than PAX3 alone (8
) and transforms primary cells (64
). Several chromosomal translocations in ALL occur at the 11q23 locus, and all convey a poor prognosis that is usually associated with a high rate of treatment failure and relapse. These breakpoints affect the MLL gene (also called HRX, ALL, or Htrx1) that is disrupted midway through the coding region (18
). A well-documented translocation arises from the fusion of MLL to the AFX gene on chromosome X (13
Importantly, the fusions of AFX and FKHR to their breakpoint partners occur at identical amino acid positions within the Forkhead proteins. The resulting fusion proteins contain the N-terminal DNA binding region of the fusion partner and the C-terminal transactivation domain of the Forkhead protein. The MLL-AFX fusion protein expresses residues 148 to 501 of AFX (57
). This preserves the NLS of AFX identified in this study (residues 180 to 221) but deletes the T28 phosphorylation site. We have shown that deletion of residues N terminal to S193 causes a loss of transcriptional regulation by insulin (42
). In addition, potential loss of S193 phosphorylation in the context of the fusion protein would allow constitutive import into the nucleus. Therefore, both the deregulation of Forkhead activity and a constitutive nuclear localization likely contribute to the oncogenic properties of these fusion proteins.