A large body of work has implicated Sp1 sites in the transcriptional control of both housekeeping and tissue-specific genes (3
). Indeed, the Sp/KLF superfamily of nuclear proteins is believed to constitute a transcriptional network that integrates disparate intracellular and extracellular signals to fine-tune gene expression during cell proliferation, growth, and differentiation (3
). Binding affinities, promoter composition, cellular context, posttranslational modifications, and interacting cofactors are the major contributors to functional specificity of individual Sp/KLF proteins (3
). With respect to cofactors, protein-protein interactions have been described both among Sp/KLF superfamily members and between them and other nuclear factors (3
). An illustrative example of the latter is the dual role of CBP or p300 in superactivating KLF1 and promoting its association with the SWI-SNF chromatin-remodeling complex (38
). To begin to elucidate the mechanisms and factors underlying KLF7 activity, we performed a genetic screen for interacting partners and identified a novel gene product that was named MoKA. Three independent sets of experimental evidence support the notion that MoKA is involved in positively modulating KLF7 action in vivo. First, a yeast two-hybrid screen, a GST pull-down assay, and protein copurification from mammalian cells all demonstrated physical interaction between MoKA and KLF7. Second, transient transfections and reporter gene assays correlated the protein-protein interaction with MoKA superactivation of KLF7. Third, in situ hybridizations documented the overlapping Klf7
patterns of gene expression in embryonic and adult tissues. Functional assays also implicated KLF7 and MoKA in the transcriptional control of the p21WAF1/Cip1
gene in addition to identifying structural motifs responsible for different MoKA functions. They include interaction with KLF7 via an F-box motif and intracellular shuttling by canonical NLSs and noncanonical NESs.
The F-box is a 50-residue-long protein-protein interaction motif that was originally described in components of the SFC ubiquitin-ligase complex and, more recently, in proteins involved in several other cellular activities (15
). The F-box is usually found in the N-terminal portion of proteins and is often coupled at the C terminus with other protein-protein interaction motifs, most commonly leucine-rich and WD repeats (15
). Homotypic interactions at the N termini link F-box-containing proteins to the core ubiquitin-ligase complex, whereas heterotypic interactions at the C termini bind them to phosphorylated substrates destined for degradation (34
). We have excluded that MoKA interaction may target KLF7 to the ubiquitination machinery by two complementary sets of experiments. It is also important that MoKA diverges from prototypical F-box-containing proteins in two major ways. First, the MoKA-KLF7 interaction is heterotypic in that the latter protein contains no F-box motif. Indeed, our results suggest that a leucine zipper motif in KLF7 is the most likely candidate to mediate the interaction with the F-box of MoKA. Second, MoKA does not contain any of the coupled protein-protein interaction motifs thus far described in canonical F-box proteins. This last observation does not obviously exclude the possibility that structural motifs other than leucine-rich sequences and WD repeats may enable MoKA to interact in the nuclear and/or cytoplasmic compartments with different proteins, including members of the Sp/KLF superfamily. For example, MoKA may regulate the activity of KLF7 and other transcription factors through multiple protein-protein interactions that ultimately direct their intracellular distribution. These events may be also connected with MoKA subcellular redistribution during the cell cycle, as preliminary data with synchronized cells seem to suggest. The availability of anti-MoKA antibodies will enable us to test this and other hypotheses.
The ability of MoKA to shuttle between the nucleus and cytoplasm of asynchronously growing cells is under the control of NLSs and NESs located in the C-and N-terminal thirds of the protein, respectively. Although the NES consensus sequence was originally defined as a leucine-rich motif of the type LX1-3
LXL (where X can be any amino acid), recent evidence indicates that other hydrophobic residues can substitute for the second to fourth leucines in functional NESs (22
). The NESs of MoKA display the same substitutions and therefore adhere to the noncanonical sequence; nonetheless, they can effectively compete with the canonical NLS of GAL4. The presence of both NESs and NLSs, as well as the distribution of the transfected protein in both nucleus and cytoplasm, imply that other factors are involved in controlling MoKA shuttling. Posttranslational modifications could conceivably be one of such modulating factors. For example, serine dephosphorylation has been shown to promote transcription factor NFAT1 relocation from the cytoplasm to the nucleus as the result of a conformational switch that masks the NES while concomitantly exposing the NLSs (26
). A similar mechanism may be operating in MoKA where a serine-rich sequence lies within the two NLSs. Indeed, a computer-aided search for canonical phosphorylation sites suggests that a number of these residues could be potentially targeted. Indeed, the larger size of MoKA overexpressed in COS7 cells than in bacterial cells is a strong indication of posttranslational modifications. Another possibility is that binding of KLF7 in close proximity to the NESs may trigger nuclear accumulation of the protein. Preliminary experiments do not, however, support this last model, in that comparable patterns of MoKA subcellular distribution have been seen, irrespective of whether the protein is overexpressed alone or together with KLF7. This preliminary finding is also consistent with the idea that functional interaction between MoKA and KLF7 is not limited by the concentration of the former protein in the nucleus. This idea is based on the finding that the internally deleted protein without NESs, which localizes exclusively to the nucleus, superstimulates KLF7 activity to nearly the same extent as the full-length MoKA, which is distributed in both the nucleus and the cytoplasm (Fig. , , and ). Work in progress is examining the mechanism of superstimulation of KLF7 activity and the biological significance of MoKA intracellular shuttling.
Four lines of evidence support a functional connection between KLF7 and MoKA and the cell cycle. First, overexpression of KLF7 in stably transfected cells results in p21 accumulation and growth arrest (17
). Second, KLF7 stimulates p21WAF1/Cip1
promoter activity by itself and together with MoKA. Third, KLF7 binds to the proximal promoter region of the endogenous p21WAF1/Cip1
gene. Lastly, Klf7
are coexpressed in cells that are in the process of exiting the cell cycle. In the testes, overlapping expression of MoKA
in postmeiotic spermatids coincides with sperm maturation and/or chromatin remodeling. In the embryonic nervous system, MoKA
are coexpressed in postmitotic neuroprogenitors, such as those in the ventral horn of the neural tube. Like Klf7
is actively transcribed in other tissues and at other developmental stages as well indicating independent involvement of the two proteins in other regulatory pathways. Along these lines, MoKA
is actively transcribed in both postmitotic motor neurons (together with Klf7
) and proliferating progenitors (without Klf7
) of the spinal cord. Our current effort is aimed at deciphering structure-function relationships between MoKA and KLF7 in vitro and in genetically targeted mice as well as in defining their involvement in cell cycle progression and p21 regulation.