PEST-dependent cytoplasmic retention of v-Rel by I(kappa)B-alpha: evidence that I(kappa)B-alpha regulates cellular localization of c-Rel and v-Rel by distinct mechanisms.

Association of c-Rel with the inhibitor of kappaB-alpha (IkappaB-alpha) protein regulates both cellular localization and DNA binding. The ability of v-Rel, the oncogenic viral counterpart of avian c-Rel, to evade regulation by p40, the avian IkappaB-alpha protein, contributes to v-Rel-mediated oncogenesis. The yeast two-hybrid system was utilized to dissect Rel:IkappaB-alpha interactions in vivo. We find that distinct domains in c-Rel and v-Rel are required for association with p40. Furthermore, while the ankyrin repeat domain of p40 is sufficient for association with c-Rel, both the ankyrin repeat domain and the PEST domain are required for association with v-Rel. Two amino acid differences between c-Rel and v-Rel that are principally responsible for PEST-dependent association of v-Rel with p40 were identified. These same amino acids were principally responsible for PEST-dependent cytoplasmic retention of v-Rel by p40. The presence of mutations in c-Rel that were sufficient to confer PEST-dependent association of the mutant c-Rel protein with p40 did not increase the weak oncogenicity of c-Rel. However, the introduction of these two c-Rel-derived amino acids into v-Rel markedly reduced the oncogenicity of v-Rel. Deletion of the NLS of either c-Rel or v-Rel did not abolish association with p40, but did confer PEST-dependent association of c-Rel with p40. Surprisingly, deletion of the nuclear localization signal in v-Rel did not affect oncogenicity by v-Rel. Analysis of several mutant c-Rel and v-Rel proteins demonstrated that association of Rel proteins with p40 is necessary but not sufficient for cytoplasmic retention. These results are not consistent with the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by the same mechanism. Rather, these results support the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by distinct mechanisms.     

Loss of IκBα-Mediated Control over Nuclear Import and DNA Binding Enables Oncogenic Activation of c-Rel

The IκBα protein is able both to inhibit nuclear import of Rel/NF-κB proteins and to mediate the export of Rel/NF-κB proteins from the nucleus. We now demonstrate that the c-Rel–IκBα complex is stably retained in the cytoplasm in the presence of leptomycin B, a specific inhibitor of Crm1-mediated nuclear export. In contrast, leptomycin B treatment results in the rapid and complete relocalization of the v-Rel–IκBα complex from the cytoplasm to the nucleus. IκBα also mediates the rapid nuclear shuttling of v-Rel in an interspecies heterokaryon assay. Thus, continuous nuclear export is required for cytoplasmic retention of the v-Rel–IκBα complex. Furthermore, although IκBα is able to mask the c-Rel-derived nuclear localization sequence (NLS), IκBα is unable to mask the v-Rel-derived NLS in the context of the v-Rel–IκBα complex. Taken together, our results demonstrate that IκBα is unable to inhibit nuclear import of v-Rel. We have identified two amino acid differences between c-Rel and v-Rel (Y286S and L302P) which link the failure of IκBα to inhibit nuclear import and DNA binding of a mutant c-Rel protein to oncogenesis. Our results support a model in which loss of IκBα-mediated control over c-Rel leads to oncogenic activation of c-Rel.     

Characterization of the nuclear import and export functions of Ikappa B(epsilon)

Control over the nuclear localization of nuclear factor kappaB/Rel proteins is accomplished in large part through association with members of the inhibitor of kappaB (IkappaB) protein family. For example, the well studied IkappaBalpha protein actively shuttles between the nucleus and the cytoplasm and both inhibits nuclear import and mediates nuclear export of NF-kappaB/Rel proteins. In contrast, the IkappaBbeta protein can inhibit nuclear import of NF-kappaB/Rel proteins but does not remove NF-kappaB/Rel proteins from the nucleus. To further understand how the IkappaB proteins control the nuclear-cytoplasmic distribution of NF-kappaB/Rel proteins, we have characterized the nuclear import and nuclear export functions of IkappaBepsilon. Our results indicate that the IkappaBepsilon protein, like the IkappaBalpha protein, actively shuttles between the nucleus and the cytoplasm. Similar to IkappaBalpha, nuclear import of IkappaBepsilon is mediated by its ankyrin repeat domain and is not blocked by the dominant-negative RanQ69L protein. However, the nuclear import function of the IkappaBepsilon ankyrin repeat domain is markedly less efficient than that of IkappaBalpha, with the result that nuclear shuttling of IkappaBepsilon between the nucleus and the cytoplasm is significantly slower than IkappaBalpha. Nuclear export of IkappaBepsilon is mediated by a short leucine-rich nuclear export sequence (NES)-like sequence ((343)VLLPFDDLKI(352)), located between amino acids 343 and 352. This NES-like sequence is required for RanGTP-dependent binding of IkappaBepsilon to CRM1. Nuclear accumulation of IkappaB(epsilon) is increased by either leptomycin B treatment or alanine substitutions within the IkappaBepsilon-derived NES. A functional NES is required for both efficient cytoplasmic retention and post-induction control of c-Rel by IkappaBepsilon, consistent with the notion that IkappaBepsilon-mediated nuclear export contributes to control over the nucleocytoplasmic distribution of NF-kappaB/Rel proteins.     

Regulation of yeast Yak1 kinase by PKA and autophosphorylation-dependent 14-3-3 binding

Yak1 is a member of an evolutionarily conserved family of Ser/Thr protein kinases known as dual-specificity Tyr phosphorylation-regulated kinases (DYRKs). Yak1 was originally identified as a growth antagonist, which functions downstream of Ras/PKA signalling pathway. It has been known that Yak1 is phosphorylated by PKA in vitro and is translocated to the nucleus upon nutrient deprivation. However, the regulatory mechanisms for Yak1 activity and localization are largely unknown. In the present study, we investigated the role of PKA and Bmh1, a yeast 14-3-3 protein, in regulation of Yak1. We demonstrate that PKA-dependent phosphorylation of Yak1 on Ser295 and two minor sites inhibits nuclear localization of Yak1. We also show that intramolecular autophosphorylation on at least four Ser/Thr residues in the non-catalytic N-terminal domain is required for full kinase activity of Yak1. The most potent autophosphorylation site, Thr335, plays an essential role for Bmh1 binding in collaboration with a yet unidentified second binding site in the N-terminal domain. Bmh1 binding decreases the catalytic activity of Yak1 without affecting its subcellular localization. Since the binding of 14-3-3 proteins to Yak1 coincides with PKA activity, such regulatory mechanisms might allow cytoplasmic retention of an inactive form of Yak1 under high glucose conditions.