The High Mobility Group Box Transcription Factor Nhp6Ap Enters the Nucleus by a Calmodulin-dependent, Ran-independent Pathway

Abstract

A gradient of Ran⅐GTP typically regulates traffic through the nuclear pore by modulating association of receptors with cargo.However, here we demonstrate that the yeast high mobility group box transcription factor Nhp6Ap enters the nucleus via a novel nuclear localization signal recognized by calcium calmodulin in a process that does not require Ran. Calmodulin is strictly required for the nondiffusional nuclear entry of Nhp6Ap. Calmodulin and DNA exhibit mutually exclusive binding to NHP6A, indicating that the directionality of Nhp6Ap nuclear accumulation may be driven by DNA-dependent dissociation of calmodulin. Our findings demonstrate that calmodulin can serve as a molecular switch triggering nuclear entry with subsequent dissociation of calmodulin binding upon interaction of cargo with chromatin. This pathway appears to be evolutionarily conserved; mammalian high mobility group box transcription factors often have two nuclear localization signals: one a classical Ran-dependent signal and a second that binds calmodulin. The finding that Nhp6Ap nuclear entry requires calmodulin but not Ran indicates that Nhp6Ap is a good model for studying this poorly understood but evolutionarily conserved calmodulin-dependent nuclear import pathway.Proteins enter the nucleus through nuclear pore complexes (NPCs).2 Those smaller than ϳ40 -50 kDa can traverse these pores by passive diffusion (1-5). Many larger proteins require soluble receptors, termed importins or karyopherins, which bind cargo in the cytoplasm, accompany it through the NPC, and release it in the nucleus. Most cargo proteins contain short nuclear localization signals (NLS), motifs recognized by the various receptor proteins.The small GTPase Ran drives the accumulation of cargo proteins in the nucleus by regulating the association and dissociation of NLS-containing proteins with importin/karyopherins.In the nucleus, most Ran is GTP-bound, whereas it is largely GDP-bound in the cytoplasm. Cargo proteins bind importins in the presence of Ran-GDP in the cytoplasm, and when an importin bound to cargo arrives in the nucleus, Ran-GTP induces cargo release (6). Thus, the Ran⅐GTP gradient across the NPC drives the accumulation of most NLS-containing cargo in the nucleus.Some Ran-independent nuclear import pathways have been described, although most remain poorly understood (for review see Ref. 7). One of the most intriguing of these involves the movement of calmodulin across the nuclear pore (8 -10). Calmodulin nuclear import was argued to be a facilitated mechanism that was blocked by the calmodulin antagonist peptide M13 and did not require cytosolic factors or an ATP-regenerating system (8, 10). In the presence of calcium, calmodulin was also shown to be able to facilitate movement of large molecules into the nucleus. This latter property suggested a Ca 2ϩ -inducible nuclear import function for calmodulin that might operate during cellular activation. The calmodulin-mediated pathway was proposed to be redundant with the canonic Ran⅐GTP-driven nuclear entry pathway...

Multiple Nuclear Localization Signals Function in the Nuclear Import of the Transcription Factor Nrf2

“…For example, the glucocorticoid receptor possesses two nuclear localization signals, one of which (NL1) appears to mediate the nuclear translocation of the unliganded receptor and requires binding to importin ␣, whereas the other (NL2) appears to mediate slower translocation of the receptor, a phenomenon that is agonist-specific and independent of binding to importin ␣ (62). Mammalian high mobility group box transcription factors also possess two nuclear localization signals, one that is a classical RanGTP-dependent signal and a second one that binds calmodulin (68). In our work, mutations in any of the nuclear localization signals in Nrf2 resulted in its failure to localize to the nucleus as well as in its failure to transactivate ARE-driven reporter gene constructs, indicating that all three nuclear localization sequences in Nrf2 are critical for the nuclear import and function of this transcription factor.…”

GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

“…Other proteins are even imported without Ran (31,32), and there are even examples of proteins, such as ␤-catenin, whose transport seems to require neither importins nor Ran as these proteins can bind directly to the nuclear pore (33). We found that Npa3 can also bind at least one nuclear pore protein, Nup133, and although Nup133 is not one of the several phenylalanine-glycine repeat nucleoporins that have so far been implicated in the binding and translocation of import complexes, it is possible that Npa3 can directly bind nuclear pore proteins and therefore does not depend on the classical nuclear import pathway.…”

Structural Studies of Soybean Calmodulin Isoform 4 Bound to the Calmodulin-binding Domain of Tobacco Mitogen-activated Protein Kinase Phosphatase-1 Provide Insights into a Sequential Target Binding Mode

“…For example, Ca 2ϩ -CaM has been shown to stimulate the nuclear localization of many proteins (58). In a recent report concerning the Ca 2ϩ -CaM-dependent nuclear entrance of the transcription factor Nhp6Ap it has been found that an unknown third protein is recruited through Ca 2ϩ -CaM binding and that a ternary complex is formed to facilitate transport across the nuclear pore (59). Consequently, the identification of proteins that can strongly and selectively interact with the N-lobe of Ca 2ϩ -CaM will be key to understanding the links between the MAPK cascade and the Ca 2ϩ signaling pathways in plants.…”