Previous work demonstrated that, in response to DNA damage, JMY accumulates in the nucleus and promotes p53-mediated apoptosis. We find that this DNA damage–induced nuclear accumulation requires a nuclear localization sequence in JMY buried within a cluster of actin monomer-binding sites. We also find that monomeric actin regulates JMY's nuclear import by blocking interaction with the import machinery. These results argue that nuclear accumulation of JMY may be triggered by DNA damage–induced actin assembly. Consistent with this idea, we observed that DNA damage induces robust actin polymerization, nearly doubling the filamentous actin concentration in the cytoplasm and significantly reducing the concentration of monomeric actin. This observation fits with previous studies demonstrating DNA damage–induced actin assembly (Levee et al., 1996
) and identifying a Rho-dependent pathway that links DNA damage to actin filament assembly (Croft et al., 2005
Is it possible that JMY's localization is dependent on binding to filamentous actin? We showed previously that, in vitro, JMY binds monomeric actin but does not appreciably copellet with filamentous actin or cap filament ends (Zuchero et al., 2009
). In cells JMY does not localize strongly to actin-rich structures such as stress fibers, nor does depolymerizing actin filaments with LatB affect JMY localization. Together these results argue that actin filaments do not directly influence JMY's subcellular localization.
The localization of both JMY and the MRTF family member MAL is determined by competition between actin monomers and the nuclear import machinery, but regulation of the two proteins is not identical. For example, when MAL is concentrated in the nucleus, treatment of cells with latrunculin B causes it to move into the cytoplasm (Vartiainen et al., 2007
; ), consistent with the fact that MAL shuttles continuously between the two compartments and can be trapped in the cytoplasm by association with newly liberated actin monomers. In contrast, latrunculin does not deplete JMY from nuclei of cells with damaged DNA (Supplemental Figure S3B), consistent with our observation that JMY does not cycle between nucleus and cytoplasm (). Why does MAL cycle between nucleus and cytoplasm, whereas JMY does not? The difference could reflect tighter inhibition of nuclear import by the WH2–actin complexes of JMY than that of the RPEL–actin complexes of MAL, or it could reflect stable association of JMY with nuclear partners, perhaps components of the DNA damage response (Shikama et al., 1999
; Demonacos et al., 2001
The lack of continuous cycling means that once JMY is in the nucleus its localization is insensitive to changes in cytoplasm actin. In this way JMY's nuclear localization performs like a toggle switch: a transient dip in monomeric actin concentration produces prolonged nuclear accumulation. This is probably important for its cellular function. For example, JMY promotes initiation of apoptosis and, early in this process, the concentration gradient of Ran-GTP between the nucleoplasm and cytoplasm collapses, trapping many NLS-containing proteins in the cytoplasm bound to Impα/β (Wong et al., 2009
). If JMY shuttled back and forth, it would not remain in the nucleus of apoptotic cells. In later stages of apoptosis the concentration of monomeric actin increases (Suarez-Huerta et al., 2000
), which would also trap rapidly shuttling JMY molecules in the cytoplasm.
Once in the nucleus, does actin also contribute to JMY-mediated transcription? Coutts et al. (2009)
found that JMY targeted to the nucleus with an NLS stimulates p53-dependent transcription of a reporter gene, and LatB blocks this. This suggests that the role of JMY in transcription either requires actin polymerization or is inhibited by actin monomers. Now that actin- and Arp2/3–binding mutants of JMY have been characterized, it will be interesting to test whether JMY promotes actin polymerization in the nucleus and whether it requires this for its role in activating p53. We show here that different actin-binding mutations can perturb the localization of JMY, so it will be essential to take this into consideration when comparing the phenotypes of mutants.
How is JMY's actin assembly activity regulated in cells? Recent cell biological experiments suggest that, in vivo, JMY activity is regulated by interaction with inhibitory binding partners. One obvious potential regulator is the nuclear import machinery, specifically the importin α/β complex (Supplemental Figure S4, D and E). The inhibitory effect of Impβ, however, relies on direct competition with monomeric actin, which has a cytoplasmic concentration at least 20-fold higher than that of Impβ (Gordon et al., 1976
; Görlich et al., 1994
). It is therefore extremely unlikely that importins regulate JMY activity in vivo.
Of interest, JMY requires its polyproline domain for retention in the cytoplasm ( and Supplemental Figure S3A). Most proteins that activate the Arp2/3 complex contain a polyproline domain N-terminal to the WCA region (Rottner et al., 2010
), and it is believed that polyproline domains facilitate the recruitment of profilin-bound actin monomers to Arp2/3 activators (Witke, 2004
). Because the polymerizable pool of actin monomers in cells is largely bound to profilin (Kaiser et al., 1999
), without the polyproline sequence it is possible that occupancy of the WH2 domains by actin is low, making this mutant equivalent to PWWWCA[A*B*C*]. Thus JMY could potentially be regulated by a protein that binds directly to its polyproline domain.
In addition to JMY and MAL, we identified 26 other actin-binding proteins with putative NLSs. It will be interesting to test whether any of these plays a role in actin assembly in the nucleus. Of particular interest are the 14 with overlap between ABD and NLS. Although most do not appear to shuttle in an actin-dependent manner, it is possible that their actin-regulatory activities are regulated by importin binding (and RanGTP). This could allow for control of their activities in the interphase nucleus or around mitotic chromosomes, similar to spindle factors, including the microtubule-organizing proteins TPX2 (Gruss et al., 2001
) and NuMA (Nachury et al., 2001
; Wiese et al., 2001
), and nuclear lamins (Tsai et al., 2006
). Future work will test the role of these proteins in coupling actin dynamics with nuclear and mitotic events.