The data presented here identify the Hsp110 family member Apg-2 as a new interaction partner of ZO-1 that regulates the function of ZO-1 in the control of the transcription factor ZONAB. Our observations indicate that Apg-2 plays a role in the regulation of epithelial proliferation and the response to heat shock and that the ZO-1–ZONAB signaling pathway becomes activated during the cellular stress response in epithelial cells. Our results suggest a model according to which heat shock-induced binding of Apg-2 to ZO-1 stimulates dissociation of ZONAB from the junctional adaptor followed by its nuclear translocation and activation of its transcriptional activity.
Apg-2 binds to the SH3 domain of ZO-1. This interaction seems to be specific because no significant binding was observed with several other SH3 domains. However, the interaction between the Hsp and the junctional protein does not occur constitutively but is regulated. Only little Apg-2 is associated with ZO-1 under control conditions. On heat shock, however, Apg-2 is redistributed, not up-regulated, resulting in accumulation in nucleoli and at intercellular junctions. The junctional accumulation is likely to be because of the increased association with ZO-1, which does not exclude that it might also bind to other junctional components, perhaps involving other regions than the ATPase domain. For example, it is conceivable that Apg-2 stabilizes tight junctions during stress conditions. Since the ATPase domain interacts with ZO-1, it is possible that such a stabilizing function involves the peptide binding domain of the heat-shock protein.
How heat shock induces the Apg-2 redistribution is not known. Because ZO-1 binds to the ATPase domain, it is possible that ATP binding or hydrolysis regulates the interaction between Apg-2 and ZO-1. However, we have so far not been able to detect a difference in the in vitro binding to ZO-1 of Apg-2 loaded with either ATP or ADP (our unpublished data). Whether Apg-2 has any specific binding partners in nucleoli is also not clear. Nevertheless, the ATPase domain is sufficient to mediate localization to both junctions as well as nucleoli in response to heat shock (our unpublished data), suggesting that an interaction mediated by the ATPase domain also occurs in nucleoli. Because the interaction between Apg-2 and the SH3 domain of ZO-1 can be reconstituted with recombinant proteins, it seems unlikely that a posttranslational modification is directly required for binding in vivo. However, it is possible that an inactivating modification needs to be removed or that an interaction that prevents junctional recruitment needs to be dissociated.
Binding of Apg-2 to the SH3 domain of ZO-1 competes with the interaction between ZONAB and ZO-1, resulting in stimulation of the transcriptional activity of the transcription factor. Because ZONAB activation has been related to proliferation, and, in particular, to G1/S phase progression, it is possible that the herein observed requirement of Apg-2 for efficient G1/S phase progression is in part because of the inhibition of ZONAB function by Apg-2 depletion. It is unlikely, however, that this is the only reason for the observed effect on G1/S phase progression. For example, there is a considerable nuclear pool of Apg-2, suggesting that it might also have nuclear interaction partners that are relevant for proliferation. It is thus possible that Apg-2 affects proliferation by modulating different cellular mechanisms and signaling pathways.
Such a model of Apg-2 function would not be without precedent because heat-shock proteins are often multifunctional and differentially interact with different partners depending on their subcellular localization. For example, the same isoforms of Hsp90 function in the cytoplasm, the nucleus, and even extracellularly (Picard, 2004
). Hsp90 binds to a variety of different proteins at different subcellular sites and thereby regulates different signaling pathways, gene expression, and proliferation (Pratt and Toft, 2003
). However, also Hsp70 family members associate with signaling proteins and have been linked to the regulation of proliferation, and the proliferative state of a cell often affects not only their expression but also their localization (Helmbrecht et al., 2000
). Many heat-shock proteins are thus multifunctional in terms of the interactions they engage in as well as the types of cellular processes they modulate at different subcellular and extracellular locations.
The finding that heat shock induces activation of the transcriptional activity of ZONAB indicates that the ZO-1–ZONAB pathway not only functions during proliferation but also during the cellular response to certain stresses. Although it is currently not known whether other types of stress also affect the transcriptional activity of ZONAB, it is possible that conditions that interfere with junctional integrity, for example, such as reduced availability of energy or oxidative stress also induce ZONAB activation (Welsh et al., 1985
; McAbee and Weigel, 1987
; Bacallao et al., 1994
; Ebnet et al., 2001
; Kale et al., 2003
; Bailey et al., 2004
). It will therefore be important to determine whether and how other stress conditions affect Apg-2 localization and expression, and how this affects ZONAB activity. Furthermore, stress conditions such as shear stress and oxidative stress are known to activate β-catenin signaling (Norvell et al., 2004
; Essers et al., 2005
; Harris and Levine, 2005
). Hence, cross-talk between ZONAB and other stress-induced signaling pathways such as the mitogen-activated protein kinase pathways or β-catenin signaling will have to be analyzed.
YB-1/DbpB, another Y-box factor, is activated in response to genotoxic stress and participates in DNA repair (Holm et al., 2002
; Kohno et al., 2003
). Thus, Y-box factors do not just regulate transcription in response to cellular stress but play a more general role. The nucleic acid binding domain of Y-box factors is a cold shock domain, an evolutionarily well conserved type of nucleic acid binding domain that also exists in bacteria in cold shock-induced proteins (Matsumoto and Wolffe, 1998
). Although the nucleic acid binding domain is the only structural conservation between Y-box factors and bacterial cold shock proteins, the function of these proteins in the cellular stress response of bacteria and eukaryotes is intriguing.
Environmental stress often induces pathways that are important for proliferation and that become activated in carcinogenesis. Both Apg-2 () and ZONAB (Balda et al., 2003
) are required for normal proliferation and regulate entry into S phase. In hepatocellular carcinomas, Apg-2 as well as the human ZONAB homologue DbpA are often overexpressed (Hayashi et al., 2002
; Gotoh et al., 2004
), suggesting that ZONAB signaling becomes activated. This is further supported by the isolation of both proteins as overexpressed markers in pancreatic cancer cells (Nakatsura et al., 2001
). It will thus be important to determine the role of the Apg-2–ZO-1–ZONAB signaling pathway in the development and progression of different types of cancers and to evaluate this pathway as a possible target for cancer therapy.