EBP50 and the related protein E3KARP (NHE3 kinase A regulatory protein), are known to regulate the activity of the Na+
exchanger NHE3 ( Weinman et al. 1995
; Yun et al. 1997
; Lamprecht et al. 1998
). This regulation involves direct binding between an internal sequence within the cytoplasmic COOH terminus of NHE3 and residues 149–358 of EBP50 ( Weinman et al. 1998
; Yun et al. 1998
). EBP50 binds ezrin, and ezrin is known to bind the regulatory subunit of PKA in overlay assays ( Dransfield et al. 1997
). Therefore, one hypothesis is that EBP50 recruits PKA to the apical membrane in close proximity with NHE3 ( Lamprecht et al. 1998
; Yun et al. 1998
). While this model is attractive, there are no conclusive data establishing a role for ezrin as an A kinase anchoring protein in cells.
In addition to NHE3, CFTR and the βAR also directly associate with EBP50 ( Hall et al. 1998a
, Hall et al. 1998b
; Short et al. 1998
; Wang et al. 1998
). While the function of the CFTR–EBP50 interaction is not yet known, binding of β2
AR to EBP50 is implicated in regulation of cellular pH by modulation of NHE3. Specifically, in fibroblasts, agonist activation of βARs stimulated association of the receptor with EBP50, and prevented EBP50 from regulating NHE3 ( Hall et al. 1998b
). However, these experiments have not been replicated in polarized cells, where NHE3 is known to reside at the apical cell surface ( Biemesderfer et al. 1997
; Janecki et al. 1998
). Furthermore, NHE3 is not expressed in airway epithelial cells, indicating that the functions of EBP50 may be tissue-specific. EBP50 may function to target proteins to the apical cell surface, and may also serve as a scaffold to organize apical membrane proteins into regulatory complexes. A logical approach to elucidating the function of EBP50 is the identification of additional proteins in EBP50 protein complexes.
Our data establish that YAP65 associates with EBP50 via the COOH terminus, and the COOH terminus of YAP65 is necessary and sufficient for association with EBP50 ( , , and ). In addition, pull-down assays using the YAP65wt peptide and GST fusion proteins of PDZ1 and PDZ2, clearly demonstrated preferential binding of YAP65 to PDZ2 ( C). Furthermore, the YAP65 COOH-terminal peptide was unable to compete for binding between GST-EBP50 and CFTR-CT ( A). These results strongly support the conclusion that YAP65 is recruited to EBP50 complexes by association with PDZ2 of EBP50.
Our localization studies clearly demonstrate that endogenous YAP65 is preferentially localized at the apical membrane of 16HBE14o− cells and in well differentiated primary cultures from human nasal epithelial cells ( and ). The expression of GFP-YAP65 chimeras in 16HBE14o− cells supports our localization studies, since GFP-YAP65 was also accumulated at the apical membrane ( C). Furthermore, our results indicate that YAP65 is tethered at the apical membrane by association with EBP50, since GFP-YAP65/−4, which lacks the PDZ2 interaction motif, was no longer localized at the apical membrane ( C). Since we were able to coimmunoprecipitate EBP50 and GFP-YAP65, but not GFP-YAP65/−4 ( B), we conclude that association with EBP50 functions to recruit YAP65 to the apical compartment in polarized cells. Together with the overexpression studies, our ability to coimmunoprecipitate endogenous EBP50 and YAP65 from 16HBE14o− cells ( A) suggests that the two proteins stably associate. However, we cannot rule out the possibility that the binding of YAP65 to other apical membrane PDZ proteins is also involved in the targeting of YAP65. Two additional PDZ proteins, E3KARP and PDZK1, share significant sequence identity with the EBP50 PDZ domains ( Yun et al. 1995
; Kocher et al. 1998
), and both proteins may associate with YAP65 in vitro (Kultgen, P., and S.L. Milgram, unpublished results). Thus, it is possible that YAP65 associates with several apical membrane PDZ proteins including EBP50, and that together these proteins are responsible for the compartmentalization of YAP65 in the subapical compartment. The generation of additional reagents to carefully examine the expression and subcellular distributions of E3KARP and PDZK1 will be needed to resolve this question.
Additional Proteins in EBP50–YAP65 Complexes
YAP65 contains multiple protein–protein interaction domains, and the binding of YAP65 and EBP50 would allow for the recruitment of additional proteins to the subapical compartment. If YAP65 functions as a dimer, as suggested by the presence of the predicted coiled coil, YAP65 could facilitate the formation of EBP50 multimers, thus, generating a larger protein complex. Dimerization of YAP65 might also be involved in mediating, or regulating, its association with other proteins. Since YAP65 contains one or two WW domains (depending on alternative mRNA splicing; Espanel and Sudol 1999
) it is likely that additional proteins associate with YAP65. Two novel proteins, WBP1 and WBP2, have been identified which associate with the first WW domain of human YAP65 in vitro ( Chen et al. 1997
; Linn et al. 1997
). Thus, it will be critical to determine whether one or both of these proteins associate with YAP65 in cells, and what functional roles these interactions may play.
YAP65 was cloned using an anti–idiotype antibody directed against the protooncogene c-Yes, a member of the Src family of NRTKs. Although the two proteins were clearly shown to associate in vitro, our GST-EBP50 pull-down experiments demonstrate for the first time that YAP65 and c-Yes stably associate in cells ( B). There is considerable overlap in the distributions of YAP65 and c-Yes in our cell culture model systems ( ). Furthermore, a significant fraction of the endogenous c-Yes expressed in 16HBE14o− cells was redistributed from the apical membrane in cells overexpressing GFP-YAP65/−4 ( ). Collectively, these data suggest that in epithelial cells one function of YAP65 is to target c-Yes to the apical cell surface. Thus, similar to AKAPs (A kinase anchoring proteins) and RACKs (receptors for activated C kinase), which interact with protein kinase A and C, respectively ( Mochly-Rosen et al. 1991
; Colledge and Scott 1999
; YAP65 may restrict the activity and enhance the specificity of c-Yes in epithelia. In addition, it is possible that YAP65 may also modulate the activity of the Src family kinase by a yet unknown mechanism. Additional experiments, both in cells and in vitro, will be required to fully understand the function of these interactions.
By comparing the amount of c-Yes in the input sample, and the amount found associated with GST-EBP50 affinity columns and coimmunoprecipitations, we estimate that 15–20% of the c-Yes expressed in 16HBE14o− cells was copurified together with YAP65. These results are not surprising since many other proteins are known to associate with Src family kinases, and these protein associations are regulated dynamically by extracellular stimuli (for review see Thomas and Brugge 1997
). Although we clearly see colocalization of EBP50, YAP65, and c-Yes in the well differentiated primary nasal epithelial cultures and 16HBE14o− cells, we also find c-Yes in other cellular compartments ( ). In kidney epithelial cells stably overexpressing epitope-tagged ezrin, c-Yes was found in ezrin immunoprecipitates ( Crepaldi et al. 1997
). Since ezrin and EBP50 are known to associate ( Reczek et al. 1997
), we have not ruled out the possibility that a portion of the activity associated with EBP50 was actually bound by ezrin. However, a direct association between ezrin and c-Yes has not been demonstrated.
In biochemical assays, YAP65 was also shown to bind the SH3 domain of c-Src and c-Yes, although the affinity for c-Yes was greater ( Sudol 1994
). Since c-Src is expressed in 16HBE14o− cells ( A), we also probed GST-EBP50 affinity resins with c-Src antisera, but did not find evidence for c-Src associated with EBP50. Therefore, our data suggest that the YAP65–EBP50 complex specifically associates with c-Yes at the apical cell surface. However, YAP65 likely associates with multiple proteins that contain SH3 domains, so it would not be surprising to find small amounts of c-Src contained within the EBP50 protein complexes. Our GFP-YAP65 cell lines will be very useful for determining whether additional proteins associate with the YAP65 proline-rich motif.
Potential Functions of EBP50-YAP65-Yes Kinase Interactions
Our previous work and the new results reported here identify several members of the EBP50 protein complex, including an adaptor protein (YAP65) and an NRTK (c-Yes). Although we focused on airway epithelia, EBP50, YAP65 and c-Yes are widely expressed in epithelia, and the makeup of EBP50 protein complexes will differ based on the specific genes expressed in each epithelium. One possible function for apical membrane c-Yes is modulation of ion channels. Src family kinases play a critical role in modulating ion transport through voltage and ligand-gated ion channels (for review see Thomas and Brugge 1997
). Furthermore, Src family kinase–mediated regulation of ion transport may be facilitated by interactions of the kinases with submembranous scaffolding proteins. For example, the association of PSD95 and Fyn in neurons facilitates tyrosine phosphorylation of the N
-aspartate receptor ( Tezuka et al. 1999
). In addition, the direct binding of p56lck to hDlg is thought to be involved in modulation of Shaker Type Kv1.3 K+
channels ( Hanada et al. 1997
). Src family kinases may be involved in the regulation of Na+
exchanger function ( Krump et al. 1997
; Tsuganezawa et al. 1998
). Since EBP50 directly binds NHE3 and indirectly associates with c-Yes, it is intriguing to speculate that these interactions are important for modulation of NHE3. Although NHE3 is not expressed in airway epithelial cells ( Brant et al. 1995
), Src family kinases may modulate the activity of other apical membrane conductances in these cells. Since exogenously applied c-Src can regulate the gating of CFTR in heterologous expression systems ( Fischer and Machen 1996
), it will be important to determine whether c-Yes functions as a regulator of CFTR in vivo.
Src family kinases are also involved in the control of gene expression ( Boyer et al. 1997
; Li et al. 1998
). In addition, a recent report indicates that YAP65 may serve as a transcriptional coactivator in some cells, and exogenously expressed YAP65 was observed in the nucleus of NIH 3T3 fibroblasts ( Yagi et al. 1999
). Although we do not see YAP65 in the nucleus of polarized airway epithelial cells grown on permeable supports, it is possible that YAP65 is capable of translocation from the cytoplasm to the nucleus in response to extracellular stimuli. Such regulation may be analogous to that of β-catenin, a protein at the epithelial adherens junction, that translocates from the cell membrane to the nucleus to regulate gene expression ( Huber et al. 1996
; Simcha et al. 1998
). Therefore, proteins contained within EBP50 complexes may be involved in transducing signals from the apical cell surface to the nucleus in response to external stimuli. Further characterization of proteins contained within EBP50 protein complexes in different cell types and the identification of extracellular stimuli that modulate the composition of these complexes will be critical for understanding the regulation of membrane proteins present at the apical cell surface in epithelia.