These studies demonstrate that Akt phosphorylates Cx43 on Ser369 and Ser373 and that an Akt-phosphorylated substrate motif antibody recognizes Cx43 from EGF-treated cells where Akt is activated. They also provide evidence for an interaction of full-length, endogenously phosphorylated Cx43 with 14-3-3 θ, in agreement with the interaction of the C-terminal domain of Cx43 with the NH2
-terminal half of 14-3-3 θ that was identified in the yeast two-hybrid system (Jin 1998
) and our previous in vitro
binding studies with fusion proteins of the C-terminal domain of Cx43 (Park et al. 2006
). Since a GST fusion protein containing the C-terminal domain of Cx45 (a connexin that does not have 14-3-3 binding sequences) and the CT of Cx43 with a Ser373Ala mutation did not bind to His-tagged 14-3-3 θ (), the data support an interaction that is specific for Cx43 and that requires phosphorylation of Ser373 of Cx43. Previous studies (Park et al. 2006
) demonstrated that GST Cx43CT also interacted with His-14-3-3 ζ and “mode-1” phosphorylated binding sites in 14-3-3 substrates are known to interact with other 14-3-3 isoforms (Yaffe et al. 1997
Akt plays an important role in intracellular signaling pathways that are activated by an exposure to extracellular growth factors, as a downstream target of PI3 kinase, and regulates cell proliferation and survival, cell differentiation, and glucose homeostasis (Dummler and Hemmings, 2007
). Akt is also constitutively activated in many tumor cells due to amplification of the p110α PI3K catalytic subunit or deletion/mutation of the PTEN (phosphatase and tensin homolog) gene that regulates PI3 kinase (Sansal and Sellers 2004
). The identification of additional Akt substrates may provide some insight into cellular responses elicited by Akt activation. The sequences recognized by Akt and 14-3-3 overlap and Akt phosphorylates a number of proteins to promote their interactions with 14-3-3 (Kovacina et al. 2003
). In our study, Akt phosphorylated Cx43 in vitro
() at the predicted target sites, Ser373 and Ser369 (), and removal of these sites, in the truncated GST Cx43CT fusion protein, prevented effective phosphorylation by Akt (). The identification of Akt-phosphorylated sites and phosphorylated “mode-1” 14-3-3-binding sites on Cx43 that was phosphorylated endogenously in EGF-treated Rat-1 cells provides further support for the interaction of these proteins in vivo
() and lends credence to the possibility of Akt-mediated phosphorylation of Cx43 in vivo
. Whether Akt-mediated phosphorylation or phosphorylation by another kinase such as PKA (Yogo et al. 2006
; Giepmans 2004
) promotes an interaction of Cx43 with 14-3-3 in vivo
remains a subject for further study.
The validity of an interaction of Cx43 with 14-3-3 was also supported by the recognition of endogenously phosphorylated Cx43 in cell lysates from EGF-treated cells by a GST-14-3-3 fusion protein in GST pull-down studies, by an apparent binding interaction of Cx43 with 14-3-3 θ in an overlay assay (), and by the co-localization of Cx43 with 14-3-3 θ by immunofluorescence microscopy (). In the overlay assay the endogenously phosphorylated Cx43 protein appeared to be a better substrate for 14-3-3 binding than the bacterially phosphorylated fusion protein. If the fusion protein was phosphorylated at the Ser369 or the Ser372 site, a PKC (protein kinase C) site (Saez et al. 1997
) this would likely reduce phosphorylation at the adjacent Ser373 site. Phosphorylation of both Ser372 and Ser373 would sterically hinder binding of the Cx43 Ser373 peptide to 14-3-3, since the dually phosphorylated Cx43 peptide (phosphorylated at Ser372 and Ser373) could not be accommodated well in the 14-3-3 substrate binding pocket in our model of rat/mouse 14-3-3 θ (Park et al. 2006
Previously we demonstrated that preventing the activation of Akt in cells pretreated with a PI3 kinase inhibitor did not prevent the disruption of Cx43 function that was induced by an exposure to LPA (lysophosphatidic acid) (Park et al. 2006
). This suggested that an Akt-mediated interaction with 14-3-3 was not required for the growth factor-induced disruption of Cx43 function. The confocal images of Rat-1 cells co-stained for Cx43 and 14-3-3 θ or with Cx43 and Akt provided several clues to a possible functional significance of this interaction, since the proteins were co-localized at the periphery of gap junctional plaques (). This is where newly synthesized Cx43 is reported to be incorporated into existing gap junctional plaques (Gaietta et al. 2002
). Although we cannot rule out the possibility that steric hindrance prevented the interaction with Akt or 14-3-3 at the center of the plaques, we do not think this was the case. The Cx43 antibody had access to Cx43 throughout the plaque and the co-localization with 14-3-3 and Akt was only detected at the periphery. The data would suggest that these interactions with Cx43 are taking place either “concomitant with” or “prior to” the insertion of Cx43 hemichannels into the plasma membrane and their subsequent incorporation into gap junctional plaques. Recent studies by Shaw et al. (2007)
support a targeted microtubule-mediated delivery of newly synthesized Cx43 to adherens junctions at regions of cell-cell contact, where gap junctional plaques are located.
Co-localization of Cx43 with 14-3-3 θ was detected in untreated Rat-1 cells as well as in EGF-treated cells, consistent with a more constitutive role for the interaction. This was demonstrated by the presence of the phospho-Ser 14-3-3-binding motif on Cx43 from both untreated and EGF-treated cells (), the apparent binding of His-14-3-3 θ to Cx43 in whole cell lysates obtained from untreated and EGF-treated cells in an overlay assay ( and data not shown), and by the co-localization of the proteins in untreated and EGF-treated cells (, lower panels). Importantly, the validity of the interaction is strongly supported by data demonstrating that endogenously phosphorylated Cx43 bound to 14-3-3 θ in the overlay assay and in GST pull-downs. Many studies of the binding interactions of 14-3-3 proteins have been carried out in cells that over-express 14-3-3 and the reputed binding partner. Over-expression of the proteins and/or expression in compartments where they might not normally be expressed could suggest interactions that may not occur at normal levels of protein expression. The interaction of Cx43 with 14-3-3 θ at the endogenous levels of protein expression and with protein phosphorylation that occurred in live cells provides more convincing evidence for an interaction in vivo.
Cx43 is widely expressed in different tissue types and has been shown to have a critical role in the development and physiology of the heart, in male fertility, and in the control of body size (Plum et al. 2000
). The interaction with 14-3-3 proteins appears to be Cx43-specific and may be an example of a connexin regulatory step that provides added quality control during connexin processing that is not generalized to other connexins. A possible function for this interaction is in the assembly of Cx43 monomers into oligomers in a manner that may be monitored by a mechanism that allows for the escape of the Cx43 multimers from the endoplasmic reticulum (ER), following the binding of 14-3-3 to one or more phosphorylated sites on Cx43. Arginine-based sorting signals (R-X-R) can mediate ER-localization of a protein until they are masked by the proof reading of multimeric complexes by 14-3-3 proteins (Michelsen et al. 2005
). This has been demonstrated for the major histocompatibility complex (MHC) invariant chain (Khalil et al. 2003
; Kuwana et al. 1998
) and for the Kir6.2 KATP
channel protein (Nufer and Hauri 2003
; O'Kelly et al. 2002
; Yoo et al. 2005
; Yuan et al. 2003
). The R-P-R sequence at Arg374-Pro-Arg376 is adjacent to Ser373 of Cx43 and the phosphorylation of Ser373 and a subsequent interaction with 14-3-3 may mask an ER retention signal and allow for the forward trafficking of Cx43 and the escape of multimers from the ER.
It is also of interest to note that interactions with PDZ domain-containing proteins have been shown to mask an Arg-based signal at the C-terminus of the NR1-3 subunit of the N-methyl-D-aspartate receptor (Standley et al. 2000
). Cx43 as well as Cx40, Cx45, Cx46, and Cx50 interact at their C-termini with the PDZ domain-containing protein, ZO-1 (Giepmans and Moolenaar 1998
; Jin et al. 2004
; Laing et al. 2001
; Nielsen et al. 2003
). Cx45 also has a dibasic motif near the C-terminal domain that could act as an ER retention signal and Cx45 also interacts with ZO-1. Thus, more than one mechanism for proofreading the formation of multimers may exist for Cx43 and for other connexins. In the case of Cx43, it seems likely that an interaction with 14-3-3 at the phosphorylated Ser373 site and an interaction with PDZ domain-containing proteins at flanking sequences would be mutually exclusive, due to steric considerations.
Some activated Akt was detected in untreated cells and the confocal images in demonstrate the physical proximity of Cx43 and Akt in the untreated cells. As demonstrated by the appearance of the phosphorylated Akt-substrate motif on a protein that migrated as Cx43 (), the interaction of Cx43 with Akt appeared to be enhanced in Rat-1 cells by a treatment with EGF and Akt activation. The motif antibody to “mode-1” phospho-Ser 14-3-3-binding sites recognized Cx43 from both untreated and EGF-treated Rat-1 cells () and Cx43 was also co-localized with 14-3-3 θ in untreated and EGF-treated cells (). Although an extensive analysis of protein co-localization was not carried out as a part of this study, an exposure to EGF appeared to significantly enhance the co-localization of Cx43 with Akt, while the co-localization of Cx43 with 14-3-3 appeared to be more constitutive.
Interactions of Cx43 with 14-3-3 dimers could occur in an inter- or intra-molecular manner. 14-3-3 dimers could cross-link Cx43 monomers within a connexon by binding to two phospho-Ser373 sites on different molecules, or cross-link Cx43 molecules in adjacent connexons. If the phospho-Ser244 site, which does not support a binding interaction on its own, were able to bind to a second 14-3-3 monomer through an induced proximity following the binding of one monomer to phospho-Ser373, this could result in an intra-molecular interaction (Yaffe 2002
). 14-3-3 could also serve as a molecular scaffold to bring another protein to Cx43. Both PKCγ and Erk5 have been shown to interact with 14-3-3 in a manner that regulates their function (Nguyen et al. 2004
; Zheng et al. 2004
) and these kinases interact with and phosphorylate Cx43 (Cameron et al. 2003
; Nguyen et al. 2003
A number of kinases that target Cx43 and their target sites have been identified (Lampe and Lau 2004
; Warn-Cramer and Lau 2004
) and in some cases, specific antibody reagents have been developed to allow assessment of the phosphorylation state of Cx43 in vivo
. Earlier studies by Yogo et al. (2006)
implicated the Ser265, Ser368, Ser369, and Ser373 sites of Cx43 as targets for PKA in rat granulosa cells stimulated with follicle-stimulating hormone (FSH). The studies reported here support a role for Akt as an additional Ser/Thr kinase that can target Cx43 in the C-terminal domain, at the Ser373 and Ser369 sites and provide evidence that Cx43 phosphorylated endogenously is recognized by an antibody to Akt-phosphorylated substrates. They also suggest that Cx43 interacts with 14-3-3 in vivo
, an interaction that might be involved in the forward trafficking of Cx43 multimers by masking an Arg-based ER retention signal near Ser373. A recent study by Thomas et al. (2005)
has demonstrated differences in the biogenesis of Cx43 and Cx26. These connexins have common secretory pathways and common intermediates, however they differ in their dependence on microtubules for transport to the plasma membrane and in their mobilities within the membrane. These authors suggested that some of these differences might be due to differences in the binding of these connexins to scaffold proteins.
An interaction with 14-3-3 proteins may also alter Cx43's association with other interacting proteins, such as ZO-1 (which interacts at the C-terminus of Cx43, near Ser373 (Giepmans and Moolenaar 1998
; Jin et al. 2004
; Laing et al. 2001
)), CIP85 (which interacts with a proline-rich region (Pro253-Pro256 (Lan et al. 2005
)) near a second possible site for 14-3-3 interaction, Ser244), or tubulin or CIP150 (which interact with the juxtamembrane region of the CT domain of Cx43 (Akiyama et al. 2005
; Giepmans et al. 2001b
) near Ser244). Cx43 trafficking appears to be complex, and, as recent studies have shown, involves a number of proteins and the transport of Cx43 multimers along microtubules to adherens junctions, prior to an incorporation into existing gap junctional plaques (Shaw et al. 2007
). The studies presented here contribute to an understanding of how Cx43 is regulated by phosphorylation and phosphorylation-dependent protein interactions and they highlight the need for additional studies of the potential role for Cx43's interaction with 14-3-3 in Cx43 trafficking.