We have previously demonstrated that PKCγ serves as an oxidative stress sensor through proper control of gap junctions in the lens (Lin et al., 2003a
; Lin and Takemoto, 2005
; Zampighi et al., 2005
; Lin et al., 2006
). We have also observed that the PKCγ H101Y C1B mutant is not activated by oxidative stress, e.g. H2
(Lin and Takemoto, 2005
). In this current study we have tested three SCA14 PKCγ mutants which have modeled structures indicating disruption of the C1B Zn-finger stress switch. We have further provided an additional link between PKCγ C1B domain stress switch → gap junction control → caspase activation. Our data conclusively demonstrate that defective PKCγ C1B domains can cause 1) dysfunction of endogenous PKCγ (i.e., a dominant effect), 2) loss of gap junction control in lens epithelial cells, and 3) increased susceptibility to caspase-3-linked apoptosis.
PKCγ is a unique isoform of classical PKC which has an exposed C1B domain which binds diacylglycerol (DAG) and is oxidized to form disulfide bonds when the cells are exposed to hydrogen peroxide or other oxidative stress (Lin and Takemoto 2005
; Lin et al., 2003a
; Ananthanarayanan et al., 2003
). Inactive PKCγ is always associated with 14-3-3 protein in the cytosol by binding to the PKCγ C1B domain where most SCA14 mutations occur (Nguyen et al., 2004
). Release of 14-3-3 and oxidation within the C1B domain of the PKCγ lead to membrane translocation and activation of PKCγ (Nguyen et al., 2004
; Lin and Takemoto 2005
). Structural modeling results predict that H101Y and G128D SCA14 mutations caused Zinc-finger conformational changes (Chen et al., 2003
; Xu et al., 1997
, and ). Our predicted model of the S119P mutation also suggests a dramatically altered C1B domain structure (). We previously demonstrated the expression of exogenous wild type rat PKCγ with full enzyme activity (Lin et al., 2003b
). In this report we demonstrate that PKCγ S119P and G128D mutations have lower basal enzyme activities when compared to the wild type PKCγ. We further demonstrate that all three of the PKCγ C1B mutants lacked responses to H2
(). Expression of the PKCγ C1B mutants caused a lack of oxidative stress responses of the remaining endogenous wild type PKCγ (). The data suggest that C1B mutations might disrupt the association of endogenous PKCγ with other associated proteins, such as 14-3-3 proteins, which, in turn, causes inactivation of wild type endogenous PKCγ. This dysfunction of the endogenous PKCγ could lead to alteration of cell signaling pathways, such as responses of gap junctions to stress.
The passage of apoptotic signals through open gap junctions is linked to oxidative stress-induced cell death (Naus et al., 2001
; Frantseva, 2002
). Inhibition of gap junctions prevents cell death (Farahani et al., 2005
; de Pina-Benabou et al., 2005
; Krysko et al., 2005
). We, for the first time, show that PKCγ C1B mutations have a negative effect on endogenous wild type PKCγ. The consequence of these mutations are: 1) altered control of gap junctions as determined by Cx43 and Cx50 phosphorylation and gap junction plaques (Fig. and ), and ) increased caspase-3 linked apoptosis (). Gap junction inhibition experiments with AGA further confirmed that PKCγ C1B domain mutations induced increased cell susceptibility to H2
-linked caspase-3 apoptosis through improper control of gap junctions.
Gap junction plaque formation is a dynamic process caused by multiple factors (Gaietta et al., 2002). Gap junction plaques assemble and disassemble at a rate of about every 2-5hr (Lauf et al., 2002
; Berthoud et al., 2004; Musil et al., 2000
), but little is known about what forces disassembly during this dynamic process. We have previously shown that activation of endogenous PKCγ results in disassembly of Cx43 and Cx50 gap junction plaques (Lin and Takemoto 2005
). We show here that overexpression of PKCγ C1B mutants results in failure of endogenous PKCγ to respond to oxidative stress (e.g. H2
) (). The dysfunction of endogenous PKCγ caused by the mutant PKCγ prevents the dynamic process of gap junction plaque disassembly before or after oxidative stress. This would result in increases in functional cell surface gap junction plaques in the cells with the PKCγ C1B mutants.
Apoptotic caspase-3 activation is a common event in cell death. During ischemia or stroke, cell death signals may pass through gap junction channels to adjacent cells (Velaquez et al., 2003
; Contreras et al., 2004
; Thompson et al., 2006
). This gap junction “Bystander effect” accounts for cell death (Farahani et al., 2005
). In our study, overexpression of PKCγ C1B mutations induced cells to be more susceptible to apoptosis by H2
as determined by caspase-3 activation. This effect was abolished by AGA inhibition of gap junctions. Since AGA caused a greater decrease in Cx50 (), we suggest that altered Cx50 gap junctions in N/N1003A cells contribute to passage of apoptotic cell signals to adjacent cells which, in turn, stimulates activation of caspase-3 and causes cells to be more susceptible to oxidative stress. Gap junction inhibition results suggest that protection of cells from oxidative stress may be restored by lowering Cx50 gap junction levels in lens epithelial cells.
In summary, PKCγ C1B mutants lack PKCγ stress sensing activity. Overexpression of these mutants results in dysfunction of endogenous PKCγ, which, in turn, leads to altered control of gap junctions by PKCγ during oxidative stress. Altered gap junctions subsequently result in failure to be protected from further exposure to damaged signals which, in turn, causes cellular caspase-3 activation and subsequent cell apoptosis.