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Gαi3-Dependent Inhibition of JNK Activity on Intracellular Membranes.

Bastin G, Yang JY, Heximer SP - Front Bioeng Biotechnol (2015)

Bottom Line: The activity of one MAPK family class, c-Jun N-terminal kinases (JNKs), has been traditionally linked to the activation of G-protein coupled receptors (GPCRs) at the plasma membrane.Together, these data support the existence of unique intracellular signaling complexes that control JNK activity deep within the cell.This work highlights some of the cellular pathways that are regulated by these intracellular complexes and identifies potential strategies for their regulation in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Heart and Stroke, Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto , Toronto, ON , Canada.

ABSTRACT
Heterotrimeric G-protein signaling has been shown to modulate a wide variety of intracellular signaling pathways, including the mitogen-activated protein kinase (MAPK) family. The activity of one MAPK family class, c-Jun N-terminal kinases (JNKs), has been traditionally linked to the activation of G-protein coupled receptors (GPCRs) at the plasma membrane. Using a unique set of G-protein signaling tools developed in our laboratory, we show that subcellular domain-specific JNK activity is inhibited by the activation of Gαi3, the Gαi isoform found predominantly within intracellular membranes, such as the endoplasmic reticulum (ER)-Golgi interface, and their associated vesicle pools. Regulators of intracellular Gαi3, including activator of G-protein signaling 3 (AGS3) and the regulator of G-protein signaling protein 4 (RGS4), have a marked impact on the regulation of JNK activity. Together, these data support the existence of unique intracellular signaling complexes that control JNK activity deep within the cell. This work highlights some of the cellular pathways that are regulated by these intracellular complexes and identifies potential strategies for their regulation in mammalian cells.

No MeSH data available.


Related in: MedlinePlus

Activated Gαi3 reduces the levels of phospho(p)-JNK in HEK 293 cells. Relative changes in endogenous p-JNK/total JNK ratio were quantified in the presence of Gαi3-CFP (WT) and (RC) by Western Blotting. The left panel show a representative Western blot of p-JNK/total JNK regulation by Gαi3 (WT) and (RC) expression. The right panel shows the quantification of p-JNK/total JNK ratio in four independent experiments (one-way ANOVA with Tukey post hoc test *P < 0.01).
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Figure 4: Activated Gαi3 reduces the levels of phospho(p)-JNK in HEK 293 cells. Relative changes in endogenous p-JNK/total JNK ratio were quantified in the presence of Gαi3-CFP (WT) and (RC) by Western Blotting. The left panel show a representative Western blot of p-JNK/total JNK regulation by Gαi3 (WT) and (RC) expression. The right panel shows the quantification of p-JNK/total JNK ratio in four independent experiments (one-way ANOVA with Tukey post hoc test *P < 0.01).

Mentions: We next examined JNK activity, an intracellular signaling pathway that we expected to be sensitive to changes in Gαi3 activity. Indeed, the prediction, based on previous reports where JNK was activated downstream of various GPCRs, was that increased Gαi3 activity would increase the level of intracellular JNK activation as determined by Western blotting for phosphorylated JNK (p-JNK). Surprisingly, however, the relative activity level of Gαi3 in our system inversely correlated with the observed p-JNK levels. Specifically, Gαi3 (RC) mediated a profound reduction of p-JNK in our system compared to Gαi3 (WT) (Figure 4). As expected, both Gαi3 clones showed a reduced level of p-JNK relative to empty vector controls (data not shown). Together, these data suggested the novel premise that Gαi3 signaling inhibits intracellular JNK activation. Since, AGS3 functions as a GDI on intracellular membrane pools, we next examined whether AGS3-mediated stabilization of GDP-bound (inactive) Gαi3 may also regulate JNK activation. Consistent with our new model, AGS3 expression markedly increased the levels of p-JNK observed in our cells relative to YFP control (Figure 5). These data suggest that there exists a tonic level of endogenous Gαi-mediated JNK inhibition in HEK293 cells that can be modulated by the expression of AGS3 or other similar GDI partners. In support of this, we also found that pertussis toxin increased p-JNK levels in a dose-dependent fashion (data not shown). Finally, we examined the effect of another potent Gαi3 inhibitor, RGS4, on the regulation of JNK activity in our system (Figure 5). Endogeneous Gαi3 with wild-type RGS4 expression resulted in a modest decrease of p-JNK compared to expression of its catalytically inactive EN-AA mutant. At first glance, these data seemed inconsistent with the observations for AGS3 and Gαi3 above. However, a more compelling story emerged when we examined the effects of the RGS4 palmitoylation site mutants Cys2A and Cys12A on intracellular JNK signaling. Notably, for the Cys2 mutant, when RGS4 was nearly exclusively localized to the plasma membrane (i.e., unable to target the intracellular membrane pool), there was a marked decrease in p-JNK levels. By contrast, for the Cys12A mutant when RGS4 was nearly exclusively localized to intracellular membranes, there was a marked increase in p-JNK levels to those even exceeding the levels observed for the catalytically inactive (EN-AA) RGS4 construct. Taken together, these data suggest that total JNK signaling in a cell or tissue represents a combination of JNK pools that likely includes cytosolic, nuclear, plasma membrane, and intracellular fractions. Wild-type RGS4, by virtue of its ability to target and inhibit multiple intracellular signaling pools showed a much different effect on JNK signaling compared to either of the two individual palmitoylation site mutants. It should be noted, however, that we cannot rule out the possibility that mutation of Cys2 and its effects on RGS4 stability, via preventing N-end rule degradation of the protein may also have contributed to its ability to regulate intracellular JNK activity. A mechanistic model showing the spatial distribution of JNK, Gαi3, and its regulators is presented in Figure 6.


Gαi3-Dependent Inhibition of JNK Activity on Intracellular Membranes.

Bastin G, Yang JY, Heximer SP - Front Bioeng Biotechnol (2015)

Activated Gαi3 reduces the levels of phospho(p)-JNK in HEK 293 cells. Relative changes in endogenous p-JNK/total JNK ratio were quantified in the presence of Gαi3-CFP (WT) and (RC) by Western Blotting. The left panel show a representative Western blot of p-JNK/total JNK regulation by Gαi3 (WT) and (RC) expression. The right panel shows the quantification of p-JNK/total JNK ratio in four independent experiments (one-way ANOVA with Tukey post hoc test *P < 0.01).
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Related In: Results  -  Collection

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Figure 4: Activated Gαi3 reduces the levels of phospho(p)-JNK in HEK 293 cells. Relative changes in endogenous p-JNK/total JNK ratio were quantified in the presence of Gαi3-CFP (WT) and (RC) by Western Blotting. The left panel show a representative Western blot of p-JNK/total JNK regulation by Gαi3 (WT) and (RC) expression. The right panel shows the quantification of p-JNK/total JNK ratio in four independent experiments (one-way ANOVA with Tukey post hoc test *P < 0.01).
Mentions: We next examined JNK activity, an intracellular signaling pathway that we expected to be sensitive to changes in Gαi3 activity. Indeed, the prediction, based on previous reports where JNK was activated downstream of various GPCRs, was that increased Gαi3 activity would increase the level of intracellular JNK activation as determined by Western blotting for phosphorylated JNK (p-JNK). Surprisingly, however, the relative activity level of Gαi3 in our system inversely correlated with the observed p-JNK levels. Specifically, Gαi3 (RC) mediated a profound reduction of p-JNK in our system compared to Gαi3 (WT) (Figure 4). As expected, both Gαi3 clones showed a reduced level of p-JNK relative to empty vector controls (data not shown). Together, these data suggested the novel premise that Gαi3 signaling inhibits intracellular JNK activation. Since, AGS3 functions as a GDI on intracellular membrane pools, we next examined whether AGS3-mediated stabilization of GDP-bound (inactive) Gαi3 may also regulate JNK activation. Consistent with our new model, AGS3 expression markedly increased the levels of p-JNK observed in our cells relative to YFP control (Figure 5). These data suggest that there exists a tonic level of endogenous Gαi-mediated JNK inhibition in HEK293 cells that can be modulated by the expression of AGS3 or other similar GDI partners. In support of this, we also found that pertussis toxin increased p-JNK levels in a dose-dependent fashion (data not shown). Finally, we examined the effect of another potent Gαi3 inhibitor, RGS4, on the regulation of JNK activity in our system (Figure 5). Endogeneous Gαi3 with wild-type RGS4 expression resulted in a modest decrease of p-JNK compared to expression of its catalytically inactive EN-AA mutant. At first glance, these data seemed inconsistent with the observations for AGS3 and Gαi3 above. However, a more compelling story emerged when we examined the effects of the RGS4 palmitoylation site mutants Cys2A and Cys12A on intracellular JNK signaling. Notably, for the Cys2 mutant, when RGS4 was nearly exclusively localized to the plasma membrane (i.e., unable to target the intracellular membrane pool), there was a marked decrease in p-JNK levels. By contrast, for the Cys12A mutant when RGS4 was nearly exclusively localized to intracellular membranes, there was a marked increase in p-JNK levels to those even exceeding the levels observed for the catalytically inactive (EN-AA) RGS4 construct. Taken together, these data suggest that total JNK signaling in a cell or tissue represents a combination of JNK pools that likely includes cytosolic, nuclear, plasma membrane, and intracellular fractions. Wild-type RGS4, by virtue of its ability to target and inhibit multiple intracellular signaling pools showed a much different effect on JNK signaling compared to either of the two individual palmitoylation site mutants. It should be noted, however, that we cannot rule out the possibility that mutation of Cys2 and its effects on RGS4 stability, via preventing N-end rule degradation of the protein may also have contributed to its ability to regulate intracellular JNK activity. A mechanistic model showing the spatial distribution of JNK, Gαi3, and its regulators is presented in Figure 6.

Bottom Line: The activity of one MAPK family class, c-Jun N-terminal kinases (JNKs), has been traditionally linked to the activation of G-protein coupled receptors (GPCRs) at the plasma membrane.Together, these data support the existence of unique intracellular signaling complexes that control JNK activity deep within the cell.This work highlights some of the cellular pathways that are regulated by these intracellular complexes and identifies potential strategies for their regulation in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Heart and Stroke, Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto , Toronto, ON , Canada.

ABSTRACT
Heterotrimeric G-protein signaling has been shown to modulate a wide variety of intracellular signaling pathways, including the mitogen-activated protein kinase (MAPK) family. The activity of one MAPK family class, c-Jun N-terminal kinases (JNKs), has been traditionally linked to the activation of G-protein coupled receptors (GPCRs) at the plasma membrane. Using a unique set of G-protein signaling tools developed in our laboratory, we show that subcellular domain-specific JNK activity is inhibited by the activation of Gαi3, the Gαi isoform found predominantly within intracellular membranes, such as the endoplasmic reticulum (ER)-Golgi interface, and their associated vesicle pools. Regulators of intracellular Gαi3, including activator of G-protein signaling 3 (AGS3) and the regulator of G-protein signaling protein 4 (RGS4), have a marked impact on the regulation of JNK activity. Together, these data support the existence of unique intracellular signaling complexes that control JNK activity deep within the cell. This work highlights some of the cellular pathways that are regulated by these intracellular complexes and identifies potential strategies for their regulation in mammalian cells.

No MeSH data available.


Related in: MedlinePlus