Regulation of protein phosphorylation by kinases and phosphatases is involved in many signaling pathways in mammalian cells. In contrast to prokaryotes and lower eukaryotes a role for the reversible ...phosphorylation of histidine residues is just emerging. The β subunit of heterotrimeric G proteins, the metabolic enzyme adenosine 5'-triphosphate-citrate lyase (ACL), and the Ca2+-activated K+ channel KCa3.1 have been identified as targets for nucleoside diphosphate kinase (NDPK) acting as protein histidine kinase and the so far only identified mammalian protein histidine phosphatase (PHPT-1). Herein, we describe the analysis of the phosphorylation and dephosphorylation of histidine residues by NDPK and PHPT-1. In addition, experimental protocols for studying the consequences of heterotrimeric G protein activation via NDPK/Gβγ mediated phosphorelay, the regulation of ACL activity and of KCa3.1 conductivity by histidine phosphorylation will be presented.
Heterotrimeric G proteins are key regulators of signaling pathways in mammalian cells. Beyond G protein-coupled receptors, the amount and mutual ratio of specific G protein α, β, and γ subunits ...determine the G protein signaling. However, little is known about mechanisms that regulate the concentration and composition of G protein subunits at the plasma membrane. Here, we show a novel cross-talk between stimulatory and inhibitory G protein α subunits (Gα) that is mediated by G protein βγ dimers and controls the abundance of specific Gα subunits at the plasma membrane. Firstly, we observed in heart tissue from constitutively Gαi2- and Gαi3-deficient mice that the loss of Gαi2 and Gαi3 was accompanied by a slight increase in the protein content of the nontargeted Gαi isoform. Therefore, we analyzed whether overexpression of selected Gα subunits conversely impairs endogenous G protein α and β subunit levels in cardiomyocytes. Integration of overexpressed Gαi2 subunits into heterotrimeric G proteins was verified by co-immunoprecipitation. Adenoviral expression of increasing amounts of Gαi2 led to a reduction of Gαi3 (up to 90 %) and Gαs (up to 75 %) protein levels. Likewise, increasing amounts of adenovirally expressed Gαs resulted in a linear 75 % decrease in both Gαi2 and Gαi3 protein levels. In contrast, overexpression of either Gαi or Gαs isoform did not influence the amount of Gαo and Gαq, both of which are not involved in the regulation of adenylyl cyclase activity. The mRNA expression of the disappearing endogenous Gα subunits was not affected, indicating a posttranslational mechanism. Interestingly, the amount of endogenous G protein βγ dimers was not altered by any Gα overexpression. However, the increase of Gβγ level by adenoviral expression prevented the loss of endogenous Gαs and Gαi3 in Gαi2 overexpressing cardiomyocytes. Thus, our results provide evidence for a novel mechanism cross-regulating adenylyl cyclase-modulating Gαi isoforms and Gαs proteins. The Gα subunits apparently compete for a limited amount of Gβγ dimers, which are required for G protein heterotrimer formation at the plasma membrane.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Heterotrimeric G proteins are key regulators of signaling pathways in mammalian cells. Beyond G protein-coupled receptors, the amount and mutual ratio of specific G protein alpha, beta, and γ ...subunits determine the G protein signaling. However, little is known about mechanisms that regulate the concentration and composition of G protein subunits at the plasma membrane. Here, we show a novel cross-talk between stimulatory and inhibitory G protein alpha subunits (Galpha) that is mediated by G protein betaγ dimers and controls the abundance of specific Galpha subunits at the plasma membrane. Firstly, we observed in heart tissue from constitutively Galphai2- and Galphai3-deficient mice that the loss of Galphai2 and Galphai3 was accompanied by a slight increase in the protein content of the nontargeted Galphai isoform. Therefore, we analyzed whether overexpression of selected Galpha subunits conversely impairs endogenous G protein alpha and beta subunit levels in cardiomyocytes. Integration of overexpressed Galphai2 subunits into heterotrimeric G proteins was verified by co-immunoprecipitation. Adenoviral expression of increasing amounts of Galphai2 led to a reduction of Galphai3 (up to 90 %) and Galphas (up to 75 %) protein levels. Likewise, increasing amounts of adenovirally expressed Galphas resulted in a linear 75 % decrease in both Galphai2 and Galphai3 protein levels. In contrast, overexpression of either Galphai or Galphas isoform did not influence the amount of Galphao and Galphaq, both of which are not involved in the regulation of adenylyl cyclase activity. The mRNA expression of the disappearing endogenous Galpha subunits was not affected, indicating a posttranslational mechanism. Interestingly, the amount of endogenous G protein betaγ dimers was not altered by any Galpha overexpression. However, the increase of Gbetaγ level by adenoviral expression prevented the loss of endogenous Galphas and Galphai3 in Galphai2 overexpressing cardiomyocytes. Thus, our results provide evidence for a novel mechanism cross-regulating adenylyl cyclase-modulating Galphai isoforms and Galphas proteins. The Galpha subunits apparently compete for a limited amount of Gbetaγ dimers, which are required for G protein heterotrimer formation at the plasma membrane.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B ...were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro . To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B ...were stably expressed in H10
cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular
GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G s α and G i α proteins. However, co-expression of G s α led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was
confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited
a decreased cAMP formation in response to G s α. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gβγ dimers. The overexpression of NDPK
B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gβ subunits. In summary, our data demonstrate a
specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B·Gβγ
complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.
Full text
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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