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PP2A/B56 and GSK3/Ras suppress PKB activity during Dictyostelium chemotaxis.

Rodriguez Pino M, Castillo B, Kim B, Kim LW - Mol. Biol. Cell (2015)

Bottom Line: Our follow-up research uncovered that B56 preferentially associated with GDP forms of RasC and RasD, but not with RasG in vitro, and psrA(-) cells displayed inefficient activation of multiple Ras species, decreased random motility, and inefficient chemotaxis toward cAMP and folic acid gradient.Surprisingly, psrA(-) cells displayed aberrantly high basal and poststimulus phosphorylation of Dictyostelium protein kinase B (PKB) kinase family member PKBR1 and PKB substrates.In summary, B56 constitutes inhibitory circuits for PKBA and PKBR1 and thus heavily affects Dictyostelium chemotaxis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Florida International University, Miami, FL 33199.

No MeSH data available.


Related in: MedlinePlus

dnGSK3- and LiCl-mediated regulation of phosphorylation of PKBR1 is Ras dependent. (A) rasD− cells showed normal basal and comparable poststimulus phosphorylation of PKBR1. rasD− cells expressing dnGSK3 displayed no significant increase in the basal phosphorylation levels of PKBR1 and PKBA. In response to cAMP stimulation, rasD− cells expressing dnGSK3 exhibited comparable levels of PKBR1 phosphorylation compared with rasD− cells (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B and C) rasD− and rasC− cells treated with LiCl showed no significant changes in the basal and poststimulus phosphorylation levels of PKBR1 (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. Error bars represent SD.
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Figure 9: dnGSK3- and LiCl-mediated regulation of phosphorylation of PKBR1 is Ras dependent. (A) rasD− cells showed normal basal and comparable poststimulus phosphorylation of PKBR1. rasD− cells expressing dnGSK3 displayed no significant increase in the basal phosphorylation levels of PKBR1 and PKBA. In response to cAMP stimulation, rasD− cells expressing dnGSK3 exhibited comparable levels of PKBR1 phosphorylation compared with rasD− cells (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B and C) rasD− and rasC− cells treated with LiCl showed no significant changes in the basal and poststimulus phosphorylation levels of PKBR1 (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. Error bars represent SD.

Mentions: Recent reports suggested that GSK3 affects Ras adaptation (Kölsh et al., 2012; Sun et al., 2013). Considering that Ras proteins are abnormally regulated in gsk3− cells and they are PKBR1 activators, it is plausible that GSK3 inhibits PKBR1 through Ras proteins. To determine whether RasD is necessary for GSK3-mediated PKBR1 regulation, we overexpressed dnGSK3 in rasD− cells. When the PKBR1 activation was examined in rasD− cells overexpressing dnGKS3, no such drastic increase in PKBR1 phosphorylation as observed in wild-type or psrA− cells expressing dnGSK3 was observed: no detectable level of active PKBR1 was observed from cells without stimulation, but no statistically significant difference in PKBR1 phosphorylation was observed in response to cAMP stimulation (+p > 0.05; Figure 9A). Increased total GSK3 levels of dnGSK3-expressing rasD− cells compared with the control cells are shown (Figure 9A). Furthermore, when rasD− or rasC− cells were treated with LiCl, no significant increase in PKBR1 phosphorylation was observed compared with nontreated cells (+p > 0.05; Figure 9, B and C) in contrast to LiCl-treated wild-type and psrA− cells, which exhibited higher levels of PKBR1 activation (Figure 8, A and B). Altogether these results suggest that GSK3 affects PKBR1 activity via a RasD- or RasC-dependent mechanism.


PP2A/B56 and GSK3/Ras suppress PKB activity during Dictyostelium chemotaxis.

Rodriguez Pino M, Castillo B, Kim B, Kim LW - Mol. Biol. Cell (2015)

dnGSK3- and LiCl-mediated regulation of phosphorylation of PKBR1 is Ras dependent. (A) rasD− cells showed normal basal and comparable poststimulus phosphorylation of PKBR1. rasD− cells expressing dnGSK3 displayed no significant increase in the basal phosphorylation levels of PKBR1 and PKBA. In response to cAMP stimulation, rasD− cells expressing dnGSK3 exhibited comparable levels of PKBR1 phosphorylation compared with rasD− cells (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B and C) rasD− and rasC− cells treated with LiCl showed no significant changes in the basal and poststimulus phosphorylation levels of PKBR1 (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. Error bars represent SD.
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Related In: Results  -  Collection

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Figure 9: dnGSK3- and LiCl-mediated regulation of phosphorylation of PKBR1 is Ras dependent. (A) rasD− cells showed normal basal and comparable poststimulus phosphorylation of PKBR1. rasD− cells expressing dnGSK3 displayed no significant increase in the basal phosphorylation levels of PKBR1 and PKBA. In response to cAMP stimulation, rasD− cells expressing dnGSK3 exhibited comparable levels of PKBR1 phosphorylation compared with rasD− cells (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B and C) rasD− and rasC− cells treated with LiCl showed no significant changes in the basal and poststimulus phosphorylation levels of PKBR1 (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. Error bars represent SD.
Mentions: Recent reports suggested that GSK3 affects Ras adaptation (Kölsh et al., 2012; Sun et al., 2013). Considering that Ras proteins are abnormally regulated in gsk3− cells and they are PKBR1 activators, it is plausible that GSK3 inhibits PKBR1 through Ras proteins. To determine whether RasD is necessary for GSK3-mediated PKBR1 regulation, we overexpressed dnGSK3 in rasD− cells. When the PKBR1 activation was examined in rasD− cells overexpressing dnGKS3, no such drastic increase in PKBR1 phosphorylation as observed in wild-type or psrA− cells expressing dnGSK3 was observed: no detectable level of active PKBR1 was observed from cells without stimulation, but no statistically significant difference in PKBR1 phosphorylation was observed in response to cAMP stimulation (+p > 0.05; Figure 9A). Increased total GSK3 levels of dnGSK3-expressing rasD− cells compared with the control cells are shown (Figure 9A). Furthermore, when rasD− or rasC− cells were treated with LiCl, no significant increase in PKBR1 phosphorylation was observed compared with nontreated cells (+p > 0.05; Figure 9, B and C) in contrast to LiCl-treated wild-type and psrA− cells, which exhibited higher levels of PKBR1 activation (Figure 8, A and B). Altogether these results suggest that GSK3 affects PKBR1 activity via a RasD- or RasC-dependent mechanism.

Bottom Line: Our follow-up research uncovered that B56 preferentially associated with GDP forms of RasC and RasD, but not with RasG in vitro, and psrA(-) cells displayed inefficient activation of multiple Ras species, decreased random motility, and inefficient chemotaxis toward cAMP and folic acid gradient.Surprisingly, psrA(-) cells displayed aberrantly high basal and poststimulus phosphorylation of Dictyostelium protein kinase B (PKB) kinase family member PKBR1 and PKB substrates.In summary, B56 constitutes inhibitory circuits for PKBA and PKBR1 and thus heavily affects Dictyostelium chemotaxis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Florida International University, Miami, FL 33199.

No MeSH data available.


Related in: MedlinePlus