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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

Introducing dnGSK3 increased PKBA and PKBR1 activities in psrA− cells. (A) Wt cells expressing the dnGSK3 exhibited higher levels of basal phosphorylation of PKBR1, which persisted upon cAMP stimulation (*, p < 0.05). No such significant changes were observed for PKBA phosphorylation (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B) psrA− expressing the dnGSK3 showed high basal and persistent poststimulus phosphorylation of PKBR1 (three independent experiments, ∼2.5-fold higher basal and twofold increase in 15-s poststimulus levels; +, p < 0.05) and exaggerated poststimulus phosphorylation of PKBA. The phospho-PKBs levels were normalized to Coomassie-stained total proteins. Error bars represent SD.
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Figure 7: Introducing dnGSK3 increased PKBA and PKBR1 activities in psrA− cells. (A) Wt cells expressing the dnGSK3 exhibited higher levels of basal phosphorylation of PKBR1, which persisted upon cAMP stimulation (*, p < 0.05). No such significant changes were observed for PKBA phosphorylation (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B) psrA− expressing the dnGSK3 showed high basal and persistent poststimulus phosphorylation of PKBR1 (three independent experiments, ∼2.5-fold higher basal and twofold increase in 15-s poststimulus levels; +, p < 0.05) and exaggerated poststimulus phosphorylation of PKBA. The phospho-PKBs levels were normalized to Coomassie-stained total proteins. Error bars represent SD.

Mentions: As mentioned earlier, several studies showed that GSK3 affects PKBs (Teo et al., 2010; Kölsh et al., 2012; Sun et al., 2013). Considering the existence of conflicting reports using gsk3− cells, we analyzed wild-type and psrA− cells expressing dnGSK3 or treated with LiCl. Wild-type cells expressing dnGSK3 showed ­significantly elevated basal and poststimulus levels of PKBR1 phosphorylation (Figure 7A), and psrA− cells expressing dnGSK3 exhibited even higher levels of basal and poststimulus PKBR1 phosphorylation and significantly increased PKBA activation in response to cAMP stimulation (Figure 7B). Increased total GSK3 level of dnGSK3-expressing cells compared with the parental cells are shown as a control (Figure 7, A and B).


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

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

Introducing dnGSK3 increased PKBA and PKBR1 activities in psrA− cells. (A) Wt cells expressing the dnGSK3 exhibited higher levels of basal phosphorylation of PKBR1, which persisted upon cAMP stimulation (*, p < 0.05). No such significant changes were observed for PKBA phosphorylation (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B) psrA− expressing the dnGSK3 showed high basal and persistent poststimulus phosphorylation of PKBR1 (three independent experiments, ∼2.5-fold higher basal and twofold increase in 15-s poststimulus levels; +, p < 0.05) and exaggerated poststimulus phosphorylation of PKBA. The phospho-PKBs 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 7: Introducing dnGSK3 increased PKBA and PKBR1 activities in psrA− cells. (A) Wt cells expressing the dnGSK3 exhibited higher levels of basal phosphorylation of PKBR1, which persisted upon cAMP stimulation (*, p < 0.05). No such significant changes were observed for PKBA phosphorylation (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B) psrA− expressing the dnGSK3 showed high basal and persistent poststimulus phosphorylation of PKBR1 (three independent experiments, ∼2.5-fold higher basal and twofold increase in 15-s poststimulus levels; +, p < 0.05) and exaggerated poststimulus phosphorylation of PKBA. The phospho-PKBs levels were normalized to Coomassie-stained total proteins. Error bars represent SD.
Mentions: As mentioned earlier, several studies showed that GSK3 affects PKBs (Teo et al., 2010; Kölsh et al., 2012; Sun et al., 2013). Considering the existence of conflicting reports using gsk3− cells, we analyzed wild-type and psrA− cells expressing dnGSK3 or treated with LiCl. Wild-type cells expressing dnGSK3 showed ­significantly elevated basal and poststimulus levels of PKBR1 phosphorylation (Figure 7A), and psrA− cells expressing dnGSK3 exhibited even higher levels of basal and poststimulus PKBR1 phosphorylation and significantly increased PKBA activation in response to cAMP stimulation (Figure 7B). Increased total GSK3 level of dnGSK3-expressing cells compared with the parental cells are shown as a control (Figure 7, A and B).

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