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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

psrA− cells exhibited aberrantly high PKBR1 activity and PKB substrate phosphorylation. (A) Compared with Wt cells, psrA− cells exhibited an abnormally high basal level of phosphorylated PKBR1 at the AL site, which persisted in response to cAMP stimulation. The levels of basal PKBR1 phosphorylation in psrA− cells are consistently higher (approximately three times) than those of wild-type cells (three independent experiments; **, p < 0.01). The levels of basal PKBA phosphorylation of Wt and psrA− cells showed no statistically significant difference (+, p > 0.05). PKBA also displayed persistent activation in response to cAMP stimulation in psrA− cells. The phospho-PKB levels were normalized to Coomassie-stained total proteins. (B) psrA− cells also displayed aberrantly higher basal and poststimulus levels of phosphorylated PKBR1 at the HM site in response to cAMP stimulation. (C) Phosphorylation levels of PKB substrates in psrA− and Wt cells were detected using the anti–phospho Akt substrate antibody. psrA− cells displayed significantly increased basal level of phosphoproteins. Some of these proteins seem to be psrA− cell specific as marked (*), and the others could be assigned to the previously reported PKBR1/PKBA substrate proteins (pp350, pp280 for TalinB, pp200/pp180 for GefN, pp140 for GacG/PakA, pp110 for GefS/PI5K, pp65/67 for GacQ; Cai et al., 2010). (D) Comparable levels of PKBR1 messages were detected from wild-type and psrA− cells by RT-PCR. Ig7 messages were shown as loading control. Error bars represent SD.
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Figure 5: psrA− cells exhibited aberrantly high PKBR1 activity and PKB substrate phosphorylation. (A) Compared with Wt cells, psrA− cells exhibited an abnormally high basal level of phosphorylated PKBR1 at the AL site, which persisted in response to cAMP stimulation. The levels of basal PKBR1 phosphorylation in psrA− cells are consistently higher (approximately three times) than those of wild-type cells (three independent experiments; **, p < 0.01). The levels of basal PKBA phosphorylation of Wt and psrA− cells showed no statistically significant difference (+, p > 0.05). PKBA also displayed persistent activation in response to cAMP stimulation in psrA− cells. The phospho-PKB levels were normalized to Coomassie-stained total proteins. (B) psrA− cells also displayed aberrantly higher basal and poststimulus levels of phosphorylated PKBR1 at the HM site in response to cAMP stimulation. (C) Phosphorylation levels of PKB substrates in psrA− and Wt cells were detected using the anti–phospho Akt substrate antibody. psrA− cells displayed significantly increased basal level of phosphoproteins. Some of these proteins seem to be psrA− cell specific as marked (*), and the others could be assigned to the previously reported PKBR1/PKBA substrate proteins (pp350, pp280 for TalinB, pp200/pp180 for GefN, pp140 for GacG/PakA, pp110 for GefS/PI5K, pp65/67 for GacQ; Cai et al., 2010). (D) Comparable levels of PKBR1 messages were detected from wild-type and psrA− cells by RT-PCR. Ig7 messages were shown as loading control. Error bars represent SD.

Mentions: Considering that psrA− cells have generally low Ras activity, we hypothesized that PKBA and PKBR1 activation may also be compromised in psrA− cells. For examination of PKBA and PKBR1 activation, aggregation-competent cells were stimulated with cAMP and the phosphorylation levels of PKBA and PKBR1 at the activation loop (AL) site were examined as previously described (Cai et al., 2010). On cAMP stimulation, wild-type cells exhibited phosphorylation at the AL site in PKBR1 and PKBA between 15 and 30 s, which decreased back to the basal level at 60 s after cAMP stimulation. In contrast, a higher basal level of phosphorylated PKBR1, but not PKBA, was observed in psrA− cells compared with Wt cells (Figure 5A). Furthermore, psrA− cells displayed higher basal phosphorylation of T470 of the HM of PKBR1 (p < 0.05; Figure 5B). Total protein levels stained with Coomassie were used as a loading control.


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

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

psrA− cells exhibited aberrantly high PKBR1 activity and PKB substrate phosphorylation. (A) Compared with Wt cells, psrA− cells exhibited an abnormally high basal level of phosphorylated PKBR1 at the AL site, which persisted in response to cAMP stimulation. The levels of basal PKBR1 phosphorylation in psrA− cells are consistently higher (approximately three times) than those of wild-type cells (three independent experiments; **, p < 0.01). The levels of basal PKBA phosphorylation of Wt and psrA− cells showed no statistically significant difference (+, p > 0.05). PKBA also displayed persistent activation in response to cAMP stimulation in psrA− cells. The phospho-PKB levels were normalized to Coomassie-stained total proteins. (B) psrA− cells also displayed aberrantly higher basal and poststimulus levels of phosphorylated PKBR1 at the HM site in response to cAMP stimulation. (C) Phosphorylation levels of PKB substrates in psrA− and Wt cells were detected using the anti–phospho Akt substrate antibody. psrA− cells displayed significantly increased basal level of phosphoproteins. Some of these proteins seem to be psrA− cell specific as marked (*), and the others could be assigned to the previously reported PKBR1/PKBA substrate proteins (pp350, pp280 for TalinB, pp200/pp180 for GefN, pp140 for GacG/PakA, pp110 for GefS/PI5K, pp65/67 for GacQ; Cai et al., 2010). (D) Comparable levels of PKBR1 messages were detected from wild-type and psrA− cells by RT-PCR. Ig7 messages were shown as loading control. Error bars represent SD.
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Figure 5: psrA− cells exhibited aberrantly high PKBR1 activity and PKB substrate phosphorylation. (A) Compared with Wt cells, psrA− cells exhibited an abnormally high basal level of phosphorylated PKBR1 at the AL site, which persisted in response to cAMP stimulation. The levels of basal PKBR1 phosphorylation in psrA− cells are consistently higher (approximately three times) than those of wild-type cells (three independent experiments; **, p < 0.01). The levels of basal PKBA phosphorylation of Wt and psrA− cells showed no statistically significant difference (+, p > 0.05). PKBA also displayed persistent activation in response to cAMP stimulation in psrA− cells. The phospho-PKB levels were normalized to Coomassie-stained total proteins. (B) psrA− cells also displayed aberrantly higher basal and poststimulus levels of phosphorylated PKBR1 at the HM site in response to cAMP stimulation. (C) Phosphorylation levels of PKB substrates in psrA− and Wt cells were detected using the anti–phospho Akt substrate antibody. psrA− cells displayed significantly increased basal level of phosphoproteins. Some of these proteins seem to be psrA− cell specific as marked (*), and the others could be assigned to the previously reported PKBR1/PKBA substrate proteins (pp350, pp280 for TalinB, pp200/pp180 for GefN, pp140 for GacG/PakA, pp110 for GefS/PI5K, pp65/67 for GacQ; Cai et al., 2010). (D) Comparable levels of PKBR1 messages were detected from wild-type and psrA− cells by RT-PCR. Ig7 messages were shown as loading control. Error bars represent SD.
Mentions: Considering that psrA− cells have generally low Ras activity, we hypothesized that PKBA and PKBR1 activation may also be compromised in psrA− cells. For examination of PKBA and PKBR1 activation, aggregation-competent cells were stimulated with cAMP and the phosphorylation levels of PKBA and PKBR1 at the activation loop (AL) site were examined as previously described (Cai et al., 2010). On cAMP stimulation, wild-type cells exhibited phosphorylation at the AL site in PKBR1 and PKBA between 15 and 30 s, which decreased back to the basal level at 60 s after cAMP stimulation. In contrast, a higher basal level of phosphorylated PKBR1, but not PKBA, was observed in psrA− cells compared with Wt cells (Figure 5A). Furthermore, psrA− cells displayed higher basal phosphorylation of T470 of the HM of PKBR1 (p < 0.05; Figure 5B). Total protein levels stained with Coomassie were used as a loading control.

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