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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

The GDP-bound or inactive form of Ras preferably associated with B56. (A) Stimulation of whole-cell lysate with GTPγS increased Flag-RasD and Flag-RasC binding to GST-Raf1-RBD, but exhibited the opposite pattern with GST-B56. (B) Dominant-negative forms of recombinant Flag-RasD(S17N) and Flag-RasC(S18N) displayed strong binding to GST-B56 but not the constitutive active forms of Flag-RasD(G12T) and Flag-RasC(G13T). The constitutive active Ras mutants showed stronger binding to GST-Raf1-RBD as expected. The dominant-negative mutants showed much weaker binding to GST-Raf1-RBD. Representative data from three independent experiments are shown.
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Figure 2: The GDP-bound or inactive form of Ras preferably associated with B56. (A) Stimulation of whole-cell lysate with GTPγS increased Flag-RasD and Flag-RasC binding to GST-Raf1-RBD, but exhibited the opposite pattern with GST-B56. (B) Dominant-negative forms of recombinant Flag-RasD(S17N) and Flag-RasC(S18N) displayed strong binding to GST-B56 but not the constitutive active forms of Flag-RasD(G12T) and Flag-RasC(G13T). The constitutive active Ras mutants showed stronger binding to GST-Raf1-RBD as expected. The dominant-negative mutants showed much weaker binding to GST-Raf1-RBD. Representative data from three independent experiments are shown.

Mentions: Given that the GST pull-down assays were performed with whole-cell lysates from unstimulated cells, in which the majority of the Ras proteins are inactive, it is likely that GDP-Ras proteins were included in the GST-B56 pull-down complex. To determine whether activated GTP-Ras proteins can also associate with B56, the Flag-Ras–containing lysates were treated with GTP-γ-S and then incubated with GST-B56, GST-Raf1–Ras binding domain (RBD), or GST-Byr2-RBD proteins. On incubation with GTP-γ-S, more Ras proteins associated with GST-RBD, but significantly fewer Ras proteins were included in the GST-B56 pull-down complex (Figure 2A). In addition, the recombinant proteins of constitutively active or dominant-negative mutant Flag-RasD and Flag-RasC were generated in Escherichia coli and were incubated with either GST-B56 or GST-RBD proteins. Consistent with GTP-γ-S experiments, the constitutively active Ras mutants displayed less binding to GST-B56, and the dominant-negative Ras mutants exhibited enhanced association with GST-B56 (Figure 2B).


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

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

The GDP-bound or inactive form of Ras preferably associated with B56. (A) Stimulation of whole-cell lysate with GTPγS increased Flag-RasD and Flag-RasC binding to GST-Raf1-RBD, but exhibited the opposite pattern with GST-B56. (B) Dominant-negative forms of recombinant Flag-RasD(S17N) and Flag-RasC(S18N) displayed strong binding to GST-B56 but not the constitutive active forms of Flag-RasD(G12T) and Flag-RasC(G13T). The constitutive active Ras mutants showed stronger binding to GST-Raf1-RBD as expected. The dominant-negative mutants showed much weaker binding to GST-Raf1-RBD. Representative data from three independent experiments are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: The GDP-bound or inactive form of Ras preferably associated with B56. (A) Stimulation of whole-cell lysate with GTPγS increased Flag-RasD and Flag-RasC binding to GST-Raf1-RBD, but exhibited the opposite pattern with GST-B56. (B) Dominant-negative forms of recombinant Flag-RasD(S17N) and Flag-RasC(S18N) displayed strong binding to GST-B56 but not the constitutive active forms of Flag-RasD(G12T) and Flag-RasC(G13T). The constitutive active Ras mutants showed stronger binding to GST-Raf1-RBD as expected. The dominant-negative mutants showed much weaker binding to GST-Raf1-RBD. Representative data from three independent experiments are shown.
Mentions: Given that the GST pull-down assays were performed with whole-cell lysates from unstimulated cells, in which the majority of the Ras proteins are inactive, it is likely that GDP-Ras proteins were included in the GST-B56 pull-down complex. To determine whether activated GTP-Ras proteins can also associate with B56, the Flag-Ras–containing lysates were treated with GTP-γ-S and then incubated with GST-B56, GST-Raf1–Ras binding domain (RBD), or GST-Byr2-RBD proteins. On incubation with GTP-γ-S, more Ras proteins associated with GST-RBD, but significantly fewer Ras proteins were included in the GST-B56 pull-down complex (Figure 2A). In addition, the recombinant proteins of constitutively active or dominant-negative mutant Flag-RasD and Flag-RasC were generated in Escherichia coli and were incubated with either GST-B56 or GST-RBD proteins. Consistent with GTP-γ-S experiments, the constitutively active Ras mutants displayed less binding to GST-B56, and the dominant-negative Ras mutants exhibited enhanced association with GST-B56 (Figure 2B).

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