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Published on: September 20, 2011
PP2A/B56 and GSK3/Ras suppress PKB activity during Dictyostelium chemotaxis
Marbelys Rodriguez Pino1, Boris Castillo1, Bohye Kim1
1Department of Biological Sciences, Florida International University, Miami, FL 33199.
Abstract:
We have previously shown that the Dictyostelium protein phosphatase 2A regulatory subunit B56, encoded by psrA, modulates Dictyostelium cell differentiation through negatively affecting glycogen synthase kinase 3 (GSK3) function. 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. Expression of constitutively active Ras mutants or inhibition of GSK3 in psrA(-) cells increased activities of both PKBR1 and PKBA, but only the PKBR1 activity was increased in wild-type cells under the equivalent conditions, indicating that either B56- or GSK3-mediated suppressive mechanism is sufficient to maintain low PKBA activity, but both mechanisms are necessary for suppressing PKBR1. Finally, cells lacking RasD or RasC displayed normal PKBR1 regulation under GSK3-inhibiting conditions, indicating that RasC or RasD proteins are essential for GSK3-mediated PKBR1 inhibition. In summary, B56 constitutes inhibitory circuits for PKBA and PKBR1 and thus heavily affects Dictyostelium chemotaxis.
Insights
Dictyostelium protein phosphatase 2A regulatory subunit B56 (B56) impacts cell differentiation and chemotaxis by inhibiting protein kinase B (PKB) and PKBR1. This study reveals B56
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Dictyostelium protein phosphatase 2A regulatory subunit B56 (B56) modulates cell differentiation by inhibiting glycogen synthase kinase 3 (GSK3).
- B56's role in regulating Ras signaling and downstream kinases in Dictyostelium remains incompletely understood.
Purpose of the Study:
- To investigate the interaction of B56 with Ras proteins.
- To elucidate the role of B56 in regulating protein kinase B (PKB) and PKBR1 activity.
- To determine the impact of B56 on Dictyostelium cell motility and chemotaxis.
Main Methods:
- In vitro binding assays to assess B56-Ras interactions.
- Analysis of Ras species activation, cell motility, and chemotaxis in wild-type and psrA(-) Dictyostelium cells.
- Phosphorylation assays to measure PKB and PKBR1 activity under various genetic and chemical conditions.
Main Results:
- B56 preferentially associated with GDP-bound RasC and RasD.
- psrA(-) cells exhibited impaired Ras activation, reduced motility, and defective chemotaxis.
- psrA(-) cells showed elevated PKBR1 and PKB substrate phosphorylation, indicating dysregulated kinase activity.
- Both B56 and GSK3 are necessary for suppressing PKBR1 activity, while only one is needed for PKBA.
- RasC and RasD are crucial for GSK3-mediated inhibition of PKBR1.
Conclusions:
- B56 plays a critical role in regulating PKBA and PKBR1 activity, thereby influencing Dictyostelium chemotaxis.
- B56-mediated inhibition of PKBR1 is partially dependent on GSK3 and RasC/RasD signaling.
- Understanding B56's regulatory pathways provides insights into Dictyostelium cell behavior and signaling networks.
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