Figure 6. Starvation stimulates release of PP2A catalytic subunit from inhibitory protein Alpha4.
(a) Recombinant PP2AC–Alpha4 complex is inactive. Phosphatase assay was carried out in vitro using increasing amounts of recombinant PP2AC (3–90 nM) co-purified with Alpha4. Recombinant PP2AC-PRL65 complex (20 nM) was used as a control. Coomassie brilliant blue staining of recombinant PP2AC–Alpha4 is shown below. (b) Starvation induces dissociation of PP2AC from Alpha4. 293T cells were starved or incubated in complete media containing 1 μM Torin1 for 2 h. Cell lysate was prepared, crosslinked with DSP and incubated with the indicated antibodies to pull down endogenous PP2A complexes for immunoblot analysis. Quantitation of co-IPed proteins relative to full media is shown on the right. (fold change±s.d., n=3. two-tail Student’s t-test, *P<0.05) (c) Alpha4 overexpression reduces PRL65 binding to PP2AC. 293T cells were transfected with Flag-S-Alpha4. Cells were lysed, and incubated with antibodies to pull down endogenous PP2AC. Immunoblotting was carried out to monitor the amount of PRL65 interacting with PP2AC. Panel on the right shows quantitation of PRL65 relative to untransfected cells. (fold change±s.d., n=3) (d) Alpha4 overexpression blocks ULK1 S637 dephosphorylation. MEF cells stably overexpressing Flag-S-Alpha4 were incubated in complete or starvation media for the indicated amount of time.