Figure 6. Mutations in conserved residues of the PPP1R15 core functional domain enfeeble its activation by actin.
(A) Traces of the velocity of eIF2aP dephosphorylation by wildtype and the indicated mutant PPP1R15B-PP1G complexes in the presence of the indicated concentrations of actin. (B) As in ‘A’ but with wildtype and mutant PPP1R15A-PP1G complexes. Note that the concentration of binary complex varied from 15–45 nM in the assays shown. It was purposely titrated to attain a velocity of dephosphorylation (in the absence of actin) comparable to ∼1/5 of that achieved by the wildtype enzyme in the presence of saturating concentration of actin. This ensures comparable activity of the wildtype and mutant enzymes in the absence of actin.
Figure 6—figure supplement 1. Mutations in conserved residues of PPP1R15A compromise function in vivo.
(A) Dual channel flow cytometric analysis of CHOP::GFP cells transfected with mCherry, fusion of wildtype PPP1R15A to mCherry or fusion of the indicated mutant derivatives of PPP1R15A to mCherry. Where indicated the cells were treated with the ER stress-inducing agent tunicamycin (TUN) to activate the ISR marker gene CHOP::GFP. (B) The bar diagram quantifies CHOP::GFP activation (the ISR marker) by tunicamycin in the mCherry marked cells (shown is the median ± InterQuartile Range of the GFP intensity of cells in quadrants 1 and 2, from ‘A’ above). It is suppressed in vivo by the wildtype and the weak F585A and I589A mutations, but the strong R571A and W575A mutations reversed the ability of PPP1R15A to suppress the ISR.