Fig. 2.

Induction of p35 activator protein by IFN-γ is required for Ser⁸⁸⁶ phosphorylation by Cdk5. (A) IFN-γ induces p35 activator protein. (Left) Lysates from U937 cells treated with IFN-γ for 4 h were immunoblotted. (Right) p35 and β-actin mRNA was determined by RT-PCR of total cellular RNA. (B) IFN-γ induces Cdk5 binding to p35. Lysates from U937 cells treated with IFN-γ for 4 h were coimmunoprecipitated with anti-Cdk5 antibody and probed with anti-p35 (Left) and anti-p39 (Right) antibodies. (C) Cdk5/p35 phosphorylates EPRS at Ser⁸⁸⁶. Lysates from U937 cells treated with IFN-γ for 1 h were immunoprecipitated and immunoblotted as shown. (D) Knockdown of p35 blocks EPRS phosphorylation. (Upper) siRNAs were transfected into U937 cells and incubated with ³²P-orthophosphate and IFN-γ for 4 h. Lysates were immunoprecipitated with anti-EPRS antibody, and EPRS phosphorylation was determined by autoradiography. (Lower) Total EPRS was detected with anti-EPRS antibody. (E) Knockdown of p35 activator protein blocks two-site EPRS phosphorylation. U937 cells were transfected with siRNA against p35 and then treated with IFN-γ as in Fig. 1D. (Left) Knockdown efficiency and EPRS phosphorylation were determined by immunoblot analysis.(Right) Lysates were incubated with anti-Cdk5 antibody, and precipitates were used for in vitro phosphorylation of Cdk5-specific peptide substrate. Aliquots were spotted on phosphocellulose P-81, and the incorporation of ³²P into peptide was determined by scintillation counting (mean ± SEM; n = 3 experiments). (F) ERK2, p35, and Cdk5 are required for GAIT complex–mediated translational suppression. U937 cells were transfected with the siRNAs shown and incubated with IFN-γ for up to 24 h. Lysates were added to RRL for in vitro translation of capped, polyadenylated luciferase (Luc) reporter transcript bearing the Cp GAIT element in the presence of ³⁵S-Met. T7 gene 10 RNA lacking the GAIT element was cotranslated as a specificity control. Luc translation was quantified by densitometry, normalized by T7 gene 10, and expressed as percentage of control (mean ± SEM; n = 3 experiments).