(A) The ability of IMP2 to drive MEF proliferation requires an acidic charge at IMP2 residue 164. Imp2−/− MEFs were engineered to express doxycycline-inducible cDNAs encoding Flag tagged IMP2 wildtype (SS), Ser162Ala/Ser164Ala (AA), Ser162Ala/Ser164Asp (AD), Ser162Asp/Ser164Asp (DD), Ser162Asp/Ser164Ala (DA) or empty flag vector (Flag). Doxycycline was adjusted to achieve Flag-IMP2 variant expression approximating the level of IMP2 found in wildtype Imp2+/+ MEFs, indicated as WT in the immunoblot. The MEFs expressing the six Flag tagged IMP2 variants were plated in replicate and grown in the presence of the adjusted level of doxycycline. Cell number was determined daily. Each point on the curves for SS, AD and DD do not differ from each other but differ from those for Flag, AA and DA starting at day 2, p<0.01. (B) Genome-wide proteomics reveals that the abundance of the IGFBP2 and Grb14 polypeptides in Imp2−/− MEFs expressing Flag-IMP2 with an Ala at residue 164 is similar to that in MEFs lacking IMP2. Whole cell extracts prepared from the six doxycycline-treated MEFs expressing Flag-IMP2 variants or empty Flag vector shown Figure 6A were analyzed by genome wide TMT proteomics. The values for each of the 7964 polypeptides (detected in all six lines) in the (slower growing) Imp2−/− MEFs expressing Flag-IMP2-AA, Flag-IMP2-DA and Flag-vector (KO) were summed and divided by the sum of that polypeptide in MEFs expressing Flag-IMP2 wildtype (SS), Flag-IMP2-AD and Flag-IMP2-DD. This ratio was then sorted from highest to lowest value and the values are shown for the polypeptides showing 6 highest ratios. The abundance of the Flag-IMP2 polypeptide variants and the endogenous rpL31 polypeptide, the latter meant to reflect overall polypeptide abundance, are also shown. The entire data set is in Supplementary file 3. Extracts of the MEFs described in Figure 6A,B were subjected to SDS-PAGE and immunoblotted as indicated. (C) The phosphorylation of IMP2(Ser164) and concurrent dual phosphorylation of IMP2(Ser162/Ser164) are post-translationally regulated by mTOR. 293E cells were grown continuously in DMEM with 10% FCS (columns 1, 4, 5) were treated with torin1, 200 nM (column 5) or switched to DPBS containing glucose, 25 mM and pyruvate,1mM (column 4) 3 hr prior to extraction some cells. Other 293 cells were subjected to overnight serum withdrawal (columns 2,3) followed by addition of insulin 100 nM for 30 min. (column 2) prior to extraction. The extracts were subjected to SDS-PAGE and immunoblot as indicated. (D) Nascent IMP2 polypeptides bound to puromycin are co-translationally phosphorylated at Ser162 and Ser164 by mTOR. Twenty min. after addition of torin1 (200 nM) to rapidly growing RD cells, puromycin was added at 10 ug/ml, and the cells were extracted 10 min. later. The extracts were blotted for IMP2 and puromycin immunoprecipitates were blotted for IMP2 and IMP2(Ser162P) and IMP2(Ser164P). (E) The modification of Ser162 does not affect the phosphorylation of Ser164 and vice versa. 293 cells were transiently transfected with vectors encoding Flag-tagged wildtype IMP2 (SS) or the Flag-IMP2 mutants Ser162Ala (AS), Ser162Asp (DS), Ser164Ala (SA) or Ser164Asp (SD). Flag immunoprecipitates were separated by SDS-PAGE and immunobloted as indicated.