Figure 2. Nuclear ERKs dislodge Rb from... | Rockefeller University Press

Figure 2.

$Figure 2. Nuclear ERKs dislodge Rb from lamin A complexes. (A, left) Nuclear localization of the different HA-tagged ERK2-NLS forms as determined in nuclear (nuc) and cytoplasmic (cyt) fractions of 293T cells transfected with 1 µg of the indicated constructs: control cells transfected with vector DNA (−), ERK2 wt (wt), ERK2 DK (DK), ERK2 unphosphorylatable mutant (AEF), and ERK2 insert region deletion mutant (ΔINS). (right) The purity of the fractions was ascertained using lamin A and Rho GDI as nuclear and cytoplasmic markers, respectively. (B) Nuclear ERK2 disrupts lamin A–Rb interaction. NIH3T3 cells were transfected with 1 µg each of the indicated ERK2-NLS constructs, grown until confluence, and kept in 0.5% CS for 18 h. Cellular lysates were immunoprecipitated for lamin A, and immunoprecipitates (IP) and the corresponding total lysates (TL) were probed by immunoblotting for the indicated proteins (α protein of interest). (C) Effects of nuclear ERK2 on lamin A–Rb interaction analyzed by FRET in U2OS cells using the acceptor-photobleaching method. Cells were transiently cotransfected with 5 µg ECFP–lamin A plus 5 µg each of the different plasmids as indicated. Data show quantification of protein–protein interactions calculated as the percentage of CFP fluorescence increments after YFP photobleaching in 30–50 cells. *, P < 0.05 and **, P < 0.01. Results show means ± SEM. (D) ERK1 disrupts lamin A–Rb interaction as effectively as ERK2. NIH3T3 cells were transfected with 1 µg each of HA-tagged ERK1-NLS and ERK2-NLS and processed as in B. Cellular lysates were immunoprecipitated with an antibody against lamin A or with preimmune serum (PI). Immunoprecipitates and the corresponding total lysates were probed by immunoblotting for the indicated proteins. (E) ERK2 displaces Rb from lamin A in vitro. GST and GST–lamin 247–355 were loaded with YFP-Rb from transfected U2OS cell extracts, incubated with the indicated amounts of purified His-tagged ERK2 for 1 h, and tested for their interaction by Western blotting. Molecular masses (given in kilodaltons) are shown in parentheses after the protein name.$

Nuclear ERKs dislodge Rb from lamin A complexes. (A, left) Nuclear localization of the different HA-tagged ERK2-NLS forms as determined in nuclear (nuc) and cytoplasmic (cyt) fractions of 293T cells transfected with 1 µg of the indicated constructs: control cells transfected with vector DNA (−), ERK2 wt (wt), ERK2 DK (DK), ERK2 unphosphorylatable mutant (AEF), and ERK2 insert region deletion mutant (ΔINS). (right) The purity of the fractions was ascertained using lamin A and Rho GDI as nuclear and cytoplasmic markers, respectively. (B) Nuclear ERK2 disrupts lamin A–Rb interaction. NIH3T3 cells were transfected with 1 µg each of the indicated ERK2-NLS constructs, grown until confluence, and kept in 0.5% CS for 18 h. Cellular lysates were immunoprecipitated for lamin A, and immunoprecipitates (IP) and the corresponding total lysates (TL) were probed by immunoblotting for the indicated proteins (α protein of interest). (C) Effects of nuclear ERK2 on lamin A–Rb interaction analyzed by FRET in U2OS cells using the acceptor-photobleaching method. Cells were transiently cotransfected with 5 µg ECFP–lamin A plus 5 µg each of the different plasmids as indicated. Data show quantification of protein–protein interactions calculated as the percentage of CFP fluorescence increments after YFP photobleaching in 30–50 cells. *, P < 0.05 and **, P < 0.01. Results show means ± SEM. (D) ERK1 disrupts lamin A–Rb interaction as effectively as ERK2. NIH3T3 cells were transfected with 1 µg each of HA-tagged ERK1-NLS and ERK2-NLS and processed as in B. Cellular lysates were immunoprecipitated with an antibody against lamin A or with preimmune serum (PI). Immunoprecipitates and the corresponding total lysates were probed by immunoblotting for the indicated proteins. (E) ERK2 displaces Rb from lamin A in vitro. GST and GST–lamin 247–355 were loaded with YFP-Rb from transfected U2OS cell extracts, incubated with the indicated amounts of purified His-tagged ERK2 for 1 h, and tested for their interaction by Western blotting. Molecular masses (given in kilodaltons) are shown in parentheses after the protein name.