Increased Ezrin Expression and Activation by CDK5 Coincident with Acquisition of the Senescent Phenotype  Hai-Su Yang, Philip W Hinds  Molecular Cell  Volume 11, Issue 5, Pages 1163-1176 (May 2003) DOI: 10.1016/S1097-2765(03)00135-7

Figure 1 Altered Expression and Distribution of Ezrin Accompanies pRb Expression in SAOS-2 Cells (A) RNA from control or pRb-expressing SAOS-2 cells was used in RT-PCR to detect expression of ezrin or GAPDH control mRNA. (B) Protein lysates of control or pRb-expressing SAOS-2 cells were prepared at the indicated times after transfection and subjected to immunoblot analysis for ezrin, moesin, radixin, or GAPDH. (C). Indirect immunofluorescence was used to detect ezrin expression and localization following transfection with vector, RB, RB + CDK5, or RB + dominant-negative CDK5 (DNK5). Ezrin expression was assayed at 2, 5, or 8 days after transfection as indicated. Molecular Cell 2003 11, 1163-1176DOI: (10.1016/S1097-2765(03)00135-7)

Figure 2 Phosphorylation of Ezrin by CDK5 In Vitro (A) Lysates from SAOS-2 cells transfected with empty vector (V), with RB, or with CDK5 + p35 were immunoprecipitated with anti-CDK5 antibody. Immunoblot analysis was then carried out using anti-ezrin antibody. “2,” “5,” and “10” indicate days after transfection prior to cell lysis. (B) Cell lysates were incubated with immobilized GST-ezrin1-309 (GST-NE), GST-ezrin310-585 (GST-CE), or with GST control (last lane). Bound proteins were analyzed with anti-CDK5 antibody. (C) CDK5 was immunoprecipitated from cell lysates transfected with p35 and either CDK5 or dominant-negative CDK5 (DNK), and used in an in vitro kinase assay with histone H1, recombinant GST-N-ezrin (GST-NE), or GST-C-ezrin (GST-CE) as indicated. Arrows indicate migration of phospho-H1 or -GST-NE. (D) Lysates from cells transfected with p35/CDK5 were immunoprecipitated with anti-CDK5 antibody followed by an in vitro kinase assay using histone H1, GST-ezrin1-309(GST-NE), GST-ezrin1-309T235A, GST-ezrin1-309T235D, or GST substrates as indicated. Molecular Cell 2003 11, 1163-1176DOI: (10.1016/S1097-2765(03)00135-7)

Figure 3 Phosphorylation of Ezrin In Vivo (A) SAOS-2 cells were transfected with Flag-tagged wt (FE), T235A, T235D, T567A, or T567D ezrin in the presence of either pRb or CDK5/p35 constructs. The transfected cells were labeled with [32P]orthophosphate prior to lysis (top) or lysed directly (bottom). Lysates were immunoprecipitated with anti-Flag antibody (top) and analyzed by SDS-PAGE and autoradiography to reveal phosphorylation of ezrin in vivo. (Bottom) Lysates were used directly for immunoblot to assess level of expression of FE and mutant forms. (B) SAOS-2 cells were transfected with RB or with p35 plus either CDK5 (CDK5) or dominant-negative CDK5 (DNK5) or were treated with CDK5 inhibitor, roscovitine (10 μM). Lysates were subjected to immunoprecipitation with anti-ezrin antibody followed by immunoblot with either anti-phospho-T235 antibody (top) or anti-ezrin (bottom) to ascertain total ezrin level. (C) Protein lysates of control or pRb-expressing SAOS-2 cells for 2, 5, or 10 days were immunoblotted with anti-phospho-ezrin (T567)/radixin (T564)/moesin (T558) antibody and anti-GAPDH antibody. (D) IMR90 cells were lysed at early passage (EP), middle passage (MP), or after undergoing senescence (S), and subjected to immunoblot analysis for phosphorylation of T235 by using anti-phospho-T235 antibody. (E) The expression and localization of ezrin phosphorylated at T235 was detected with anti-phospho-T235 antibody by indirect immunofluorescence, following transfection with vector, RB for 10 days; RB + CDK5, or RB + DNK5 for 5 days. Molecular Cell 2003 11, 1163-1176DOI: (10.1016/S1097-2765(03)00135-7)

Figure 4 Effect of T235 Mutation on Ezrin Self-Association (A) GST-N-ezrin (GST-NE) or GST-C-ezrin (GST-CE) was added to 35S-labeled, in vitro translated wt ezrin, T235A or T235D variants (lanes 2, 4, 6, 8, 10, and 12). After washing, the complex was analyzed by SDS-PAGE and autoradiography. Level of input, in vitro translated protein is shown in lanes 1, 3, 5, 7, 9, and 11. Binding of GST-NE to the T235D mutant ezrin was readily detected (lane 6). (B) Lysates of SAOS-2 cells transfected with Flag-tagged wt, T235A, or T235D ezrin and with (+) or without (−) p35/CDK5 were subjected to SDS-PAGE and immunoblot with anti-Flag antibody. (C) Lysates from (B) above were incubated with GST-N-ezrin (GST-NE) followed by immunoblot with anti-Flag antibody. Expression of CDK5/p35 increased binding of wt but not mutant forms of ezrin to GST-NE. Molecular Cell 2003 11, 1163-1176DOI: (10.1016/S1097-2765(03)00135-7)

Figure 5 Localization of Ezrin Mutants in SAOS Cells SAOS-2 cells were transfected with empty vector (C) or with Flag-tagged wt (FE), T235A, T235D, T567A, T567D, T235A/T567A (TATA), or T235D/T567D (TDTD) ezrin as indicated. Cells were fixed and subjected to indirect immunofluorescence using anti-Flag antibody. Molecular Cell 2003 11, 1163-1176DOI: (10.1016/S1097-2765(03)00135-7)

Figure 6 Effect of Ezrin Expression on Cell Shape and Senescence (A) SAOS-2 cells were transfected with pBabe-puro plus empty vector (pSVE), pRb expression vector, wt ezrin (FE), T235A, T235D, T567A, T567D, T235A/T567A (TATA), or T235D/T567D (TDTD) mutant ezrin as indicated. Cells selected with puromycin for 10 days were stained to detect expression of senescence-associated β-galactosidase (blue). Photomicrographs (60× magnification) of representative fields are shown to illustrate effects of pRb or ezrin on cell morphology. (B) The area of individual cells in photomicrographs from (A) above was measured using NIH Image. Average cell area in square pixels is depicted graphically with standard error indicated for each transfectant. Molecular Cell 2003 11, 1163-1176DOI: (10.1016/S1097-2765(03)00135-7)

Figure 7 Effect of “Knock Down” of Endogenous Expression of Ezrin, Moesin, and Radixin on Cell Shape and Senescence (A) Immunoblot for ezrin, moesin, and radixin was performed with lysates from SAOS-2 cells transfected with pBabe-puro, pBS/U6 vector, pBS/U6 ezrin SiRNA (EZ), pBS/U6 moesin SiRNA (MO), or pBS/U6 radixin SiRNA (RD) at 5 days posttransfection. (B) SAOS-2 cells were transfected with pBabe-puro plus pBS/U6, pRb expression vector, pRb with EZ, MO, RD, or combinations of these siRNA constructs as indicated. Cells selected with puromycin for 10 days were stained to detect expression of senescence-associated β-galactosidase (blue). Photomicrographs (60× magnification) of representative fields are shown to illustrate effects of pRb or different ERM RNAi constructs on cell morphology. (C) The area of individual cells in photomicrographs from (B) above was measured using NIH Image. Average cell area in square pixels is depicted graphically with standard error indicated for each transfectant. Molecular Cell 2003 11, 1163-1176DOI: (10.1016/S1097-2765(03)00135-7)