Cellular senescence requires CDK5 repression of Rac1 activity

Abstract

Cellular senescence is a tumor-suppressive process characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated beta-galactosidase (SA-beta-Gal). We report here a role for CDK5 in induction of senescent cytoskeletal changes. CDK5 activation is upregulated in senescing cells. The increased activity of CDK5 further reduces GTPase Rac1 activity and Pak activation. The repression of the activity of the GTPase Rac1 by CDK5 is required for expression of the senescent phenotype. CDK5 regulation of Rac1 activity is necessary for actin polymerization accompanying senescent morphology in response to expression of pRb, activated Ras, or continuous passage. Inhibition of CDK5 attenuates SA-beta-Gal expression and blocks actin polymerization. These results point to a unique, nonneuronal role for CDK5 in regulation of Rac1 activity in senescence, illuminating the mechanisms underlying induction of senescence and the senescent shape change.

FIG. 1.

Roscovitine blocks senescence. (A) SAOS-2 cells were transfected with pBabe-puromycin and pSVE or pSVE-RB, and cells were selected with puromycin. Transfected cells were treated with roscovitine 2- to 10-days posttransfection. MDA-MB-468 cells were infected with Ad-GFP or Ad-RB and treated 1- to 7-days postinfection with roscovitine. IMR90 cells were infected with pBabe-puromycin or pBabe-RasV12 and treated 5- to 10-days posttransfection with roscovitine. Passaged (psg) senescent IMR90 and WI38 cells were treated with roscovitine for 48 h. After being treated with roscovitine, all cells were assayed for SA-β-Gal expression, and the percentage of SA-β-Gal-positive cells relative to the total number of cells was determined. DMSO, dimethyl sulfoxide. (B) The change in morphology among cycling presenescent, senescent, and roscovitine-treated senescent cells is shown. All images were obtained by using phase contrast microscopy at a magnification of ×60.

FIG.2.

A requirement for CDK5 in senescence. (A) Lysates harvested from cells undergoing senescence by a variety of stimuli were immunoblotted for pRb or CDK5 or immunoprecipitated for CDK5 to perform an in vitro kinase assay using PSD-95 as the substrate. SAOS-2 cells were transfected with pBabe-puromycin and pSVE or pSVE-RB; MDA-MB-468 cells were infected with Ad-GFP or Ad-RB and lysates harvested 2, 4, and 7 days after pRb reintroduction. Lysates from IMR90 cells infected with pBabe-puromycin or pBabe-RasV12 were harvested 10 days after infection and puromycin selection. IMR90 cells were passaged in culture, and cells were harvested for lysates at early passage (EP), middle passage (MP), or after undergoing senescence (S). (B) Cell lysates from SAOS-2 cells transfected with pRb, MDA-MB-468 cells infected with Ad-RB, and differently passaged IMR90 cells were immunoprecipitated with Pak1 antibody. Immunocomplexes were detected with anti-phospho-Pak1 antibody. Phosph-Pak, phosphorylated Pak1. (C) SAOS-2 cells were cotransfected with pBabe-puromycin and pSVE-RB and dnCDK5 at increasing concentrations. After 10 days, cells were assayed for the senescent shape change and SA-β-Gal expression. The fraction of flat or SA-β-Gal-positive cells is expressed as a percentage of the total number of cells counted. (D) SAOS-2 cells were transfected with pBabe-puromycin, pBS/U6, and pSVE or pSVE-RB or cotransfected with pSVE-RB and a CDK5 siRNA construct, pBS/U6-siCDK5, for 10-days posttransfection. Cell lysates were immunoblotted with CDK5 or CDK2 antibody. An in vitro CDK5 kinase assay as described for panel A was performed to determine CDK5 kinase activity. (E) SAOS-2 cells transfected as described for panel C were assayed for shape change (flat) or SA-β-Gal expression 10-days posttransfection, and the percentage of flat or SA-β-Gal-positive cells was determined.

FIG. 3.

CDK5 represses Rac1 activity to regulate SA-β-Gal expression. (A) SAOS-2 cells were transfected with pBabe-puromycin and pSVE-RB plus pCMV or activated RacV12 or dominant-negative RacN17. After 10 days of selection, the cells were stained for SA-β-Gal expression and photographed. (B) SAOS-2 cells were transfected with pSVE-RB or pSVE-RB and pEBG-RacV12 or pEBG-RacN17 together with either pBS/U6 CDK5 siRNA, pcDNA3-dnCDK5, or pcDNA3-CDK5. One set of transfectants was treated with roscovitine. After 10 days, the number of SA-β-Gal-positive cells was assayed and expressed as a percentage of the number of total cells.

FIG. 4.

Rac1 activity decreases in senescing cells. (A) Cells were collected at 2-, 4-, and 7-days posttransfection from SAOS-2 cells transfected with pSVE vector (V) or pSVE-RB or cotransfected with pBS/U6 CDK5 siRNA. GST-Pak pulldowns were separated by SDS-PAGE and immunoblotted for GTP-bound Rac1. As the control, an equal volume of lysates used for the pulldown was subjected to immunoblotting for total Rac1. (B) MDA-MB-468 cells were infected with Ad-RB for the indicated time in days. Lysates were incubated with GST-Pak fusion protein for Rac1 or Cdc42 activity or with GST-rhotekin fusion protein for RhoA activity. The amount of active GTP-bound Rac1, RhoA, or Cdc42 was analyzed by immunoblotting with anti-Rac1, -RhoA or -Cdc42 antibodies after GST precipitation. The total amount of Rac1, RhoA, or Cdc42 was determined by immunoblotting. (C) IMR90 fibroblasts were harvested at early passage (EP), middle passage (MP), or after undergoing senescence (S). The activation state of Rac1, RhoA, or Cdc42 was detected as described for panel B. (D) IMR90 cells infected with pBabe-puromycin or pBabe-RasV12 were untreated or treated with roscovitine. The Rac1 activity was determined by immunoblotting Rac1 in GST-Pak immunocomplexes. Puro, puromycin. (E) SAOS-2 cells were transfected with pSVE or pSVE-RB for the indicated number of days. IMR90 cells were passaged in culture, and cells were harvested for lysates at early passage, middle passage, or after undergoing senescence. To determine Pak1 kinase activity, cell lysates were immunoprecipitated with Pak1 antibody. The immunoprecipitates were then subjected to histone H4 kinase assays.

FIG. 5.

CDK5 and Rac1 regulate actin. (A) Indirect immunostaining and immunofluorescence following anti-actin antibody treatment in presenescent and senescent pSVE-RB-transfected SAOS-2 cells or passaged (Psg) IMR90 or WI38 cells untreated or treated with roscovitine (Rosc.) as described in the legend to Fig. 1. (B) Indirect immunostaining for actin in SAOS-2 cells transfected with pBabe-puro, pSVE-RB, and RacV12 or RacN17 following 2 and 7 days of selection.