Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2 - PubMed (original) (raw)

Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2

T Soucek et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of aberrant growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The tuberous sclerosis complex gene-2 (TSC2) on chromosome 16 encodes the tumor suppressor protein tuberin. We have shown earlier that loss of TSC2 is sufficient to induce quiescent cells to enter the cell cycle. Here we show that TSC2-negative fibroblasts exhibit a shortened G1 phase. Although the expression of cyclin E, cyclin A, p21, or Cdc25A is unaffected, TSC2-negative cells express much lower amounts of the cyclin-dependent kinase (CDK) inhibitor p27 because of decreased protein stability. In TSC2 mutant cells the amount of p27 bound to CDK2 is diminished, accompanied with elevated kinase activity. Ectopic expression studies revealed that the aforementioned effects can be reverted by transfecting TSC2 in TSC2-negative cells. High ectopic levels of p27 have cell cycle inhibitory effects in TSC2-positive cells but not in TSC2-negative counterparts, although the latter still depend on CDK2 activity. Loss of TSC2 induces soft agar growth of fibroblasts, a process that cannot be inhibited by high levels of p27. Both phenotypes of TSC2-negative cells, their resistance to the activity of ectopic p27, and the instability of endogenous p27, could be explained by our observation that the nucleoprotein p27 is mislocated into the cytoplasm upon loss of TSC2. These findings provide insights into the molecular mechanism of how loss of TSC2 induces cell cycle entry and allow a better understanding of its tumor suppressor function.

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Figures

Figure 1

Figure 1

Cell cycle analysis of _TSC2_-positive and _TSC2_-negative rat embryonic fibroblasts. (A) Flow cytometry analysis of DNA content of logarithmically growing _TSC2_-negative rat embryonic fibroblasts (EEF8) and _TSC2_-positive counterparts (EEF4). (B) Doubling times of the cells analyzed in A were determined by cell counting, and the duration of the distinct cell cycle phases was calculated by relating doubling times and percentage of cell cycle phase distributions. The values of three independent experiments are presented. (C) Western blot analyses of tuberin, cyclin E, cyclin A, p27, p21, and Cdc25A expression in EEF4 and EEF8 cells. (D) Protein extracts of EEF4 and EEF8 cells were assayed for CDK4-, cyclin E- and cyclin A-associated kinase activities by using glutathione _S_-transferase-retinoblastoma protein or histone H1 as substrate, respectively. (E) Immunoprecipitates (IP) performed with antituberin or anti-CDK2 antibodies were investigated for p27, cyclin E, cyclin A, tuberin, and CDK2 protein by Western blot analyses. Immunoprecipitates performed with anti-CDK4 antibody were analyzed for p27 protein by Western blotting.

Figure 2

Figure 2

p27 protein levels depend on the TSC2 status. (A) _TSC2_-negative cells (EEF8) were transfected with the empty control vector or with an expression vector containing TSC2 cDNA. After 14 days of antibiotic selection, cells were analyzed for DNA distribution on the flow cytometer and for tuberin and p27 protein expression by Western blotting. Early passages of primary embryonic fibroblasts derived from _TSC2_-positive (EEF-TSC2+/+) and tuberin-negative (EEF-_TSC2_−/−) Eker rats were analyzed for DNA distribution and tuberin and p27 expression. (B) Rat1 immortalized fibroblasts and SKNSH human neuroblastoma cells were transfected with the empty control vector or with an expression vector containing TSC2 cDNA. After 14 days of antibiotic selection cells were analyzed for DNA distribution and for tuberin and p27 protein expression. Rat1 cells also were treated with TSC2 antisense oligonucleotides for 24 hr and analyzed as described above.

Figure 3

Figure 3

TSC2 affects the regulation of p27 stability. (A) Logarithmically growing EEF4 and EEF8 cells were separated according to the different cell cycle phases by centrifugal elutriation. The obtained cell fractions were cytofluorometrically analyzed for DNA distribution after staining DNA with propidium iodide (Upper). Protein extracts of the obtained fractions were analyzed for p27 protein expression by Western blot analysis. (B) Logarithmically growing EEF4 and EEF8 cells were incubated in 100 μg/ml of cycloheximide for the indicated time periods. p27 protein levels were compared by Western blot analysis. (C) Logarithmically growing EEF4 and EEF8 cells were incubated for 4 hr with [35S]methionine and then chased in medium containing unlabeled methionine for the indicated times. Radiolabeled p27 was immunoprecipitated, separated by gel electrophoresis, and detected by autoradiography. To be able to detect p27 degradation in EEF8 cells on the same Western blot with EEF4 cells it was necessary to perform long exposures (compare with Fig. 1_C_). The presented blots are overexposed in respect to p27 expression in EEF4 cells. p27 protein started to decrease after 3-hr cycloheximide treatment of EEF4 cells (data not shown).

Figure 4

Figure 4

High ectopic levels of p27 lose their cell cycle inhibitory effects in _TSC2_-negative cells. (A) Logarithmically growing EEF4 cells (_TSC2_-positive) and EEF8 cells (_TSC2_-negative) were cotransfected either with the empty control vector and an expression vector containing GFP or the expression plasmid containing p27 cDNA and GFP. GFP-positive cells were cytofluorometrically analyzed for DNA content. The cells were further analyzed for p27 protein expression by Western blotting. The transfected p27 protein migrates faster than the endogenous p27 protein. (B) EEF8 cells were transfected with empty control vectors, with the expression vector containing the p27VPKK mutant or a dominant-negative CDK2 mutant together with a GFP-expressing plasmid. GFP-positive cells were analyzed for DNA distribution on the flow cytometer, and cell extracts were analyzed for p27 expression by Western blot detection. (C) Rat1 cells expressing high levels of Myc (positive control), primary fibroblasts (fibros, negative control), EEF4 cells, and EEF8 cells, both transfected either with the empty expression vector or with p27 were analyzed for soft agar growth, and colonies were scored after 1 week. The experiment was repeated three times, and the obtained data are presented in percentage relative to the highest value (set 100%). EEF4 and EEF8 cell extracts were analyzed for p27 expression by Western blotting.

Figure 5

Figure 5

Loss of TSC2 affects p27 localization. (A) Protein extracts of EEF8 cells were assayed for CDK2-associated kinase activity by using histone H1 as substrate. CDK2 activity in EEF8 extracts could be inhibited by the addition of 20nM recombinant p27 protein directly to the kinase assay’s reaction. (B) Immunocytochemical detection of the subcellular localization of p27 in EEF4 and in EEF8 cells. The signals in EEF8 cells have been enhanced to visualize p27 localization. Nuclei were identified by 4′,6-diamidino-2-phenylindole (DAPI) staining.

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