Regulation of the cell cycle by focal adhesion kinase - PubMed (original) (raw)
Regulation of the cell cycle by focal adhesion kinase
J H Zhao et al. J Cell Biol. 1998.
Abstract
In this report, we have analyzed the potential role and mechanisms of integrin signaling through FAK in cell cycle regulation by using tetracycline-regulated expression of exogenous FAK and mutants. We have found that overexpression of wild-type FAK accelerated G1 to S phase transition. Conversely, overexpression of a dominant-negative FAK mutant DeltaC14 inhibited cell cycle progression at G1 phase and this inhibition required the Y397 in DeltaC14. Biochemical analyses indicated that FAK mutant DeltaC14 was mislocalized and functioned as a dominant-negative mutant by competing with endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decreases of Erk activation in cell adhesion. Consistent with this, we also observed inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKDeltaC14, in transient transfection assays using primary human foreskin fibroblasts. Finally, we also found that DeltaC14 blocked cyclin D1 upregulation and induced p21 expression, while wild-type FAK increased cyclin D1 expression and decreased p21 expression. Taken together, these results have identified FAK and its associated signaling pathways as a mediator of the cell cycle regulation by integrins.
Figures
Figure 1
Inhibition of new DNA synthesis by Y397F and ΔC14. (A) HFF cells were transiently transfected with expression vectors encoding WT FAK, ΔC14, or Y397F, as indicated. The control cells (C) were transfected by pKH3 vector alone. They were then analyzed for BrdU incorporation as described in Materials and Methods. The percentage of BrdU (+)/positively transfected cells was determined by analyzing 40–50 positively transfected cells for each transfection in multiple fields. The results show mean + SE for at least three independent experiments. *P = 0.0041 and 0.036 in comparison to value from control cells for ΔC14 and Y397F, respectively. **P = 0.014 in comparison to value from control cells. (B) Similar experiments were performed with transiently transfected NIH3T3 cells. The results show mean + SE for at least three independent experiments. *P = 0.046 in comparison to value from control cells.
Figure 2
Induction of ΔC14 expression by removal of tetracycline. ΔC14 cells were incubated in media in the presence (uninduced, U) or absence (induced, I) of 0.4 μg/ml tetracycline. At the indicated times, lysates were prepared and analyzed by Western blot with α-HA or α-FAK.
Figure 3
Inhibition of new DNA synthesis by ΔC14. (A) Quiescent ΔC14 cells were stimulated with 10% CS in the presence (uninduced, U) or absence (induced, I) of 0.4 μg/ml tetracycline. 16 hr after stimulation, the cells were processed for immunofluorescent staining with α-BrdU and Hoechst dye staining, as described in Materials and Methods. (B) Similar experiments were performed for Mock cells and two clones of ΔC14 cells, as described in A. The percentage of BrdU (+) cells at 16, 24, and 36 h after stimulation was determined by analyzing ∼500 cells in multiple fields. (C) The percentage of BrdU (+) Mock (▵) and ΔC14 (○) cells at 16 h after serum stimulation in media containing indicated concentrations of tetracycline. The inset shows the induced expression of ΔC14 at various tetracycline concentrations, as detected by α-HA Western blot.
Figure 4
Effects on cell cycle progression by overexpression of FAK and mutants. (A) α-HA or α-FAK Western blot of whole cell lysates prepared from cells expressing the indicated proteins under uninduced (lanes U) and induced (lanes I) conditions. (B) α-FAK immunofluorescence (a, d, g), α-BrdU immunofluorescence (b, e, h) and Hoechst dye staining (c, f, i) of cells expressing ΔC14 (a–c), ΔC14F (d–f), and WT (g–i). (C) The percentage of BrdU (+) cells expressing the indicated proteins at 16 h after serum stimulation under uninduced (open bars) and induced (closed bars) conditions. The results show mean + SE for at least three independent experiments. *P = 0.0022 in comparison to value from uninduced cells.
Figure 5
WT FAK stimulation of cell cycle progression. The percentage of BrdU (+) Mock or WT FAK cells at 12 h after serum stimulation under uninduced (open bars) and induced (closed bars) conditions. The results show mean + SE for three independent experiments. *P = 0.0059 in comparison to value from uninduced cells.
Figure 6
Cell cycle distribution of cells expressing exogenous FAK and mutants. (A) Quiescent (0 h) cells were stimulated with 10% CS in the presence (uninduced, U) or absence (induced, I) of 0.4 μg/ml tetracycline for 16 h. The cells were then analyzed for DNA content by staining with propidium iodide followed by FACS®, as described in Materials and Methods. (B) The decrease in the proportion of cell in G1 at 16 h from that at 0 h was determined for cells expressing the indicated proteins under uninduced (open bars) and induced (closed bars) conditions. The results show mean + SE for at least three independent experiments. *P = 0.0011 and **P = 0.013 in comparison to value from uninduced cells.
Figure 7
Subcellular localization of ΔC14 and WT FAK. α-HA (a, b) and α-integrin α5 (c, d) immunofluorescent staining of cells expressing ΔC14 (a, c) and WT (b, d). Examples of focal contacts are indicated by arrowheads.
Figure 8
Biochemical analysis of FAK and mutants. Whole cell lysates were prepared from cells that had been induced to express the indicated proteins. They were immunoprecipitated by α-HA (A–D), α-Src (E, G) or α-Fyn (F, H) followed by Western blot with α-FAK (A), PY20 (B), or α-HA (E, F), or in vitro kinase assay in the presence of E4Y1 (D) or enolase (G, H), or nothing (C). The results show mean + SE of relative kinase activities (normalized to expression levels in each experiment) normalized to WT FAK from at least three independent experiments (D).
Figure 9
Effects of ΔC14 on Erk activation and Shc phosphorylation. Quiescent Mock or ΔC14 cells under uninduced (U) or induced (I) conditions were detached by trypsin/EDTA (A–D) or EDTA alone (E, F) and either maintained in suspension (Sus) or replated on fibronectin (FN), as described in Materials and Methods. Whole cell lysates were western blotted with α-HA (A) or α-Erk (C), or immunoprecipitated by α-phospho-Erk followed by in vitro kinase assay with MBP as substrate (B). (D) The average and standard deviation of relative Erk activites were obtained from three independent experiments. The relative activities were normalized to uninduced cells plated on FN. Alternatively, the lysates were immunoprecipitated by α-Shc followed by Western blotting with anti-PY (E) or anti-Shc (F). Lysates from PDGF-stimulated cells were also used as a positive control in E and F.
Figure 10
Effects of FAK constructs on BrdU incorporation and Erk activation in HFF cells. (A) HFF cells were transiently transfected with expression vectors encoding FRNK, FRNKΔC14, or pKH3 vector alone (Control). They were then analyzed for BrdU incorporation as described in Materials and Methods. The percentage of BrdU (+)/positively transfected cells was determined by analyzing 40–50 positively transfected cells for each transfection in multiple fields. The results show mean + SE for at least three independent experiments. *P = 0.001 in comparison to value from control cells. (B–E) HFF cells were transiently transfected with expression vectors encoding FRNK, FRNKΔC14, Y397F (i.e., F397), or pKH3 vector alone (Mock). They were serum starved, detached by trypsinization and then either maintained in suspension (Sus) or replated on fibronectin (FN), as described in Materials and Methods. Whole cell lysates were Western blotted with α-HA (B) or α-His (D), or immunoprecipitated by α-His followed by in vitro kinase assay with MBP as substrate (C). (E) The average and standard deviation of relative Erk activites were obtained from four independent experiments. The relative activities were normalized to MOCK cells plated on FN. *P = 0.015 and 0.052 in comparison to value from MOCK cells on FN for cells transfected with FRNK and Y397F on FN, respectively.
Figure 11
Regulation of the expression levels of components of G1/S cyclin-Cdk complexes by exogenous FAK and mutants. (A) α-p21, α-cyclin D1, α-Cdk2, α-cyclin E, α-cyclin A, or α-p27 Western blot of whole cell lysates prepared from cells expressing the indicated proteins under uninduced (lanes U) and induced (lanes I) conditions. (B) Serum-starved cells expressing ΔC14 were incubated in media containing 10% CS in the presence (U) or absence (I) of tetracycline. At the indicated times, lysates were prepared and analyzed by Western blot with α-p21, α-cyclin D1, α-Cdk2, α-cyclin E, or α-cyclin A.
References
- Cary L, Chang J, Guan J-L. Stimulation of cell migration by overexpression of focal adhesion kinase and its assocation with Src and Fyn. J Cell Sci. 1996;109:1787–1794. - PubMed
- Chan P-Y, Kanner SB, Whitney G, Aruffo A. A transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK . J Biol Chem. 1994;269:20567–20574. - PubMed
- Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death. Science. 1997;276:1425–1428. - PubMed
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