In vivo analysis of protein kinase B (PKB)/Akt regulation in DNA-PKcs-null mice reveals a role for PKB/Akt in DNA damage response and tumorigenesis

Abstract

Full activation of protein kinase B (PKB/Akt) requires phosphorylation on Thr-308 and Ser-473. It is well established that Thr-308 is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1). Ser-473 phosphorylation is mediated by both mammalian target of rapamycin-rictor complex (mTORC2) and DNA-dependent protein kinase (DNA-PK) depending on type of stimulus. However, the physiological role of DNA-PK in the regulation of PKB phosphorylation remains to be established. To address this, we analyzed basal, insulin-induced, and DNA damage-induced PKB Ser-473 phosphorylation in DNA-PK catalytic subunit-null DNA-PKcs(-/-) mice. Our results revealed that DNA-PK is required for DNA damage-induced phosphorylation but dispensable for insulin- and growth factor-induced PKB Ser-473 phosphorylation. Moreover, DNA-PKcs(-/-) mice showed a tissue-specific increase in basal PKB phosphorylation. In particular, persistent PKB hyperactivity in the thymus apparently contributed to spontaneous lymphomagenesis in DNA-PKcs(-/-) mice. Significantly, these tumors could be prevented by deletion of PKBalpha. These findings reveal stimulus-specific regulation of PKB activation by specific upstream kinases and provide genetic evidence of PKB deregulation in DNA-PKcs(-/-) mice.

FIGURE 1.

DNA-PK is dispensable for PKB Ser-473 phosphorylation upon insulin and growth factor stimulation. After overnight fasting and insulin (ins) stimulation (1 unit/kg body weight) for 20 min, tissues from 3-month-old (WT) and DNA-PKcs^-/- (KO) mice were analyzed for PKB Ser-473 phosphorylation via immunoblotting with phospho-specific antibodies. A, skeletal muscle; B, liver; C, heart; D, adipose tissue. Wild-type and DNA-PKcs^-/- MEFs (KO) were stimulated with IGF-1 (50 ng/ml) (E) or serum (10%) (F) for various times, and PKB Ser-473 phosphorylation was analyzed by immunoblotting.

FIGURE 2.

DNA-PK is dispensable for maintenance of glucose metabolism. For analysis of glucose metabolism, 3-month-old wild-type (WT) and DNA-PKcs^-/- (KO) mice after overnight fasting were treated with insulin (1 unit /kg body weight) for insulin tolerance tests (A) or glucose (2 g/kg body weight) for glucose tolerance tests (B). The graphs depict arithmetic means ± S.E. of blood glucose concentrations at the indicated time points following the treatments.

FIGURE 3.

DNA-PK is the physiological PKB Ser-473 kinase in the response to γ-irradiation. PKB and FoxO4 phosphorylation were analyzed by phospho-specific antibodies in wild-type (WT) and DNA-PKcs^-/- (KO) MEF lysates 30 min after the indicated doses ofγ-irradiation (A) or at indicated time points after 10 Gy irradiation (B). Right panels, quantification of fold increase in PKB Ser-473 phosphorylation after γ-irradiation determined by phospho-PKB (p-PKB)Ser-473/PKB ratio with respect to nonirradiated WT and KO controls. Data were tested for significance using one-way analysis of variance with the Holm-Sidak multiple comparisons procedure. ns, not significant. C, analysis of PKB and FoxO4 phosphorylation in WT and DNA-PKcs^-/- (KO) brain 30 min after 1 Gy total body γ-irradiation or sham irradiation. Right panel, quantification of fold increase in p-PKB Ser-473/actin ratio and phospho-FoxO4 (p-FoxO4) Ser-193/actin ratio after γ-irradiation with respect to sham-irradiated WT and KO controls.

FIGURE 4.

Analysis of basal PKB phosphorylation in DNA-PKcs^-/- tissues. Tissues were collected from 2-month-old random-fed WT and DNA-PKcs^-/- mice and analyzed by immunoblotting. A, top panel, PKB phosphorylation in WT and DNA-PKcs^-/- (KO) adipose tissue. Bottom panel, quantification of PKB Ser-473 phosphorylation determined by p-PKB Ser-473/PKB ratio relative to wild-type ratio. B, top panel, PKB phosphorylation in WT and DNA-PKcs^-/- (KO) brown fat. Bottom panel, quantification of PKB Ser-473 phosphorylation determined by p-PKB Ser-473/PKB ratio relative to wild-type ratio. C, top panel, PKB Ser-473, PKB Thr-308, GSK3β Ser-9, FoxO3a Thr-32, and S6K Thr-389 phosphorylation and total IRS-1 protein levels in WT and DNA-PKcs^-/- (KO) thymi. Bottom panel, quantification of PKB Ser-473 phosphorylation determined by p-PKB Ser-473/PKB ratio relative to wild-type ratio.

FIGURE 5.

Deletion of PKBα prevents spontaneous development of thymic tumors. A, incidence of thymic tumors from wild-type (WT), DNA-PKcs^-/- (KO) analyzed between 3 and 7 months age and DNA-PKcs^-/- PKBα^-/- double knock-out (DKO) mice analyzed between 2 and 15 months age. B, PKB Ser-473, FoxO4 Ser-193, and FoxO3a Thr-32 phosphorylation in WT thymus, DNA-PKcs^-/- tumor, and DNA-PKcs^-/-PKBα^-/- (DKO) thymi.

FIGURE 6.

Deletion of PKBα improves survival of DNA-PKcs^-/- mice. Kaplan-Meier survival curves for wild-type (WT), DNA-PKcs^-/-, PKBα^-/-, and DNA-PKcs^-/-PKBα^-/- double knock-out (DKO) mice analyzed by LogRank analysis and the Holm-Sidak multiple comparisons procedure. The survival of DNA-PKcs^-/-PKBα^-/-DKO mice significantly increased compared with DNA-PKcs^-/- mice (p < 0.001). Mice that survived longer than 2 months were included in the analysis. Numbers of mice analyzed are as follows: WT 52; DNA-PKcs^-/- 21; PKBα^-/- 26; and DNA-PKcs^-/-PKBα^-/- (DKO) 13.

FIGURE 7.

Deletion of PKBα prevents neoplastic expansion of DNA-PKcs^-/- thymocytes. A, histological analysis of thymi from wild-type (WT), DNA-PKcs^-/- (KO), and DNA-PKcs^-/-PKBα^-/- double knock-out (DKO) mice and thymic tumors from DNA-PKcs^-/- mice. Thymi were fixed with 4% paraformaldehyde, processed with paraffin, sectioned, and stained with hematoxylin-eosin (×400 magnification; scale bar indicates 50 μm). The insets show ×40 magnification. M, medulla; C, cortex. B, flow cytometric analysis of thymocytes from WT, DNA-PKcs^-/- (KO), and DNA-PKcs^-/-PKBα^-/- (DKO) mice for CD4 and CD8 markers.