Limits...
GIMAP5 Deficiency Is Associated with Increased AKT Activity in T Lymphocytes.

Chen XL, Serrano D, Mayhue M, Hoebe K, Ilangumaran S, Ramanathan S - PLoS ONE (2015)

Bottom Line: The underlying mechanism responsible for the disruption of quiescence in Gimap5 deficient T cells remains largely unknown.In this study, we show that loss of functional Gimap5 results in increased basal activation of mammalian target of rapamycin (mTOR), independent of protein phosphatase 2A (PP2A) or AMP-activated protein kinase (AMPK).Our results suggest that the constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway may be one of the consequences of the absence of functional GIMAP5.

View Article: PubMed Central - PubMed

Affiliation: Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, J1H 5N4, Québec, Canada.

ABSTRACT
Long-term survival of T lymphocytes in quiescent state is essential to maintain their cell numbers in secondary lymphoid organs. In mice and in rats, the loss of functional GTPase of the immune associated nucleotide binding protein 5 (GIMAP5) causes peripheral T lymphopenia due to spontaneous death of T cells. The underlying mechanism responsible for the disruption of quiescence in Gimap5 deficient T cells remains largely unknown. In this study, we show that loss of functional Gimap5 results in increased basal activation of mammalian target of rapamycin (mTOR), independent of protein phosphatase 2A (PP2A) or AMP-activated protein kinase (AMPK). Our results suggest that the constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway may be one of the consequences of the absence of functional GIMAP5.

No MeSH data available.


Related in: MedlinePlus

Gimap5 mutation results in constitutive activation of AKT.(A and C) CD4+ T cells from control and Gimap5 deficient mice (A) and rats (C) were stimulated with 5 μg/mL anti-CD3 or anti-CD3/CD28 for the indicated duration. Lysates were analyzed by Western blot with the indicated antibodies. (B and E) CD4+ T cells from the OT-II TCR-transgenic control and Gimap5sph/sph mice were simulated with OVA peptide presented by APC in the absence (B) or presence of rapamycin (E). Cell lysates were probed with specific antibodies. (D) CD4+ T cells from control and Gimap5 deficient mice were treated with 200 nM rapamycin for 30min followed by TCR crosslinking at different time points. Cell lysates were analyzed by Western blot using indicated antibodies. (A-E) Representative data from 3 independent experiments are shown. Histograms show densitometric data from 3 experiments. * p<0.05 control vs mutant cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4595448&req=5

pone.0139019.g005: Gimap5 mutation results in constitutive activation of AKT.(A and C) CD4+ T cells from control and Gimap5 deficient mice (A) and rats (C) were stimulated with 5 μg/mL anti-CD3 or anti-CD3/CD28 for the indicated duration. Lysates were analyzed by Western blot with the indicated antibodies. (B and E) CD4+ T cells from the OT-II TCR-transgenic control and Gimap5sph/sph mice were simulated with OVA peptide presented by APC in the absence (B) or presence of rapamycin (E). Cell lysates were probed with specific antibodies. (D) CD4+ T cells from control and Gimap5 deficient mice were treated with 200 nM rapamycin for 30min followed by TCR crosslinking at different time points. Cell lysates were analyzed by Western blot using indicated antibodies. (A-E) Representative data from 3 independent experiments are shown. Histograms show densitometric data from 3 experiments. * p<0.05 control vs mutant cells.

Mentions: Cross-linking of TCR/CD3 complex results in the recruitment of PI3K to the CD3 complex, to generate phosphatidylinositol (3,4,5)-triphosphate (PIP3) at the plasma membrane [44]. Phosphoinositide-dependent kinase–1 (PDK1) and AKT are recruited by PIP3 through their pleckstrin homology (PH) domains leading to the phosphorylation of AKT at Thr308, followed by the phosphorylation of Ser473 by the mTORC2 complex [45]. Activated AKT phosphorylates the TSC1/TSC2 complex, resulting in the inhibition of its GTPase-activating protein (GAP) activity and releasing the RHEB GTPase to activate mTORC1 [46]. We assessed whether the increased phosphorylation of AKT following cross-linking of TCR/CD3 complex and CD28 could explain the increased mTOR activity. In CD4+ T cells from control mice, we observed that the pAKT peaked between 2 and 10 minutes after CD3/CD28 cross-linking, and returned to the basal level by 30 minutes (Fig 5A). In contrast, phosphorylation of AKT was detected even in the absence of stimulation in CD4+ T cells from Gimap5sph/sph mice (Fig 5A). Crosslinking CD3/CD28 also increased the pAKT that returned to basal level within 30 minutes in Gimap5sph/sph CD4+ T cells. But it was still higher than that observed in the control cells. Following activation of control OTII cells with peptide/APC, phosphorylation of AKT was evident at 5 minutes and was strong even at 180 minutes (Fig 5B). However, in Gimap5 mutant OTII cells, pAKT was detected even in the absence of stimulation and showed minimal increase over time (Fig 5B). Increased basal AKT phosphorylation was also observed in CD4+ T cells from Gimap5lyp/lyp rats (Fig 5C). These results strongly indicated that loss of functional GIMAP5 protein results in elevated basal phosphorylation of AKT in T cells. A previous report has shown that the expression of FOXO1, the down stream target of AKT, is reduced in CD4+ T cells from 4-week old Gimap5sph/sph mice [31]. We observed that the phosphorylation of FOXO1 is comparable or lower in Gimap5 mutant CD4+ T cells even in the presence of hyperactivated Akt (Figs 5 and 6). Furthermore, to confirm that the activation of AKT was not an artifact, we determined the phosphorylation status of AKT and FOXO1 in the cells where mTOR was inhibited. While phosphorylation downstream of mTORC1 was inhibited following pre-treatment with rapamycin (Fig 1D, 1E and 1F), phosphorylation of AKT and FOXO1 remained unchanged (Fig 5D and 5E). These results indicate that the constitutive activation of AKT likely underlies the activation of the mTORC1 pathway in T cells lacking functional GIMAP5 protein.


GIMAP5 Deficiency Is Associated with Increased AKT Activity in T Lymphocytes.

Chen XL, Serrano D, Mayhue M, Hoebe K, Ilangumaran S, Ramanathan S - PLoS ONE (2015)

Gimap5 mutation results in constitutive activation of AKT.(A and C) CD4+ T cells from control and Gimap5 deficient mice (A) and rats (C) were stimulated with 5 μg/mL anti-CD3 or anti-CD3/CD28 for the indicated duration. Lysates were analyzed by Western blot with the indicated antibodies. (B and E) CD4+ T cells from the OT-II TCR-transgenic control and Gimap5sph/sph mice were simulated with OVA peptide presented by APC in the absence (B) or presence of rapamycin (E). Cell lysates were probed with specific antibodies. (D) CD4+ T cells from control and Gimap5 deficient mice were treated with 200 nM rapamycin for 30min followed by TCR crosslinking at different time points. Cell lysates were analyzed by Western blot using indicated antibodies. (A-E) Representative data from 3 independent experiments are shown. Histograms show densitometric data from 3 experiments. * p<0.05 control vs mutant cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4595448&req=5

pone.0139019.g005: Gimap5 mutation results in constitutive activation of AKT.(A and C) CD4+ T cells from control and Gimap5 deficient mice (A) and rats (C) were stimulated with 5 μg/mL anti-CD3 or anti-CD3/CD28 for the indicated duration. Lysates were analyzed by Western blot with the indicated antibodies. (B and E) CD4+ T cells from the OT-II TCR-transgenic control and Gimap5sph/sph mice were simulated with OVA peptide presented by APC in the absence (B) or presence of rapamycin (E). Cell lysates were probed with specific antibodies. (D) CD4+ T cells from control and Gimap5 deficient mice were treated with 200 nM rapamycin for 30min followed by TCR crosslinking at different time points. Cell lysates were analyzed by Western blot using indicated antibodies. (A-E) Representative data from 3 independent experiments are shown. Histograms show densitometric data from 3 experiments. * p<0.05 control vs mutant cells.
Mentions: Cross-linking of TCR/CD3 complex results in the recruitment of PI3K to the CD3 complex, to generate phosphatidylinositol (3,4,5)-triphosphate (PIP3) at the plasma membrane [44]. Phosphoinositide-dependent kinase–1 (PDK1) and AKT are recruited by PIP3 through their pleckstrin homology (PH) domains leading to the phosphorylation of AKT at Thr308, followed by the phosphorylation of Ser473 by the mTORC2 complex [45]. Activated AKT phosphorylates the TSC1/TSC2 complex, resulting in the inhibition of its GTPase-activating protein (GAP) activity and releasing the RHEB GTPase to activate mTORC1 [46]. We assessed whether the increased phosphorylation of AKT following cross-linking of TCR/CD3 complex and CD28 could explain the increased mTOR activity. In CD4+ T cells from control mice, we observed that the pAKT peaked between 2 and 10 minutes after CD3/CD28 cross-linking, and returned to the basal level by 30 minutes (Fig 5A). In contrast, phosphorylation of AKT was detected even in the absence of stimulation in CD4+ T cells from Gimap5sph/sph mice (Fig 5A). Crosslinking CD3/CD28 also increased the pAKT that returned to basal level within 30 minutes in Gimap5sph/sph CD4+ T cells. But it was still higher than that observed in the control cells. Following activation of control OTII cells with peptide/APC, phosphorylation of AKT was evident at 5 minutes and was strong even at 180 minutes (Fig 5B). However, in Gimap5 mutant OTII cells, pAKT was detected even in the absence of stimulation and showed minimal increase over time (Fig 5B). Increased basal AKT phosphorylation was also observed in CD4+ T cells from Gimap5lyp/lyp rats (Fig 5C). These results strongly indicated that loss of functional GIMAP5 protein results in elevated basal phosphorylation of AKT in T cells. A previous report has shown that the expression of FOXO1, the down stream target of AKT, is reduced in CD4+ T cells from 4-week old Gimap5sph/sph mice [31]. We observed that the phosphorylation of FOXO1 is comparable or lower in Gimap5 mutant CD4+ T cells even in the presence of hyperactivated Akt (Figs 5 and 6). Furthermore, to confirm that the activation of AKT was not an artifact, we determined the phosphorylation status of AKT and FOXO1 in the cells where mTOR was inhibited. While phosphorylation downstream of mTORC1 was inhibited following pre-treatment with rapamycin (Fig 1D, 1E and 1F), phosphorylation of AKT and FOXO1 remained unchanged (Fig 5D and 5E). These results indicate that the constitutive activation of AKT likely underlies the activation of the mTORC1 pathway in T cells lacking functional GIMAP5 protein.

Bottom Line: The underlying mechanism responsible for the disruption of quiescence in Gimap5 deficient T cells remains largely unknown.In this study, we show that loss of functional Gimap5 results in increased basal activation of mammalian target of rapamycin (mTOR), independent of protein phosphatase 2A (PP2A) or AMP-activated protein kinase (AMPK).Our results suggest that the constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway may be one of the consequences of the absence of functional GIMAP5.

View Article: PubMed Central - PubMed

Affiliation: Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, J1H 5N4, Québec, Canada.

ABSTRACT
Long-term survival of T lymphocytes in quiescent state is essential to maintain their cell numbers in secondary lymphoid organs. In mice and in rats, the loss of functional GTPase of the immune associated nucleotide binding protein 5 (GIMAP5) causes peripheral T lymphopenia due to spontaneous death of T cells. The underlying mechanism responsible for the disruption of quiescence in Gimap5 deficient T cells remains largely unknown. In this study, we show that loss of functional Gimap5 results in increased basal activation of mammalian target of rapamycin (mTOR), independent of protein phosphatase 2A (PP2A) or AMP-activated protein kinase (AMPK). Our results suggest that the constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway may be one of the consequences of the absence of functional GIMAP5.

No MeSH data available.


Related in: MedlinePlus