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PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis.

Lee MS, Jeong MH, Lee HW, Han HJ, Ko A, Hewitt SM, Kim JH, Chun KH, Chung JY, Lee C, Cho H, Song J - Nat Commun (2015)

Bottom Line: The activity of the phosphatase and tensin homologue (PTEN) is known to be suppressed via post-translational modification.EGFR/PI3K/AKT-mediated ubiquitination and degradation of PTEN are dependent on the MKRN1 E3 ligase.Taken together, our results demonstrate that PI3K/AKT signals enforce positive-feedback regulation by suppressing PTEN function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.

ABSTRACT
The activity of the phosphatase and tensin homologue (PTEN) is known to be suppressed via post-translational modification. However, the mechanism and physiological significance by which post-translational modifications lead to PTEN suppression remain unclear. Here we demonstrate that PTEN destabilization is induced by EGFR- or oncogenic PI3K mutation-mediated AKT activation in cervical cancer. EGFR/PI3K/AKT-mediated ubiquitination and degradation of PTEN are dependent on the MKRN1 E3 ligase. These processes require the stabilization of MKRN1 via AKT-mediated phosphorylation. In cervical cancer patients with high levels of pAKT and MKRN1 expression, PTEN protein levels are low and correlate with a low 5-year survival rate. Taken together, our results demonstrate that PI3K/AKT signals enforce positive-feedback regulation by suppressing PTEN function.

No MeSH data available.


Related in: MedlinePlus

AKT phosphorylates serine 109 of MKRN1.(a,b) The interaction between ectopically expressed MKRN1 and AKT1 was demonstrated using a co-immunoprecipitation assay. (c) A GST pull-down assay revealed the direct interaction between MKRN1 and AKT1. GST-MKRN1 purified from bacteria and in vitro translated HA-AKT1 were incubated under cell-free conditions, and GST-MKRN1 was pulled down by glutathione Sepharose beads. (d) Endogenous MKRN1 was immunoprecipitated from ME-180 cells with or without EGF treatment (100 ng ml−1), and MKRN1-bound endogenous AKT1 was immunoblotted. (e) A consensus AKT phosphorylation site is present in MKRN1. (f) AKT directly phosphorylates MKRN1 WT but not the S109A mutant. An in vitro phosphorylation assay was performed using bacterially produced GST-AKT1 and FLAG-MKRN1 (WT or S109A), which was purified from FLAG-MKRN1-transfected HEK293T cells. Purified proteins were incubated with [γ-32P]ATP, and 32P incorporation was detected by autoradiography. (g) Constitutively active, but not inactive, AKT phosphorylates MKRN1 on serine 109. H1299 cells were co-transfected with FLAG-MKRN1 (WT or S109A) and HA-tagged Myr-AKT or K179M. Phosphorylation of ectopically expressed MKRN1 was detected by an in vivo phosphorylation assay (IP panel); WCL was also analysed. (h) AKT phosphorylates MKRN1 upon EGF treatment. After serum starvation, ME-180 cells were stimulated by EGF (100 ng ml−1) in the absence or presence of MG132 (10 μM) for 4 h and were examined in an in vivo phosphorylation assay. Endogenous phospho-MKRN1 was immunoprecipitated using an anti-phosphoserine antibody (IP panel), and WCL was immunoblotted. (i) EGF-induced MKRN1 phosphorylation is inhibited by AKT ablation. ME-180 cells were transduced with control siRNA or AKT1 siRNA (siAKT1) and subsequently serum starved, followed by treatment with EGF (100 ng ml−1). Cell extracts were analysed using an in vivo phosphorylation assay. Endogenous phospho-MKRN1 is indicated in the IP panel.
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f4: AKT phosphorylates serine 109 of MKRN1.(a,b) The interaction between ectopically expressed MKRN1 and AKT1 was demonstrated using a co-immunoprecipitation assay. (c) A GST pull-down assay revealed the direct interaction between MKRN1 and AKT1. GST-MKRN1 purified from bacteria and in vitro translated HA-AKT1 were incubated under cell-free conditions, and GST-MKRN1 was pulled down by glutathione Sepharose beads. (d) Endogenous MKRN1 was immunoprecipitated from ME-180 cells with or without EGF treatment (100 ng ml−1), and MKRN1-bound endogenous AKT1 was immunoblotted. (e) A consensus AKT phosphorylation site is present in MKRN1. (f) AKT directly phosphorylates MKRN1 WT but not the S109A mutant. An in vitro phosphorylation assay was performed using bacterially produced GST-AKT1 and FLAG-MKRN1 (WT or S109A), which was purified from FLAG-MKRN1-transfected HEK293T cells. Purified proteins were incubated with [γ-32P]ATP, and 32P incorporation was detected by autoradiography. (g) Constitutively active, but not inactive, AKT phosphorylates MKRN1 on serine 109. H1299 cells were co-transfected with FLAG-MKRN1 (WT or S109A) and HA-tagged Myr-AKT or K179M. Phosphorylation of ectopically expressed MKRN1 was detected by an in vivo phosphorylation assay (IP panel); WCL was also analysed. (h) AKT phosphorylates MKRN1 upon EGF treatment. After serum starvation, ME-180 cells were stimulated by EGF (100 ng ml−1) in the absence or presence of MG132 (10 μM) for 4 h and were examined in an in vivo phosphorylation assay. Endogenous phospho-MKRN1 was immunoprecipitated using an anti-phosphoserine antibody (IP panel), and WCL was immunoblotted. (i) EGF-induced MKRN1 phosphorylation is inhibited by AKT ablation. ME-180 cells were transduced with control siRNA or AKT1 siRNA (siAKT1) and subsequently serum starved, followed by treatment with EGF (100 ng ml−1). Cell extracts were analysed using an in vivo phosphorylation assay. Endogenous phospho-MKRN1 is indicated in the IP panel.

Mentions: Given that EGF stabilizes the MKRN1 protein, possibly via activation of the PI3K/AKT axis, we investigated whether MKRN1 is a substrate for AKT. Employing co-immunoprecipitation assays using endogenous, overexpressed or recombinant proteins, an interaction between AKT1 and MKRN1 was detected (Fig. 4a–d). A search for a potential MKRN1 phosphorylation site revealed a consensus AKT phosphorylation motif including serine 109 of MKRN1 (Fig. 4e). Although the motif (RXRXXXS), including serine 109 of MKRN1, is only a partial AKT consensus motif (RXRXXS/T), an AKT substrate containing an RXRXXXS motif has been reported. Furthermore, previous structural studies have indicated that the RXRXXXS motif is a highly plausible AKT-targeting sequence3031. To determine whether AKT targets the serine 109 site of MKRN1 for phosphorylation, we substituted alanine at this site. A subsequent in vitro phosphorylation assay using32 P-labelled adenosine triphosphate (ATP) and purified GST-AKT under cell-free conditions revealed that wild-type (WT) MKRN1 was phosphorylated, but not an S109A mutant (Fig. 4f). In accordance with this finding, only Myr-AKT (the active form of AKT) but not K179M (a kinase-dead form of AKT) was able to induce phosphorylation of WT MKRN1 but not the S109A mutant (Fig. 4g). Upon EGF treatment, an anti-phosphoserine antibody specifically detected pMKRN1, and MKRN1 was stabilized (Fig. 4h). Depletion or inhibition of AKT by short interfering RNA (siRNA) suppressed EGF-driven MKRN1 phosphorylation (Fig. 4i). Finally, phospho-AKT substrate antibody detecting phosphorylation on the overexpressed MKRN1 but not on the S109A mutant by Myr-AKT1 further suggests that the serine 109 site of MKRN1 is specifically phosphorylated by AKT (Supplementary Fig. 4). These data suggest that MKRN1 might be post-translationally stabilized via AKT-mediated phosphorylation.


PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis.

Lee MS, Jeong MH, Lee HW, Han HJ, Ko A, Hewitt SM, Kim JH, Chun KH, Chung JY, Lee C, Cho H, Song J - Nat Commun (2015)

AKT phosphorylates serine 109 of MKRN1.(a,b) The interaction between ectopically expressed MKRN1 and AKT1 was demonstrated using a co-immunoprecipitation assay. (c) A GST pull-down assay revealed the direct interaction between MKRN1 and AKT1. GST-MKRN1 purified from bacteria and in vitro translated HA-AKT1 were incubated under cell-free conditions, and GST-MKRN1 was pulled down by glutathione Sepharose beads. (d) Endogenous MKRN1 was immunoprecipitated from ME-180 cells with or without EGF treatment (100 ng ml−1), and MKRN1-bound endogenous AKT1 was immunoblotted. (e) A consensus AKT phosphorylation site is present in MKRN1. (f) AKT directly phosphorylates MKRN1 WT but not the S109A mutant. An in vitro phosphorylation assay was performed using bacterially produced GST-AKT1 and FLAG-MKRN1 (WT or S109A), which was purified from FLAG-MKRN1-transfected HEK293T cells. Purified proteins were incubated with [γ-32P]ATP, and 32P incorporation was detected by autoradiography. (g) Constitutively active, but not inactive, AKT phosphorylates MKRN1 on serine 109. H1299 cells were co-transfected with FLAG-MKRN1 (WT or S109A) and HA-tagged Myr-AKT or K179M. Phosphorylation of ectopically expressed MKRN1 was detected by an in vivo phosphorylation assay (IP panel); WCL was also analysed. (h) AKT phosphorylates MKRN1 upon EGF treatment. After serum starvation, ME-180 cells were stimulated by EGF (100 ng ml−1) in the absence or presence of MG132 (10 μM) for 4 h and were examined in an in vivo phosphorylation assay. Endogenous phospho-MKRN1 was immunoprecipitated using an anti-phosphoserine antibody (IP panel), and WCL was immunoblotted. (i) EGF-induced MKRN1 phosphorylation is inhibited by AKT ablation. ME-180 cells were transduced with control siRNA or AKT1 siRNA (siAKT1) and subsequently serum starved, followed by treatment with EGF (100 ng ml−1). Cell extracts were analysed using an in vivo phosphorylation assay. Endogenous phospho-MKRN1 is indicated in the IP panel.
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Related In: Results  -  Collection

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f4: AKT phosphorylates serine 109 of MKRN1.(a,b) The interaction between ectopically expressed MKRN1 and AKT1 was demonstrated using a co-immunoprecipitation assay. (c) A GST pull-down assay revealed the direct interaction between MKRN1 and AKT1. GST-MKRN1 purified from bacteria and in vitro translated HA-AKT1 were incubated under cell-free conditions, and GST-MKRN1 was pulled down by glutathione Sepharose beads. (d) Endogenous MKRN1 was immunoprecipitated from ME-180 cells with or without EGF treatment (100 ng ml−1), and MKRN1-bound endogenous AKT1 was immunoblotted. (e) A consensus AKT phosphorylation site is present in MKRN1. (f) AKT directly phosphorylates MKRN1 WT but not the S109A mutant. An in vitro phosphorylation assay was performed using bacterially produced GST-AKT1 and FLAG-MKRN1 (WT or S109A), which was purified from FLAG-MKRN1-transfected HEK293T cells. Purified proteins were incubated with [γ-32P]ATP, and 32P incorporation was detected by autoradiography. (g) Constitutively active, but not inactive, AKT phosphorylates MKRN1 on serine 109. H1299 cells were co-transfected with FLAG-MKRN1 (WT or S109A) and HA-tagged Myr-AKT or K179M. Phosphorylation of ectopically expressed MKRN1 was detected by an in vivo phosphorylation assay (IP panel); WCL was also analysed. (h) AKT phosphorylates MKRN1 upon EGF treatment. After serum starvation, ME-180 cells were stimulated by EGF (100 ng ml−1) in the absence or presence of MG132 (10 μM) for 4 h and were examined in an in vivo phosphorylation assay. Endogenous phospho-MKRN1 was immunoprecipitated using an anti-phosphoserine antibody (IP panel), and WCL was immunoblotted. (i) EGF-induced MKRN1 phosphorylation is inhibited by AKT ablation. ME-180 cells were transduced with control siRNA or AKT1 siRNA (siAKT1) and subsequently serum starved, followed by treatment with EGF (100 ng ml−1). Cell extracts were analysed using an in vivo phosphorylation assay. Endogenous phospho-MKRN1 is indicated in the IP panel.
Mentions: Given that EGF stabilizes the MKRN1 protein, possibly via activation of the PI3K/AKT axis, we investigated whether MKRN1 is a substrate for AKT. Employing co-immunoprecipitation assays using endogenous, overexpressed or recombinant proteins, an interaction between AKT1 and MKRN1 was detected (Fig. 4a–d). A search for a potential MKRN1 phosphorylation site revealed a consensus AKT phosphorylation motif including serine 109 of MKRN1 (Fig. 4e). Although the motif (RXRXXXS), including serine 109 of MKRN1, is only a partial AKT consensus motif (RXRXXS/T), an AKT substrate containing an RXRXXXS motif has been reported. Furthermore, previous structural studies have indicated that the RXRXXXS motif is a highly plausible AKT-targeting sequence3031. To determine whether AKT targets the serine 109 site of MKRN1 for phosphorylation, we substituted alanine at this site. A subsequent in vitro phosphorylation assay using32 P-labelled adenosine triphosphate (ATP) and purified GST-AKT under cell-free conditions revealed that wild-type (WT) MKRN1 was phosphorylated, but not an S109A mutant (Fig. 4f). In accordance with this finding, only Myr-AKT (the active form of AKT) but not K179M (a kinase-dead form of AKT) was able to induce phosphorylation of WT MKRN1 but not the S109A mutant (Fig. 4g). Upon EGF treatment, an anti-phosphoserine antibody specifically detected pMKRN1, and MKRN1 was stabilized (Fig. 4h). Depletion or inhibition of AKT by short interfering RNA (siRNA) suppressed EGF-driven MKRN1 phosphorylation (Fig. 4i). Finally, phospho-AKT substrate antibody detecting phosphorylation on the overexpressed MKRN1 but not on the S109A mutant by Myr-AKT1 further suggests that the serine 109 site of MKRN1 is specifically phosphorylated by AKT (Supplementary Fig. 4). These data suggest that MKRN1 might be post-translationally stabilized via AKT-mediated phosphorylation.

Bottom Line: The activity of the phosphatase and tensin homologue (PTEN) is known to be suppressed via post-translational modification.EGFR/PI3K/AKT-mediated ubiquitination and degradation of PTEN are dependent on the MKRN1 E3 ligase.Taken together, our results demonstrate that PI3K/AKT signals enforce positive-feedback regulation by suppressing PTEN function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.

ABSTRACT
The activity of the phosphatase and tensin homologue (PTEN) is known to be suppressed via post-translational modification. However, the mechanism and physiological significance by which post-translational modifications lead to PTEN suppression remain unclear. Here we demonstrate that PTEN destabilization is induced by EGFR- or oncogenic PI3K mutation-mediated AKT activation in cervical cancer. EGFR/PI3K/AKT-mediated ubiquitination and degradation of PTEN are dependent on the MKRN1 E3 ligase. These processes require the stabilization of MKRN1 via AKT-mediated phosphorylation. In cervical cancer patients with high levels of pAKT and MKRN1 expression, PTEN protein levels are low and correlate with a low 5-year survival rate. Taken together, our results demonstrate that PI3K/AKT signals enforce positive-feedback regulation by suppressing PTEN function.

No MeSH data available.


Related in: MedlinePlus