Figure 4
Figure 4. Modulation of MYC protein abundance occurs via the PTEN phosphatidylinositol 3-kinase/AKT axis. (A) Schematic representation of upstream effectors of MYC activity. Efficient addressing of MYC to the proteasome degradation pathway is performed through an ordered pattern of phosphorylation, starting at serine 62 (S62) via the RAS/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway,19 which enhances both MYC activity and phosphorylation at threonine 58 (T58) via the GSK3β serine-threonine kinase. The double S62T58 phosphorylated form of MYC is then recognized by the PIN1 phosphatase (not depicted for clarity), which rapidly dephosphorylates S62, and allows the T58 mono-phosphorylated form to be recognized and poly-ubiquitinylated by the UbE3 ligase FBXW7 for final degradation.14,19–20 For clarity, only few regulators/effectors are displayed; notably the multiple targets of the major AKT hub (such as mTOR) are not shown. (B) Effect of PTEN gain-of-function in PTENneg T-ALL. Jurkat Tet-On cells were transiently transfected with the doxycycline-inductible expression vector PTRE-PTEN, then PTEN, MYC, p-AKT, and p-GSK3β protein levels monitored by Western blot with or without doxycycline induction (in absence of PTRE-PTEN, doxycycline does not induce a decrease of MYC protein level, see supplemental Figure 4A). MYC transcript levels were monitored by RQ-polymerase chain reaction and normalized to ABL transcripts. (C) Effect of PTEN loss-of-function in PTENpos T-ALL. DND41 cell line was transduced either with TRIP-shPTEN, a lentiviral vector that expresses EGFP and a PTEN shRNA, or with TRIP, the native lentiviral vector that only expresses EGFP used here as control. PTEN and MYC proteins and transcripts levels were then monitored by Western blot and RQ-PCR, respectively. MYC and PTEN transcript levels were normalized to ABL transcripts. (B-C) Bands intensities were quantified using TotalLab Quant software, normalized to β-actin and reported below the blots as relative values to the control assays. Data shown are representative of at least 3 independent experiments.

Modulation of MYC protein abundance occurs via the PTEN phosphatidylinositol 3-kinase/AKT axis. (A) Schematic representation of upstream effectors of MYC activity. Efficient addressing of MYC to the proteasome degradation pathway is performed through an ordered pattern of phosphorylation, starting at serine 62 (S62) via the RAS/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway,19  which enhances both MYC activity and phosphorylation at threonine 58 (T58) via the GSK3β serine-threonine kinase. The double S62T58 phosphorylated form of MYC is then recognized by the PIN1 phosphatase (not depicted for clarity), which rapidly dephosphorylates S62, and allows the T58 mono-phosphorylated form to be recognized and poly-ubiquitinylated by the UbE3 ligase FBXW7 for final degradation.14,19,20  For clarity, only few regulators/effectors are displayed; notably the multiple targets of the major AKT hub (such as mTOR) are not shown. (B) Effect of PTEN gain-of-function in PTENneg T-ALL. Jurkat Tet-On cells were transiently transfected with the doxycycline-inductible expression vector PTRE-PTEN, then PTEN, MYC, p-AKT, and p-GSK3β protein levels monitored by Western blot with or without doxycycline induction (in absence of PTRE-PTEN, doxycycline does not induce a decrease of MYC protein level, see supplemental Figure 4A). MYC transcript levels were monitored by RQ-polymerase chain reaction and normalized to ABL transcripts. (C) Effect of PTEN loss-of-function in PTENpos T-ALL. DND41 cell line was transduced either with TRIP-shPTEN, a lentiviral vector that expresses EGFP and a PTEN shRNA, or with TRIP, the native lentiviral vector that only expresses EGFP used here as control. PTEN and MYC proteins and transcripts levels were then monitored by Western blot and RQ-PCR, respectively. MYC and PTEN transcript levels were normalized to ABL transcripts. (B-C) Bands intensities were quantified using TotalLab Quant software, normalized to β-actin and reported below the blots as relative values to the control assays. Data shown are representative of at least 3 independent experiments.

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