Prohibitin 2 represents a novel nuclear AKT substrate during all‐ trans retinoic acid–induced differentiation of acute promyelocytic leukemia cells

Abstract

The AKT/PKB kinase is essential for cell survival, proliferation, and differentiation; however, aberrant AKT activation leads to the aggressiveness and drug resistance of many human neoplasias. In the human acute promyelocytic leukemia cell line NB4, nuclear AKT activity increases during all-trans retinoic acid (ATRA)-mediated differentiation. As nuclear AKT activity is associated with differentiation, we sought to identify the nuclear substrates of AKT that were phosphorylated after ATRA treatment. A proteomics-based search for nuclear substrates of AKT in ATRA-treated NB4 cells was undertaken by using 2D-electrophoresis/mass spectrometry (MS) in combination with an anti-AKT phospho-substrate antibody. Western blot analysis, an in vitro kinase assay, and/or site-directed mutagenesis were performed to further characterize the MS findings. MS analysis revealed prohibitin (PHB)-2, a multifunctional protein involved in cell cycle progression and the suppression of oxidative stress, to be a putative nuclear substrate of AKT. Follow-up studies confirmed that AKT phosphorylates PHB2 on Ser-91 and that forced expression of the PHB2(S91A) mutant results in a rapid loss of viability and apoptotic cell death. Activation of nuclear AKT during ATRA-mediated differentiation results in the phosphorylation of several proteins, including PHB2, which may serve to coordinate nuclear-mitochondrial events during differentiation.

Prohibitin 2: At a communications crossroads

“…On the other hand, the data presented by Bavelloni et al indicate that AKT1/2 also phosphorylates S91 during differentiation, and more importantly phosphorylation of S91 is required for survival in this cell model. The cellular death reported by Bavelloni et al following overexpression of a PHB2 (S91A) mutant was highly indicative of mitochondrial dysfunction and very similar to findings reported following the expression of PHB mutants lacking or containing alterations in the mitochondrial transmembrane domain (MTS), critical for mitochondrial localization (Fig. C).…”

“…In addition to S91, S176 was also phosphorylated by AKT in this model system, although S176 phosphorylation was secondary to S91, suggesting a hierarchical phosphorylation. Interestingly, in this study, while exogenous expression of a S176A mutant had little effect on cell viability, exogenous expression of a S91A phospho‐mutant resulted in a rapid and complete apoptosis of NB4 cells within 24 h after transfection . Such apoptosis is a hallmark of mitochondria catastrophe and this phenotype matched those observed in embryonic stem cells in which the PHBs were knocked‐out and cell lines where PHBs were knocked‐down using siRNA (Fig.…”

PML/RARA inhibits expression of HSP90 and its target AKT

“…Here, we show that primary blasts from APL patients express very low levels of AKT protein. It is known that PML deactivates and promotes degradation of nuclear AKT within nuclear bodies (PML‐NBs) (Salomoni & Pandolfi, ; Bavelloni et al , ). Thus, one could expect that in PML/RARA‐positive cells, where the function of PML‐NBs is impaired, nuclear AKT would promote survival over apoptosis.…”

Induction of cellular and molecular immunomodulatory pathways by vitamin A and flavonoids

“…A recent study using the human acute promyelocytic leukemia cell line, NB4, identified two substrates, the Serine/arginine-rich splicing factor 3 (SRSF3) and the prohibitin 2 (PHB2), which are associated with AKT/PBK pathway and their phosphorylation increased during in vitro activation with ATRA [28]. This, in turn, led to activation of AKT/PBK resulting in promotion of cell survival and differentiation vs. apoptosis [28]. …”