Interleukin-3 Induces the Phosphorylation of a Distinct Fraction of Bcl-2

Abstract

Bcl-2-related proteins (i.e. Bcl-2 and Bax) regulate the effector stage of apoptosis and can modulate the entry of quiescent cells into the cell cycle. Phosphorylation of Bcl-2 is presumed to modify its apoptosis-inhibitory function. By utilizing an interleukin-3 (IL-3)-dependent hematopoietic cell line, we examined the structural requirements of Bcl-2 phosphorylation and the correlation of this post-translational modification with its function. In the presence of IL-3, constitutively expressed Bcl-2 was phosphorylated on serine residue(s), and phosphorylated Bcl-2 lost its capacity to heterodimerize with Bax. Whereas the majority of Bcl-2 resided in mitochondria, phosphorylation only affected a minor pool of total Bcl-2 that selectively partitioned into a soluble fraction. Cytosolic targeting of Bcl-2 greatly increased its ratio of phosphorylation. Bcl-2 phosphorylation was reduced during IL-3 deprivation, and its phosphorylation was also delayed after transient cytokine deprivation. This pattern of phosphorylation temporally correlated with the accelerated exit and delayed reentry of Bcl-2-expressing cells into the cell cycle upon transient IL-3 deprivation and subsequent cytokine restimulation. Thus, IL-3-induced phosphorylation of a distinct pool of Bcl-2 may contribute to the inactivation of its antiproliferative function.

Unwinding the loop of Bcl-2 phosphorylation

“…Since phosphorylation on S70 by paclitaxel already caused a mobility shift, either it is accompanied by an additional phosphorylation event outside the loop domain or 'IL-3-phosphorylated' Bcl-2 is not visible on immunoblots. The latter possibility is supported by recent findings by Poommipanit et al 32 Although a majority of Bcl-2 reside in mitochondria, IL-3-induced phosphorylation only affected a minor pool of soluble Bcl-2. 32 A small amount of cytosolic Bcl-2 may not be visible on immunoblot.…”

“…The latter possibility is supported by recent findings by Poommipanit et al 32 Although a majority of Bcl-2 reside in mitochondria, IL-3-induced phosphorylation only affected a minor pool of soluble Bcl-2. 32 A small amount of cytosolic Bcl-2 may not be visible on immunoblot. In contrast, paclitaxel caused phosphorylation of a membrane-bound Bcl-2.…”

“…Thus, ERK/MAPK can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways. 59 A recent publication by Poommipanit et al 32 provides data for modification of the IL-3 model, demonstrating that free rather than mitochondrial Bcl-2 is phosphorylated. According to this study, IL-3-induced phosphorylation of a cytosolic pool of Bcl-2 may contribute to the inactivation of its antiproliferative function.…”

“…According to this study, IL-3-induced phosphorylation of a cytosolic pool of Bcl-2 may contribute to the inactivation of its antiproliferative function. 32 In mitogen-dependent hemopoietic cell lines, Bcl-2 overexpression is synergistic with MEK1 and Raf-1 activities on relieving the cytokine dependency. 47,72,73 This suggests that phosphorylation of Bcl-2 promotes mitogen-induced cell survival and proliferation.…”

Novel triterpenoid CDDO-Me is a potent inducer of apoptosis and differentiation in acute myelogenous leukemia

“…63,64 Furthermore, posttranslational modifications of Bcl-2 appear to be involved in the regulation of apoptosis. In particular, Bcl-2 phosphorylation was associated with enhanced Bcl-2 function in some studies 17,18,39,65 and with the inactivation of Bcl-2 in others. [66][67][68][69] Phosphorylation at serine 70 was also found to be required for the anti-apoptotic function of Bcl-2 to be mediated by mitochondrial PKC-␣.…”