Abstract
The antiapoptotic Bcl-2 family member Mcl-1 is a PEST protein (containing sequences enriched in proline, glutamic acid, serine, and threonine) and is subject to rapid degradation via multiple pathways. Impaired degradation leading to the maintenance of Mcl-1 expression is an important determinant of drug resistance in cancer. Phosphorylation at Thr 163 in the PEST region, stimulated by 12-O-tetradecanoylphorbol acetic acid (TPA)-induced activation of extracellular signal-regulated kinase (ERK), is associated with Mcl-1 stabilization in BL41-3 Burkitt lymphoma cells. This contrasts with the observation that Thr 163 phosphorylation in normal fibroblasts primes glycogen synthase kinase (GSK3)-induced phosphorylation at Ser 159, producing a phosphodegron that targets Mcl-1 for degradation. In the present follow-up studies in BL41-3 cells, Mcl-1 degradation was found to be independent of the GSK3-mediated pathway, providing a parallel to emerging findings showing that Mcl-1 degradation through this pathway is lost in many different types of cancer. Findings in Mcl-1-transfected CHO cells corroborated those in BL41-3 cells in that the GSK3-targeted phosphodegron did not play a major role in Mcl-1 degradation, and a phosphomimetic T163E mutation resulted in marked Mcl-1 stabilization. TPA-treated BL41-3 cells, in addition to exhibiting Thr 163 phosphorylation and Mcl-1 stabilization, exhibited an ∼10-fold increase in resistance to multiple chemotherapeutic agents, including Ara-C, etoposide, vinblastine, or cisplatin. In these cancer cells in which Mcl-1 degradation is not dependent on the GSK3/phosphodegron-targeted pathway, ERK activation and Thr 163 phosphorylation are associated with pronounced Mcl-1 stabilization and drug resistance – effects that can be suppressed by inhibition of ERK activation.
Highlights
Increased expression of Mcl-1, stimulated by growth factors and other environmental signals, promotes viability and allows the amplification and function of cell types and lineages needed by the organism [1,2,3,4]
BL41-3 cells exhibit abundant constitutive expression of endogenous Mcl-1 (,5-fold higher than ML-1 cells stimulated with tetradecanoylphorbol acetic acid (TPA) [23]), and have proven very useful for studies of its posttranslational regulation
BL41-3 cells are useful for studies of TPA/extracellular signalregulated kinase (ERK)-induced Thr 163 phosphorylation [24], because effects on the stability of the protein can be examined in the absence of substantial Mcl-1 induction
Summary
Increased expression of Mcl-1, stimulated by growth factors and other environmental signals, promotes viability and allows the amplification and function of cell types and lineages needed by the organism [1,2,3,4]. Agents that induce Mcl-1 turnover, and inhibitors designed to target the protein, can promote tumor cell death [16,17,18,19,20]. Mcl-1 was identified based on increased transcription in ML-1 human myeloblastic leukemia cells induced to differentiate upon exposure to TPA [21,22]. Exposure of BL41-3 cells to TPA results in activation of the mitogen-activated protein (MAP) kinase ERK, GAPDH (ChemiDoc). M C: BL41-3 cells were either left untreated or exposed to TPA (1 nM). Some cells were immediately exposed to CHX to monitor Mcl-1 protein decay on Day 0. The untreated cell sample (Time 0) is identical for each pair of Western blots. The blot at the bottom confirmed that pERK was reduced at 24 hours. doi:10.1371/journal.pone.0047060.g001 along with an ERK-dependent increase in Mcl-1 phosphorylation at Thr 163 and markedly slowed degradation of the Mcl-1 protein [25,26]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
