Abstract
Mcl-1 is an antiapoptotic Bcl-2 family member that is highly regulated and when dysregulated contributes to cancer. The Mcl-1 protein is phosphorylated at multiple sites in response to different signaling events. Phosphorylations at Thr163 (by ERK) and Ser159 (by glycogen-synthase kinase 3beta) have recently been shown to slow and enhance, respectively, Mcl-1 protein turnover. Phosphorylation is also known to be stimulated at other, as-yet uncharacterized sites in the G2/M phase of the cell cycle. Using an S peptide-tagged Mcl-1 T163A mutant, Ser64 was identified as a novel Mcl-1 phosphorylation site by mass spectrometry. Immunoblotting demonstrated that phosphorylation at this site was maximal in cells in G2/M phase, was enhanced by tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) treatment, was blocked by inhibitors of CDK (but not ERK or glycogen-synthase kinase 3beta), and was stimulated in vitro by CDK 1, CDK2, and JNK1. The half-life of a nonphosphorylatable S64A Mcl-1 mutant was indistinguishable from that of the wild type polypeptide. In contrast, this mutant failed to protect cells from TRAIL-mediated apoptosis, whereas reconstitution with the phosphomimetic S64E Mcl-1 mutant rendered cells TRAIL-resistant. This anti-apoptotic phenotype of the S64E Mcl-1 mutant was also associated with enhanced binding to the proapoptotic proteins Bim, Noxa, and Bak. A pharmacological CDK inhibitor that reduced Ser64 phosphorylation also sensitized cells to TRAIL cytotoxicity. Collectively, these observations not only identify G2/M-associated phosphorylation at Ser64 as a critical determinant of the antiapoptotic activity of Mcl-1 but also elucidate a novel mechanism by which CDK1/2 inhibitors can enhance the effectiveness of the cytotoxic cytokine TRAIL.
Highlights
The Bcl-2 family member Mcl-1 promotes cell viability at a critical upstream point in the apoptotic cascade [1]
Further analysis demonstrated that Ser64, which is within a “poor” PEST sequence upstream from the PEST sequence that contains Thr163 and Ser159, is conserved through a wide range of species (Fig. 2B) including dog and cat
The results of the present study demonstrate that Mcl-1 can be phosphorylated on Ser64 and establish that the inability to phosphorylate this site diminishes the antiapoptotic function of Mcl-1 without altering the half-life of the protein
Summary
Materials—The CDK inhibitor SU9156 [37] was from Alexis (San Diego, CA). The MEK inhibitors (U0126 and PD98059), the p38 MAPK inhibitor SB220025, the JNK inhibitor SP600125, the GSK-3 inhibitor BIO (GSK inhibitor IX, (2ЈZ,3ЈE)-6-bromoindirubin-3Ј-oxime), the CDK 2/5 inhibitor PNU 112455A (N4-(6-aminopyrimidin-4-yl)-sulfanilamide), the CDK inhibitors olomoucine and roscovitine, and the proteasome inhibitor MG-132 were from Calbiochem (La Jolla, CA). Phosphorylation Site Analysis—KMCH cells stably transfected with S peptide-tagged T163A Mcl-1 were cultured in 10-cm dishes. After the cells were solubilized in lysis buffer, S peptide-tagged T163A Mcl-1 was recovered on S protein-agarose as previously described [39]. For KMCH cells transfected with the S peptide-tagged Mcl-1 mutants (see below), protein S-agarose purification was used to recover tagged Mcl-1 prior to immunoblot analysis. Mcl-1 Half-life Estimation—KMCH cells stably expressing shRNA to the endogenous Mcl-1 and stably expressing S peptide-tagged Mcl-1 constructs (described above) were treated for a total of 4 h with vehicle or TRAIL During this treatment, 20 g/ml cycloheximide was added for 0 –120 min. Determination of Mcl-1 Binding Partners—KMCH cells expressing short interfering RNA-resistant S peptide-tagged mutant (S64A or S64E) and short interfering RNA against endogenous Mcl-1 were solubilized in lysis buffer containing. Statistical Analysis—Statistical analysis was performed using a Student’s t test from at least three independent experiments
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