Inhibition of Protein Phosphatase 2A (PP2A) Prevents Mcl-1 Protein Dephosphorylation at the Thr-163/Ser-159 Phosphodegron, Dramatically Reducing Expression in Mcl-1-amplified Lymphoma Cells

• Published in: The Journal of biological chemistry Vol. 289; no. 32; pp. 21950 - 21959
• Main Authors: Nifoussi, Shanna K., Ratcliffe, Nora R., Ornstein, Deborah L., Kasof, Gary, Strack, Stefan, Craig, Ruth W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.08.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Binding Sites; Burkitt Lymphoma - drug therapy; Burkitt Lymphoma - genetics; Burkitt Lymphoma - metabolism; Cell Line, Tumor; Drug Resistance, Neoplasm; Enzyme Inhibitors - pharmacology; Gene Expression; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; Marine Toxins; Molecular Bases of Disease; Myeloid Cell Leukemia Sequence 1 Protein - chemistry; Myeloid Cell Leukemia Sequence 1 Protein - genetics; Myeloid Cell Leukemia Sequence 1 Protein - metabolism; Okadaic Acid - pharmacology; Oxazoles - pharmacology; Phosphorylation - drug effects; Protein Phosphatase 2 - antagonists & inhibitors; Protein Phosphatase 2 - genetics; Proteolysis; Serine - chemistry; Tetradecanoylphorbol Acetate - pharmacology; Threonine - chemistry; Mcl-1; B-cell Lymphoma 2 (Bcl-2) Family; Cell Death; Cancer; Extracellular Signal-regulated Kinase (ERK); Protein Phosphatase 2 (PP2A)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.587873

Abundant, sustained expression of prosurvival Mcl-1 is an important determinant of viability and drug resistance in cancer cells. The Mcl-1 protein contains PEST sequences (enriched in proline, glutamic acid, serine, and threonine) and is normally subject to rapid turnover via multiple different pathways. One of these pathways involves a phosphodegron in the PEST region, where Thr-163 phosphorylation primes for Ser-159 phosphorylation by glycogen synthase kinase-3. Turnover via this phosphodegron-targeted pathway is reduced in Mcl-1-overexpressing BL41-3 Burkitt lymphoma and other cancer cells; turnover is further slowed in the presence of phorbol ester-induced ERK activation, resulting in Mcl-1 stabilization and an exacerbation of chemoresistance. The present studies focused on Mcl-1 dephosphorylation, which was also found to profoundly influence turnover. Exposure of BL41-3 cells to an inhibitor of protein phosphatase 2A (PP2A), okadaic acid, resulted in a rapid increase in phosphorylation at Thr-163 and Ser-159, along with a precipitous decrease in Mcl-1 expression. The decline in Mcl-1 expression preceded the appearance of cell death markers and was not slowed in the presence of phorbol ester. Upon exposure to calyculin A, which also potently inhibits PP2A, versus tautomycin, which does not, only the former increased Thr-163/Ser-159 phosphorylation and decreased Mcl-1 expression. Mcl-1 co-immunoprecipitated with PP2A upon transfection into CHO cells, and PP2A/Aα knockdown recapitulated the increase in Mcl-1 phosphorylation and decrease in expression. In sum, inhibition of PP2A prevents Mcl-1 dephosphorylation and results in rapid loss of this prosurvival protein in chemoresistant cancer cells.