Proteomic analysis of acute promyelocytic leukemia: PML-RARalpha leads to decreased phosphorylation of OP18 at serine 63.
In the present study, we employed 2-DE to characterize the effect of the acute promyelocytic leukemia (APL)-specific PML-RARalpha fusion protein on the proteome. Differentially expressed proteins, a number of which are related to the cell cycle function, including oncoprotein18 (OP18), heat shock protein70, glucose-regulated protein75, and peptidyl-prolyl isomerase, were identified by MS. Subsequent bioinformatic pathway discovery revealed an integrated network constituting SMARCB1, MYC, and TP53-regulated pathways. The data from the DNA microarray and proteomic experiments demonstrated the correlation between the translocation and higher expression of OP18 at mRNA and protein levels. Transient cotransfection assay revealed that PML-RARalpha is a potent activator of OP18 promoter and this transcriptional activation is retinoic acid sensitive. PML-RARalpha induction also leads to decreased phosphorylation on Ser63 residue of OP18, which is okadaic acid sensitive suggesting the involvement of a phosphatase pathway. Overexpression of a constitutively phosphorylated Ser63 mutant of OP18 in PML-RARalpha expressing APL patient, PR9, and NB4 cells led to a G2/M-phase arrest in contrast to a phosphorylation-deficient Ser63 mutant and untransfected control. Taken together, our results demonstrate the significance of decreased Ser63 phosphorylation of OP18 in PML-RARalpha-mediated effects on cell cycle.
Zada
Abdul A Peer
AA
BMT Unit, Internal Medicine IV, MLU University, Halle, Germany.
Geletu
Mulu H
MH
Pulikkan
John A
JA
Müller-Tidow
Carsten
C
Reddy
Venkateshwar A
VA
Christopeit
Maximilian
M
Hiddemann
Wolfgang D
WD
Behre
Hermann M
HM
Tenen
Daniel G
DG
Behre
Gerhard
G
D002460:Cell Line; D002999:Clone Cells; D019295:Computational Biology / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism* / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D013058:Mass Spectrometry; D009154:Mutation; D015514:Oncogene Proteins, Fusion / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D050777:Stathmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020298:U937 Cells; D019287:Zinc Sulfate
D015514:Oncogene Proteins, Fusion; D020033:Protein Isoforms; D020543:Proteome; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D050777:Stathmin; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D012694:Serine; D019287:Zinc Sulfate; D008156:Luciferases