Analysis of phosphatase and tensin homolog tumor suppressor interacting proteins by in vitro and in silico proteomics.
The phosphatase and tensin homolog (PTEN) tumor suppressor is a multifunctional protein deregulated in many types of cancer. To date, a comprehensive documentation of PTEN interacting proteins has not been performed. The goal of our study was to characterize the PTEN interactome using affinity pull-down and tandem mass spectrometry (MS/MS). Wild-type PTEN cDNA was inserted into pTRC-His2 vector to create a 6-His tagged protein, which was expressed in Escherichia coli. Lysate from a human lymphoma cell line was used in pull-down assays, utilizing affinity for nickel-agarose beads. Bound proteins were eluted with imidazole, digested and analyzed on an LCQ DecaXP ion trap mass spectrometer. The nickel affinity pull-down efficiency was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Acquired data were searched against the NCBI nr.fasta nonredundant protein database using the SEQUEST algorithm and screened using INTERACT and ProteinProphet. All experiments were performed in duplicate with 6-His-lacZ serving as control. A total of 79 proteins were identified in the wild-type 6-His-PTEN pull-down by MS/MS. We further validated a subset of the proteins present in the PTEN interactome by performing immunoprecipitation using an anti-PTEN antibody and establishing the presence of the proteins in the immunocomplex by Western blot analysis. A search of published PTEN interactions was also performed using Online Mendelian Inheritance in Man, Human Protein Reference Database, the IntAct Project database, and PubMed. This in silico analysis confirmed 42 out of 79 (53%) of the proteins identified by MS/MS. The remaining 37 proteins represent probable PTEN interactions not previously documented in public databases or reported in the literature. These results highlight the value of combining both in vitro biochemical approaches with in silico analyses for a comprehensive study of protein-protein interactions.
Crockett
David K
DK
ARUP Institute for Clinical and Experimental Pathology.
Fillmore
G Chris
GC
Elenitoba-Johnson
Kojo S J
KS
Lim
Megan S
MS
D000465:Algorithms; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D006801:Humans; D008223:Lymphoma; D008969:Molecular Sequence Data; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D025521:Tumor Suppressor Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human