Mass spectrometry study of PRL-3 phosphatase inactivation by disulfide bond formation and cysteine into glycine conversion.
The Phosphatase of Regenerating Liver-3 (PRL-3) is a cysteine-based phosphatase (CBP) that is highly over-expressed in liver metastasis in colorectal cancer and suspected to be involved in the progression from tumor to metastasis. During substrate-specificity studies based on the screening of PRL-3 phosphatase activity on several phosphorylated synthetic peptides, we observed a decrease in activity depending on sample aging and storage conditions. By liquid chromatography combined with selective alkylation and mass spectrometry, we found two main PRL-3 inactivation pathways: a disulfide bond formation between the catalytic C104 and C49, blocking the enzyme in an inactive oxidized form, or the conversion of the catalytic C104 into glycine. We also found that the disulfide formation and the cysteine into glycine conversion are catalyzed by cations present in the sample after protein purification through a nickel column. By adding a cation chelator such as EDTA and de-oxygenating the sample with argon, PRL-3 phosphatase activity was preserved. These findings suggest that PRL-3, like other CBPs, is sensitive to inactivation by catalytic cysteine oxidation and this has implications for future studies of its activity and specificity.
Orsatti
Laura
L
IRBM P. Angeletti, Via Pontina km 30.600, 00040 Pomezia, Italy. laura_orsatti@merck.com
Innocenti
Federica
F
Lo Surdo
Paola
P
Talamo
Fabio
F
Barbato
Gaetano
G
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004789:Enzyme Activation; D005998:Glycine / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*
D004220:Disulfides; D009363:Neoplasm Proteins; C484730:PTP4A3 protein, human; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine; D005998:Glycine