Paul Payette

MECHANISM OF INHIBITION OF PROTEIN-TYROSINE PHOSPHATASES BY VANADATE AND PERVANADATE

Vanadate and pervanadate (the complexes of vanadate with hydrogen peroxide) are two commonly used general protein-tyrosine phosphatase (PTP) inhibitors. These compounds also have insulin-mimetic properties, an observation that has generated a great deal of interest and study. Since a careful kinetic study of the two inhibitors has been lacking, we sought to analyze their mechanisms of inhibition. Our results show that vanadate is a competitive inhibitor for the protein-tyrosine phosphatase PTP1B, with a Ki of 0.38 ± 0.02 μM. EDTA, which is known to chelate vanadate, causes an immediate and complete reversal of the inhibition due to vanadate when added to an enzyme assay. Pervanadate, by contrast, inhibits by irreversibly oxidizing the catalytic cysteine of PTP1B, as determined by mass spectrometry. Reducing agents such as dithiothreitol that are used in PTP assays to keep the catalytic cysteine reduced and active were found to convert pervanadate rapidly to vanadate. Under certain conditions, slow time-dependent inactivation by vanadate was observed; since catalase blocked this inactivation, it was ascribed to in situ generation of hydrogen peroxide and subsequent formation of pervanadate. Implications for the use of these compounds as inhibitors and rationalization for some of their in vivo effects are considered.

A Natural Disruption of the Secretory Group II Phospholipase A2 Gene in Inbred Mouse Strains

The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. In an attempt to determine the role of sPLA2 in inflammation, we set out to generate sPLA2-deficient mice. During this investigation, we observed that in a number of inbred mouse strains, the sPLA2 gene was already disrupted by a frameshift mutation in exon 3. This mutation, a T insertion at position 166 from the ATG of the cDNA, terminates out of frame in exon 4, resulting in the disruption of the calcium binding domain in exon 3 and loss of both activity domains coded by exons 4 and 5. The mouse strains C57BL/6, 129/Sv, and B10.RIII were found to be homozygous for the defective sPLA2 gene, whereas outbred CD-1:SW mice had variable genotype at this locus. BALB/c, C3H/HE, DBA/1, DBA/2, NZB/B1N, and MRL lpr/lpr mice had a normal sPLA2 genotype. The sPLA2 mRNA was expressed at very high levels in the BALB/c mouse small intestine, whereas in the small intestine of the sPLA2 mutant mouse strains, sPLA2 mRNA was undetectable. In addition, PLA2 activity in acid extracts of the small intestine were approximately 40 times higher in BALB/c than in the mutant mice. Transcription of the mutant sPLA2 gene resulted in multiple transcripts due to exon skipping. None of the resulting mutant mRNAs encoded an active product. The identification of this mutation should not only help define the physiological role of sPLA2 but also has important implications in mouse inflammatory models developed by targeted mutagenesis.

Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505

Cytosolic PLA2 (cPLA2) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA2 on Ser-505 by MAP kinase in response to agonist treatment, is thought to be one of the mechanisms required for activation of the enzyme in the cell. In order to obtain enough material for enzymological studies as well as to investigate the role of phosphorylation in the activation of cPLA2, the human enzyme was overexpressed in insect cells using a recombinant baculovirus. We report here on the characterization of the phosphorylation state of cPLA2 overexpressed in Sf9 cells. The level of overexpressed cPLA2 was shown to peak between 48 and 60 h post-infection, by this time the phosphorylated enzyme could easily be detected because of its reduced mobility on polyacrylamide gels. The reduced mobility or gel-shift has been shown to be due to phosphorylation of Ser-505. To determine whether this was also the case for insect cell overexpressed cPLA2, Ser-505 was replaced by Ala, and this mutant (cPLA2S505A) was expressed in Sf9 cells. Analysis of the overexpressed cPLA2S505A showed that it migrated only as the lower unshifted cPLA2 band confirming that the baculovirus overexpressed cPLA2 is extensively phosphorylated on Ser-505. Furthermore, treatment of infected Sf9 cells expressing the wild-type cPLA2 with phorbol 12-tetradecanoate 13-acetate (TPA) shifted all of the overexpressed cPLA2 to the phosphorylated Ser-505 form. When infected Sf9 cells were labelled with [32P], in addition to labelling of Ser-505 other sites were also labelled. Both cPLA2 and cPLA2S505A were purified from infected Sf9 cells and the specific activity for each of the enzymes was measured in a phosphatidylcholine vesicle fluorescence assay using 1-(10-pyrenedecanyl)arachidonyl-sn-glycero-3-phosphocholine as substrate. Under these conditions the specific activity of cPLA2 was, 2 mumol/min per mg, whereas cPLA2S505A was 7-fold less active. These findings suggest that Sf9 cells have a mechanism for phosphorylating cPLA2 similar to that found in mammalian cells which probably proceeds through a MAP kinase. Thus, insect cell overexpressed cPLA2 is a very good source for the Ser-505 phosphorylated enzyme.

Development and validation of an intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses.

We have developed an intact cell assay to be used in the direct quantitation of protein tyrosine phosphatase (PTP) activity. Utilizing the baculovirus expression system, the assay readily allows for a direct activity readout for PTPs such as PTP1B or CD45. Infected Sf9 cells expressing either full-length PTP1B, full-length CD45, CD45 catalytic domain, or hCOX-1 (mock-infected) are harvested 29 hr post-infection, at which time cells are viable and the expressed proteins are processed, as well as localized to their predicted subcellular compartments. Assays are carried out in a 96-well format, with cells expressing the PTP of interest. Cells are preincubated with or without inhibitor and challenged with substrate, and the phosphatase activity is determined spectrophotometrically by monitoring the conversion of p-nitrophenyl phosphate to p-nitrophenol at OD405. Documented PTP inhibitors have been used to validate this assay system. This study demonstrates that a direct readout of PTP activity in intact cells can be achieved, thus providing a useful cell-based screen for determining selective inhibitors of PTPs.