BIOCHEMICAL-CHARACTERIZATION AND DELETION ANALYSIS OF RECOMBINANT HUMAN PROTEIN PHOSPHATASE 2C-ALPHA

The use of protein phosphatase inhibitors has been instrumental in def ining the intracellular roles of protein phosphatase 1 (PP1), PP2A and PP2B. Identification of the role of PP2C in vivo has been hampered, i n part, by the unavailability of specific inhibitors. In order to faci litate the identification of novel and specific inhibitors of PP2C by random screening of compounds, and to further characterize this enzyme at the molecular level by site-directed mutagenesis and X-ray crystal lography, we have expressed active recombinant human PP2C alpha (rPP2C alpha) in Escherichia coli. Biochemical characterization of rPP2C alp ha showed that it could hydrolyse p-nitrophenyl phosphate (pNPP) altho ugh, in contrast with native PP2C, this was not stimulated by Mg2+. As with native PP2C, okadaic acid failed to inhibit rPP2C alpha, whereas 50 mM NaF dramatically inhibited its activity. An alignment of the am ino acid sequence of AMP-activated protein kinase (AMPK) with those of other serine/threonine protein kinases around the regulatory phosphor ylation site (subdomains VII-VIII) revealed a high degree of conservat ion. Phosphopeptides derived from this region of AMPK and containing t he almost invariant threonine (Thr(172) in AMPK) were found to be good substrates for rPP2C alpha. We also showed that rPP2C alpha can inact ivate AMPK, but only in the presence of Mg2+. To define the regions of PP2C alpha important for catalytic activity, we expressed a number of truncated proteins based on the sequence and proposed domain structur e of the PP2C alpha homologue from Paramecium tetraurelia. Deletion of 75 residues (9 kDa) from the C-terminus appeared to have little effec t on the catalytic activity using pNPP, phosphopeptides or AMPK as sub strates. This suggests that the residues important in catalysis lie el sewhere in the protein. A further deletion of the C-terminus led to a completely inactive and very poorly soluble protein.