Purification and Characterization of Two Potent Heat-Stable Protein Inhibitors of Protein Phosphatase 2A from Bovine Kidney

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Abstract
Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I-1(PP2A) and I-2(PP2A), have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I-1(PP2A) exhibited an apparent M(r) similar to 30 000 and 250 000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I-2(PP2A) exhibited an apparent M(r) similar to 20 000 and 80 000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I-1(PP2A) and I-2(PP2A) inhibited PP2A with P-32-labeled myelin basic protein, P-32-labeled histone H1, P-32-labeled pyruvate dehydrogenase complex, P-32-labeled phosphorylase, and protamine kinase as substrates. By contrast, I-1(PP2A) and I-2(PP2A) exhibited little effect, if any, on the activity of PP2A with P-32-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein kinase [Guo, H., and Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I-1(PP2A) and I-2(PP2A) had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With P-32-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I-1(PP2A) and I-2(PP2A) were noncompetitive and displayed a K-i of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I-1(PP2A) and I-2(PP2A) displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I-1(PP2A) and I-2(PP2A) are novel proteins.

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