Abstract
Protein phosphatase 2A consists of a heterotrimeric complex composed of a catalytic subunit (C) and two associated subunits (A and B). Limited tryptic digestion of the heterotrimeric ABC form resulted in the selective degradation of the Mr = 55,000 B subunit to a 48-kDa polypeptide. The cleavage sites were determined to be within a 3-7-kDa region of the COOH terminus. Proteolysis led to dissociation of the B subunit from the enzyme complex and correlated with an increase in cardiac myosin light chain, smooth muscle myosin light chain peptide, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) phosphatase activity. Purification of the digestion products and native gel electrophoresis indicated that dissociation of the B subunit was responsible for the increase in phosphatase activity. Kinetic analyses with several substrates revealed that dissociation of the B subunit resulted in a 2-7-fold increase in Vmax and a 1.6-5 fold increase in Km. Proteolytic dissociation of the B subunit increased the sensitivity of protein phosphatase 2A to inhibition by okadaic acid. Inhibition of the trypsinized enzyme was very similar to that observed for the purified AC form of protein phosphatase 2A. Incubation of the ABC complex with N-ethylmaleimide resulted in dissociation of the C subunit and generation of an AB complex. Selective release of the C subunit indicated that the B subunit interacts directly with the A subunit and that one or more free sulfhydryls are required to maintain the heterotrimeric structure of protein phosphatase 2A. Treatment of the enzyme with heparin resulted in an increase in specific activity that was due to the release of the B subunit from the complex. These results provide evidence that the B subunit binds directly to the A subunit to modulate enzyme activity and substrate specificity and that the COOH-terminal region of this protein is important for interaction with the AC complex. Dissociation of the B subunit by polyanionic substances related to heparin may represent a mechanism for regulating the activity of this enzyme.
Highlights
From the Departmentof Pharmacology and $Howard Hughes Medical Institute, University of Teras Southwestern Medical Center, Dall&, Teras 75235-9041
This study has demonstrated that the B subunit of the heterotrimericform of bovine cardiac PPZA is extremely sensitive to the effects of trypsin
Proteolysis occurred in a progressive manner with the generationof a 48-kDa polypeptide
Summary
Time Course of MLC Phosphatase Activation-To deter- and immunoblot (Fig. 4B, lane c ) , the AC form is not affected mine if proteolysis had a directeffect on phosphatase activity, by trypsin These dataindicate that trypsindigestion of ABC the activity of trypsinizedsamples were determinedusing causes proteolysis of the M , = 55,000 protein resulting in bovine cardiac myosin light chains as substrate. The K , and V,,, of trypsin-treated ABC amounts of the AC complex (Fig. 6, lune d) This indicated are increased5- and 7-fold, respectively, whencompared with that the 48-kDa protein had dissociated fromtheenzyme native ABC. Values are the mean f S.E. of at least three experiments
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