Subunit interactions control protein phosphatase 2A

Effcts of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit

Craig Kamibayashi, Robert Estes, Clive A. Slaughter, Marc C. Mumby

Research output: Contribution to journalArticle

61 Citations (Scopus)

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 is 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.

Original languageEnglish (US)
Pages (from-to)13251-13260
Number of pages10
JournalJournal of Biological Chemistry
Volume266
Issue number20
StatePublished - Dec 1 1991
Externally publishedYes

Fingerprint

Proteolysis
Protein Phosphatase 2
Ethylmaleimide
Heparin
kemptide
Enzymes
Myosin Light Chains
Phosphoric Monoester Hydrolases
Digestion
Cardiac Myosins
Smooth Muscle Myosins
Okadaic Acid
Substrate Specificity
Peptides
Enzyme activity
Substrates
Electrophoresis
Catalytic Domain
Gels
Purification

ASJC Scopus subject areas

  • Biochemistry

Cite this

Subunit interactions control protein phosphatase 2A : Effcts of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit. / Kamibayashi, Craig; Estes, Robert; Slaughter, Clive A.; Mumby, Marc C.

In: Journal of Biological Chemistry, Vol. 266, No. 20, 01.12.1991, p. 13251-13260.

Research output: Contribution to journalArticle

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