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J. Biol. Chem., Vol. 267, Issue 24, 16883-16888, 08, 1992
BE Wadzinski, BJ Eisfelder, LF Peruski Jr, MC Mumby and GL Johnson
Functional expression of recombinant wild-type phosphatase 2A catalytic
subunit has been unsuccessful in the past. A nine-amino-acid peptide
sequence (YP-YDVPDYA) derived from the influenza hemagglutinin protein was
used to modify the NH2 and/or COOH terminus of the phosphatase 2A catalytic
subunit. Addition of the nine-amino-acid sequence at the NH2 terminus
allowed recombinant phosphatase 2A expression as a predominantly cytosolic
phosphatase 2A enzyme. The 12CA5 monoclonal antibody that recognizes the
nine-amino-acid hemagglutinin peptide sequence was used to
immunoprecipitate the epitope-tagged phosphatase 2A catalytic subunit.
Assay of the immunoprecipitated epitope-tagged phosphatase 2A demonstrated
an okadaic acid-sensitive dephosphorylation of [32P] histone H1 and
[32P]myelin basic protein similar to that measured with the wild-type
enzyme. Functional phosphatase activity could be demonstrated for the
NH2-terminal modified phosphatase 2A catalytic subunit following transient
expression in COS cells or stable expression in Rat1a cells. In contrast,
the COOH-terminal-modified phosphatase 2A catalytic subunit was very poorly
expressed. The NH2-, COOH-modified subunit, having the nine-amino-acid
hemagglutinin peptide sequence encoded at both termini of the polypeptide,
was also expressed as a functional phosphatase 2A enzyme. Thus,
NH2-terminal modification of the phosphatase 2A catalytic subunit results
in a functional plasmid- expressed enzyme. The unique nine-amino-acid
epitope-tag sequence also provides a method to easily resolve the
recombinant phosphatase 2A from the endogenous wild-type gene product and
related phosphatases expressed in cells.
NH2-terminal modification of the phosphatase 2A catalytic subunit allows functional expression in mammalian cells
Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
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