The Phosphotyrosyl Phosphatase Activator of Protein Phosphatase 2A

A Novel Purification Method, Immunological and Enzymic Characterization

Authors


J. Goris, Afdeling Biochemie, Campus Gasthuisberg, Herestraat, B-3000 Leuven, Belgium
Fax:+32 16 345995.

Abstract

A simple, improved procedure for the isolation of the phosphotyrosyl phosphatase activator (PTPA) from rabbit skeletal muscle has been developed. The majority of the protein phosphatase 2A (PP2A) was separated from PTPA at an early stage in the procedure. The procedure yields approximately 1 mg essentially pure PTPA/kg rabbit skeletal muscle; it was also applied to porcine brain and the yeast Saccharomyces cerevisiae. The physico-chemical properties of PTPA obtained from all sources are very similar. The pure rabbit skeletal muscle protein was used to raise polyclonal goat antibodies and to affinity purify these antibodies. Immunological studies revealed the presence of PTPA in all mammalian tissues and cell lines examined with differences in tissue distribution, brain showing the highest concentration. PTPA could only be detected in cytosolic fractions. Using a semi-quantitative immunological assay (Western blot), the in vivo concentration could be estimated to be micromolar, which is in the same range as the PP2A target. The purified Xenopus oocyte PTPA showed only a weak cross reactivity, whereas yeast PTPA was not recognised by the antibody indicating some evolutionary diversity of the protein. In a PTPA-affinity column chromatography, the weak interaction with PP2A was independent of the presence of ATP · Mg, a necessary cofactor in the activation process. Interaction of PTPA with PP2A in a 1:1 ratio induces a low (kcat= 3 min-1) ATPase activity that is inhibited by okadaic acid, ADP and non-hydrolysable ATP analogues.

Abbreviations
PTPA

phosphotyrosyl phosphatase activator

PTPase

phosphotyrosyl phosphatase

PP2A

protein phosphatase type 2A

PP2AD

the dimeric form of PP2A

MAP kinase

mitogen-activated protein kinase

RCM lysozyme

reduced carboxamido-methylated and maleylated lysozyme

IC50

concentration necessary for 50% inhibition

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