Purification and characterization of multiple S6 phosphatases from the rat parotid gland

Abstract

S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn2+. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn(2+)-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC50 of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn2+.

Involvement of Protein Phosphatase 2A in the Interleukin-3-Stimulated Jak2-Stat5 Signaling Pathway

“…(30) This concentration of OA inhibits .90% of the PP2A activity. The reaction was terminated by the addition of 0.8 ml of 5 mM silicotungstate/5 mM H 2 SO 4 and 1.2 ml isobutanol and toluene (1:1).…”

“…(30) To prepare 32 P-labeled phosphorylase a, phosphorylase b was treated with phosphorylase kinase at 30°C for 4 h in a reaction mixture containing 50 mM Tris-HCl, pH 8.2, 10 mM MgCl 2 , 0.3 mM CaCl 2 , 15 mg phosphorylase kinase, 15 mg phosporylase b, and 0.2 mM g 32 P-ATP (500-800 cpm/pmol). The reaction mixture was applied to an AG1X-8 column to remove free ATP.…”

Protein phosphatase 2A interacts with the Src kinase substrate p130CAS

“…Immunoprecipitates were washed four times with 25 mM Tris-HCl (pH 7.5) plus 0.5% NP-40, and incubated with or without PP2A in the reaction mixture for 1 h at 308C. The reaction mixtures contained 50 mM TrisHCl (pH 7.5), 0.1 mM EDTA, 0.1 mM DTT, 1 mM ca eine and 100 mg BSA (Yokoyama, 1995). Immunoprecipitates were then washed with 0.5% NP-40 plus phosphate-bu ered saline four times and proteins were analysed by SDS ± PAGE and Western blotting analysis using phosphoserine, Cas and PP2A antibodies.…”