Autophosphorylation-activated protein kinase phosphorylates and inactivates protein phosphatase 2A.

Abstract

Purified preparations of a distinct autophosphorylation-activated protein kinase from bovine kidney phosphorylated and inactivated purified preparations of protein phosphatase 2A2 (PP2A2) by about 80% with the autophosphorylation-activated protein kinase, protamine kinase, and 32P-labeled myelin basic protein as substrates. Analysis of incubations performed in the presence of 0.2 mM [-32P]ATP by autoradiography following SDS/PAGE and by FPLC gel permeation chromatography on Superose 12 demonstrated that the catalytic subunit of PP2A2 was phosphorylated in the incubation mixtures containing the kinase and phosphatase. Up to 0.3 mol of phosphate groups was incorporated per mol of the catalytic subunit of PP2A2 following incubation with the kinase. This phosphorylation was enhanced about 5-fold in the presence of 0.4 FM microcystin-LR. In addition, up to 1 mol of phosphate groups was incorporated per mol of the PP2A2 subunit of apparent Mr 60,000 when microcystin-LR was included. Analysis by thin-layer chromatography indicated that PP2A2 catalyzed an autodephosphorylation reaction which was inhibited by microcystin-LR. Phospho amino acid analysis showed that the catalytic subunit of PP2A2 was phosphorylated on threonine residues by the autophosphorylation-activated protein kinase. Together with previous observations, the results suggest that inactivation of PP2A by phosphorylation catalyzed by the autophosphorylation-activated protein kinase could contribute to the marked increase in the phosphorylation of cellular proteins in response to insulin and other mitogens.Protein phosphatase 2A (PP2A) is a protein-serine/threonine-phosphatase which acts on the enzymes and proteins that regulate the rates of glycogen metabolism, glycolysis/ gluconeogenesis, cholesterol synthesis, aromatic amino acid metabolism, protein synthesis, gene transcription, and numerous other processes (1, 2). Two forms, PP2A1 and PP2A2, have been found in the cytosol of nearly all tissues examined (1, 2). A form of PP2A2 has also been purified to apparent homogeneity from extracts of bovine kidney mitochondria (3). PP2A1 and PP2A2 contain a catalytic subunit of apparent Mr 36,000 and a subunit of apparent Mr 60,000. PP2A1 also contains a subunit of apparent Mr 55,000. The catalytic subunit is highly conserved (4-8), and in some species two forms have been identified by cloning methods (4-8). The deduced amino acid sequences indicate that these two forms of the catalytic subunit exhibit 97% identity, with four of the seven amino acid substitutions located in the N terminus being conservative (4-8). However, the exact physiological function of PP2A is uncertain because the enzyme exhibits overlapping specificity with PP1 and PP2C. Furthermore, other than subunit-subunit interactions which modulate the specificity of PP2A (1, 2), there is little information on the regulation of this enzyme (1, 2). Nevertheless, PP2A is considered an important enzyme that is likely to occupy a critical position in the control of diverse metabolic pathways and...

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…Threonine phosphorylation occurs via the activity of an 'autophosphorylation-activated protein kinase', the identity of which is currently unknown. Similar to what is observed for Tyr307, PP2A can auto-dephosphorylate these threonine site(s) in vitro [49].…”

Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen

“…The fact that tyrosine 307 is accessible to v-Src phosphorylation suggests that one or both of the threonines might also be accessible for phosphorylation. Furthermore, the inhibition of catalytic activity by both tyrosine and threonine phosphorylation is consistent with a potential mechanism wherein a phosphorylated carboxy-terminus (on either tyrosine or threonine) occupies the enzyme active site (Guo and Damuni, 1993;Chen et al, 1992).…”

“…It remains to be determined, however, what the molecular consequences of this interaction are for the regulation of PP2A. Inhibition of PP2A activity in vitro by phosphorylation of the catalytic subunit on tyrosine 307 or on an unidenti®ed threonine residue(s) was reported (Guo and Damuni, 1993;Chen et al, 1992). Some data suggest that these or similar modi®cations may occur in vivo in response to transformation or growth stimulation (Chen et al, 1994).…”

Role of Janus kinase-2 in insulin-mediated phosphorylation and inactivation of protein phosphatase-2A and its impact on upstream insulin signalling components

“…So far, over 15 regulatory subunits have been identified for PP-2A, which, in addition to regulating enzymic activity, are predicted to be involved in the subcellular targeting of the holoenzyme. In addition to regulatory subunits, PP-2A activity is also influenced by catalytic subunit phosphorylation [5][6][7][8] and carboxymethylation [9,10].…”