Phosphoprotein Phosphatase of Soybean Hypocotyls

Abstract

A soybean histone-type protein kinase was used to prepare 32P-labeled histone Hi as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.13.16) phorylation and dephosphorylation has been suggested to be an important mechanism in the control of metabolic processes in higher plants (12,25). Randall and his colleagues (21) have shown that phosphorylation of the pyruvate dehydrogenase complex from broccoli mitochondria results in inactivation of the enzyme. Additionally, the nuclear (17), ribosomal (20, 24) and membrane proteins (1) of higher plants are also subject to modification by phosphorylation, although the biological significance of the phosphorylation is unknown. The phosphorylation of these proteins apparently is catalyzed by several protein kinases, including histone-type and casein (or phosvitin)-type enzymes (9,12,25). Although much work has been carried out on phosphorylation of proteins and demonstration of the activities of protein kinases, little attention has been paid to phosphoprotein phosphatase (EC 3.1.3.16) which catalyzes the protein dephosphorylation reaction. There apparently has been no characterization of the phosphatase from higher plants, except a brief mention of the presence of phosphohistone phosphatase activity in pea seedlings (16). Here, we describe the purification and properties of a phosphoprotein phosphatase from soybean hypocotyls. The enzyme is distinct from the phosphatases which catalyze the hydrolysis ofnonprotein low mol wt phosphoesters. Additionally, several lines of evidence indicate that the soybean enzyme is a nonspecific phosphoprotein phosphatase acting on phosphorylated histones, protamine, and, possibly, phosphorylated casein and phosvitin. MATERIALS AND METHODSUnless otherwise stated, the chemicals and biochemical compounds used in this study were obtained from the sources as indicated in the previous paper (12). [y-32PJATP was prepared according to the method of Schendel and Wells (22). All other chemicals were of reagent grade. Soybean seeds (Glycine max var. Wayne) were germinated in the dark and the mature hypocotyls were harvested as described (12).Preparation of 32P-labeled and Nonradioactive Phosphohistone Proteins. Lysine-rich histone HI and other histone species (H2A, H2B, H3, and H4) were purified from the total calfthymus histone mixture (Sigma II or IIA) by column chromatographies on BioGel P-60 and P-10 (12, 14). A histone-type protein kinase with high specificity for histone HI was partially purified from soybean hypocotyls as described (12); the kinase was used to phosphorylate histone H1. For the preparation of 32P-labeled histone species other than histone H1, a cAMP-dependent histone kinase from rat liver cytosol (4) was used. The liver kinase was partially purified by ammonium sulfate fractionation and DEAE-Sephadex A-25 column chromatography.To prepare a large quantity of 32P-labeled histone H1, the reaction mixture in a total volume of 10 ml contained 20 mM Tris-HCI (pH 8.

Complementation of the cs dis2-11 cell cycle mutant of Schizosaccharomyces pombe by a protein phosphatase from Arabidopsis thaliana.

“…Protein phosphatase 1 related enzymes have been detected in all eukaryotic cells tested. Thus, it was not unexpected that protein phosphatases would be detected in higher plants (Bennett, 1980;Lin et al, 1980;Polya and Haritou, 1988; MacKintosh and Cohen, 1989). It was found that of the four major protein phosphatase catalytic subunits, only type 1 and type 2A activities were present in plant cell extracts at particularly high levels (MacKintosh and Cohen, 1989).…”

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

“…These genes can be grouped into three families: the family consisting of PP2A-1, PP2A-2 and the gene product containing the AP-1 sequence, all of them very related to each other and also to the PP2A proteins from other organisms; the family that includes PP2A-3, also very related to other PP2A proteins (Table 1); and the family corresponding to the gene(s) containing the AP-2 sequence, more distantly related to the previously mentioned families and also to the type 2A phosphatases from other organisms, but still closer to the type 2A family than to type 1 or 2B phosphatases. Whether or not any of these proteins correspond to plant phosphatase activities so far characterized [14,19,26] cannot be determined at present. It fact, it would be very interesting to know if the complexity of type 2A protein phosphatase gene family found in A.…”

Protein Kinase Activities in Tonoplast and Plasmalemma Membranes from Corn Roots

“…Under the conditions studied, the Tp fraction lacks any protein phosphatase activity. In this fraction, the cycle of phosphorylation and dephosphorylation may be completed by a soluble protein phosphatase (14) …”

“…When labeling by protein kinase was stopped by adding 5 mM ATP, no phosphatase activity was detected in either fraction. This observation suggests that ATP may serve as a substrate for the protein phosphatase and competes with phosphoprotein as a substrate (14). Endogenous membrane protein phosphatase (8,23) and soluble phospho-histone H 1 phosphatase (14) have been reported in plants.…”