Polyamine Stimulation of Protein Phosphorylation in Isolated Pea Nuclei

Abstract

ABSTRACIrThe phosphorylation of several proteins in isolated nuclei from Pisum sativum L. was stimulated by spermine. Although spermine increased the general protein phosphorylation by 10 to 20%, it increased the phosphorylation of a 47 kilodalton polypeptide by 150%. By comparison other polyamines, spermidine, putrescine, and cadavarine had far less effect on the phosphorylation of the 47 kilodalton or any other polypeptide. Sodium fluoride was able to inhibit the phosphorylation of the 47 kilodalton polypeptide in the control, implying the participation of protein phosphatase(s) in the phosphorylation of nuclear proteins. Spermine stimulated the phosphorylation of the 47 kilodalton polypeptide over the controls, even in the presence of NaF. This result indicates that spermine probably activates a nuclear kinase, a conclusion supported also by thiophosphorylation data. The inability of ethyleneglycol-bis(6-aminoethyl ether)-N,N'-tetraacetic acid and Compound 48/80, a calmodulin antagonist, to inhibit this spermine stimulated phosphorylation renders improbable any role ofcalcium and calmodulin in mediating this response.The role of polyamines in plant growth and development is well documented (11,25). Polyamines are known to regulate a wide variety of physiological and biochemical processes in the nuclei. Some of these include regulation of cell division (5, 16), and the activities of several enzymes involved in nucleic acid synthesis (14, 27) and repair (23). The activity of at least some of these enzymes is known to be controlled by polyamines via modulation of their phosphorylation state. In slime molds, Kuehn et al. (18) showed that polyamines regulate the phosphorylation of a certain nucleolar protein that regulates rRNA synthesis. They later identified this protein as ornithine decarboxylase and showed that polyamines act via regulating the activity of a protein kinase which in turn regulates the phosphorylation state and hence the activity of ornithine decarboxylase (2,19).We have already shown that phytochrome stimulates the phosphorylation of certain proteins in isolated nuclei from peas (9). In pea seedlings, phytochrome has also been shown to regulate polyamine biosynthesis by modulating the activity of arginine decarboxylase, one ofthe first enzymes in polyamine biosynthesis (6). In animal systems, polyamines are known to regulate nuclear protein phosphorylation (4,15,18). Given the above results, we decided to test the effect of polyamines on protein phosphorylation in isolated pea nuclei.In the present paper we show that polyamines, especially spermine, stimulate the phosphorylation of certain nuclear pro-'This work was supported by the National Science Foundation Grant PCM 8402526. teins in peas. We also present evidence that this stimulation is probably due to an increased activity of a protein kinase. Since the maximum stimulation of phosphorylation is of a 47 kD polypeptide most of the work in this paper is on the regulation of phosphorylation of the 47 kD polypeptide containing nuclear prote...

Partial Purification and Characterization of an Enzyme from Pea Nuclei with Protein Tyrosine Phosphatase Activity

“…The pea nuclear phosphatase is inhibited by both agents. A possible significance of nuclear phosphatase inhibition by spermine may be seen in the fact that spermine treatment stimulates the phosphorylation of several pea nuclear proteins (Datta et al, 1986), including a 77-kD protein that we have found that is recognized by anti-phosphotyrosine antibodies (data not shown). The possibility that the spermine inhibition of a phosphatase could be a means of stabilizing the spermine-induced phosphorylation state of nuclear proteins deserves further investigation.…”

Characterization of Spermidine Binding to Solubilized Plasma Membrane Proteins from Zucchini Hypocotyls1

“…In both animal and plant systems it has been postulated that these compounds may play a role in the posttranslational modifications of proteins (Ä schlimann and Paulsson, 1994;Serafini-Fracassini et al, 1995), as well as in the modulation of many enzyme activities such as protein kinases, phosphatases (Datta et al, 1986;Friedman, 1986), and 1,3-␤-glucan synthase (Kauss and Jeblick, 1985). Despite the large amount of information on the covalent interactions between polyamines and proteins, little is known about the noncovalent binding of polyamines to PM proteins, even though this may represent one of the first steps in their action at the cellular level.…”

The effect of polyamines on protein kinase activities of wheat (Triticum aestivum) L. anthers

“…Moreover, these amines are now known to be associated with elements in signal transduction systems like phosphoinositide metabolism and protein kinase activities [31,23] . Polyamines were found to inhibit certain histone type kinase activities in wheat embryos and soybean hypocotyls [28,20] while kinases from corn coleoptile and pea nuclei were stimulated by polyamines [34,7] .…”

“…The summarized data and concentrations used are shown in Table 1 . The polyamines, spermine, spermidine, putrescine, and L-arginine were included in 1 mM concentrations [7] . Protein kinase activity was determined in 30 µl reaction mixtures containing substrate proteins from the crude extract in a concentration of 4 .6 mg/ml and 0 .25 nmoles adenosine 5'-[y 3 2 P] triphosphate triethyl-ammonium salt ([y3 2 P]ATP) .…”