This paper presents evidence that a polyaminedependent protein idnase (EC 2.7A 1.37) purified from nuclei ofthe slime mold Physarum polycephalum catalyzes phosphorylation of ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.
An acidic nucleolar phosphoprotein with a subunit M, of 70,000 was purified as an a parent dimer of 139,000 from isolated nuclei of the slime mo sarum polycephalum. The protein was purified without the aid of strong dissociating agents after its selective phosphorylation in isolated nuclei by a polyamine-mediated reaction. Its amino acid composition resembled that of a nucleolar phosphoprotein from Novikoff hepatoma ascites cells. The phosphoprotein stimulated rRNA synthesis 5-fold by RNA polymerase I within a nucleolar, ribosomal deoxyribonucleoprotein complex isolated from nucleoli of P. polycephalum. It was also identified as a component of the complex. It bound with high affinity and specificity to the palindromic ribosomal DNA of 38 X 106 Mr from P. polycephalum, which contained two coding sequences for 5.8S, 19S, and 26S rRNA. It also bound to three fragments of ribosomal DNA of Mr 21.2 X 106, 17.1 X 10", and 8.1 X 10", prepared by cleavage with restriction endonucleases HindIII, Pst I, and BamHI, respectively. All of these fragments included the symmetry axis of the palindromic ribosomal DNA. The phosphoprotein that had been treated with alkaline phosphataseagarose to hydrolyze the phosphate groups did not stimulate transcription and did not bind to ribosomal DNA or to the restriction fragments indicated. We have thus isolated a specific phosphoprotein with the capacity to stimulate transcription of a specific set of genes in a eukaryote. These findings suggest that this phosphoprotein may specifically regulate functions of ribosomal DNA in a manner dependent on its degree of phosphorylation.The polyamines, putrescine, spermidine, and spermine, have been implicated in the control and promotion of cell growth processes (1, 2). One favored proposal has linked the polyamines to the metabolism (1), structure (3), and function (3, 4) of the nucleic acids. Specifically, much correlative evidence implicates their involvement in stimulating rRNA synthesis although evidence for a causal relationship has not been conclusive (for reviews see refs. 1, 2, and 5).Recently the polyamines were found to selectively stimulate the phosphorylation of two nonhistone acidic proteins in isolated nucleoli from the slime mold Physarum polycephalum (6). Numerous investigations support the general notion that genetic regulatory molecules of eukaryotic cells are to be found among the nonhistone chromosomal proteins (7). The two nucleolar phosphoproteins were of major interest for further investigation of putative regulation of rRNA synthesis because the genes that code for rRNA in P. polycephalum reside on a satellite DNA that has been extensively characterized. This MATERIALS AND METHODS Preparation of Nuclei and Nucleoli. Nuclei were isolated from 48-hr shake cultures of microplasmodia (6,11). Slime-free nucleoli and nuclei were isolated by the Percoll (Pharmacia) gradient method (10) from synchronous surface cultures 18-21 hr after provision of nutrient medium (8).Purification of the 70,000 Mr Phosphoprotein. The total ...
Intact isolated nuclei and nucleoli from the slime mold Physarum polycephalum incorporated inorganic [32P]phosphate into the phenol-soluble acidic nuclear proteins when incubated for 1 h in a phosphorylation mixture consisting of [32P]phosphate (pH 6.8), glucose, Mg2+, and ATP. The polyamines spermidine and spermine, or equimolar combinations of these compounds with putrescine, at 1 mM total amine concentration, stimulated phosphorylation of these nuclear proteins compared to control experiments lacking these compounds. The magnitude of enhanced phosphorylation ranged from 6-fold to 30-fold depending on the combination of polyamines employed and the given nuclear preparation. Magnesium ion could not substitute for the polyamines in effecting this stimulation. Fractionation of the phosphorylated proteins by polyacrylamide gel electrophoresis in sodium dodecylsulfate revealed that different polypeptides among them were phosphorylated by nuclei in the presence, versus the absence, of the polyamines. In the presence of all three polyamines, each at 0.33 mM, enhanced phosphorylation was primarily due to [32P]-phosphate incorporation into four major nuclear proteins. These proteins had molecular weights of 14000, 27000, 52000, and 70000, respectively. In the absence of the polyamines, three major proteins were phosphorylated with molecular weights of 17 500, 21 200, and 25 000, respectively. The polypeptides of 52000 and 70000 molecular weight were also selectively phosphorylated in, and isolated from, intact nucleoli after incubation in phosphorylation mixtures supplemented with 1 mM total polyamines. The [32P]phospho-protein linkage of the 32P-containing substances in polyacrylamide gels was established by the capacity of pronase to eliminate these protein bands and by their insensitivity to nuclease digestion. Polyamine stimulation of the phosphorylation of these nuclear proteins was unaltered by 2-propynylamine, an irreversible inhibitor of amine oxidase. Moreover, H202 did not stimulate phosphorylation of these proteins by intact nuclei in which amine oxidase was inhibited by 2-propynylamine.
Methods are described for the detection and purification of a protein kinase from nuclei and nucleoli of Physarum polycephalum which catalyzed transfer of phosphate from [gamma-32P]ATP to a unique nonhistone protein of Mr 70 000 in a reaction that was polyamine dependent. Enzymatic phosphorylation of the nonhistone protein by the purified protein kinase was stimulated greatly, at times more than 60-fold, by the polyamines spermidine and spermine. This unique polyamine-dependent reaction was localized on the rDNA minichromosome of the nucleolus. The polyamine-dependent protein kinase, which was first partially purified with the acidic nonhistone protein fraction from isolated nucleoli, was resolved from at least six other protein kinases by phosphocellulose chromatography into a catalytic component of Mr 26 000 and a complex comprised of the catalytic component associated with a phosphate acceptor protein of Mr 70 000. The complex also catalyzed polyamine-dependent phosphorylation of the endogenous Mr 70 000 component. The resolved catalytic component catalyzed polyamine-dependent phosphorylation of a dephosphorylated Mr 70 000 nonhistone protein that had been independently isolated from nucleoli and previously demonstrated to have properties concordant with a specific regulatory role in rRNA gene transcription [Keuhn, G. D., Affolter, H. U., Atmar, V. J., Seebeck, T., Gubler, U., & Braun, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2541-2545]. These studies indicate one way that the polyamines may regulate rRNA gene transcription through the mediation of a highly specific nonhistone protein kinase.
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