Dirk Van den BROECK, Marc Van MONTAGU and Allan CAPLAN
Laboratorium voor Genetica, Universiteit Gent, K. L. Ledeganckstreat 35, B-9000 Gent, Belgium
Despite the rapidly growing body of evidence that the concentration of polyamines changes during the course of such diverse phenomena as development, cell division, stress response, and senescence, there are few, if any, studies showing whether these fluctuations are responses or determinants of the associated physiological events. Recently, it has been demonstrated that polyamines can differentially modulate the transcription of growth-associated genes (Celano et al. 1989). One way that changes in polyamine levels might alter gene expression is by stimulating phosphorylation of regulatory proteins. lt is known that many transcription factors are modulated by their phosphorylation status. Factors like myb (Luscher et al. 1990) and AT-1 (Datta and Cashmore 1989) loose DNA-binding capacities upon phosphorylation by casein kinase II (CKII). Others, like c-jun gain activity, not as a result of increased binding but because of potentiation of the transactivation domain (Binetruy et al. 1991). In order to understand better physiological controls over gene expression in rice, we have performed several preliminary studies on the phosphorylation of nuclear proteins in response to exogenous polyamines.
Nuclei of lamina from 8-week-old rice plants (Oryza sativa var. Taipei 309) were isolated according to the method of Hamilton et al. (1972). Storage of nuclei at -20°C for several months did not visibly alter phosphorylation activities in vitro.
To perform an in vitro kinase assay, the nuclear pellet (2 x 106 nuclei) was taken up in 50 μl of kinase buffer (10 mM Tris, pH 7.5, 10μM MgCl2, 1.14 M sucrose) in the absence or presence of different supplements (Ca2+, spermine, spermidine). After pre-incubation for 2 minutes at room temperature the reaction was started by the addition of 50 μM ATP, 10 μCi γ-32 ATP (specific activity 300 Ci/mmol) followed by incubation for 30 minutes at room temperature. The reaction was stopped by mixing with 50 μl of phenol (saturated with 50 mM Tris, pH 8, 20 mM EDTA). After 20 minutes, the phenol phase was isolated by centrifugation and washed twice with an equal volume of extraction buffer (0.5 M Tris, 30 mM HCl, 50 mM EDTA, 0.1 M KCl, 2% β-mercaptoethanol, 12 mg/ml polyvinylpolypyrrolidone). The aqueous phase was discarded after each wash. The proteins were finally precipitated out of the phenol by the addition of 5 volumes 0.1 M NH4OAc in methanol with an overnight incubation at -20°C. The pellet was washed once with 80% acetone, 20% water. After drying the pellets, the samples were dissolved in lysis buffer and electrophoresed on sodiuni dodecylsulfate-polyacrylamide (12%) gels. The gels were then silver stained (Ansorge 1985), dried, and autoradiographed using Kodak-AR films.
Although Ca2+ has been shown to induce phosphorylation in pea nuclei (Datta et al. 1985), we did not see any effect of Ca2+ , up to 2 mM on the phosphorylation pattern in rice nuclei (data not shown). By contrast, spermine (1 mM) and spermidine (1 mM) clearly induced the phosphorylation of five protein species. Besides the 18-, 26-, 28-, and 46-kDa phosphorylated bands seen in the control, bands of 16, 17, 55, and most especially 34 and 23 kDa appeared. Addition of both spermine and spermidine showed a strong synergistic effect. Figure I shows the silver-stained pattern (B) and spermine-induced phosphorylation (A). None of the phosphorylated proteins can be detected as a major component of the nuclear extracts.
CKII is the only kinase known to be induced by polyamines (Edelman et al. 1987). In order to show that the polyamine induced phosphorylation is due to a CKII-like activity within the nuclei of rice, we established the following kinase assay. The reaction mixture (30μl) contained 50 mM Tris, pH 7, 10 mM MgCl2, 1% casein, 10 mM ATP (1 μl γ-32R-ATP), and 5 μl nuclear extract, corresponding to 2 X 105 nuclei.
After 30 minutes, the reaction was stopped by spotting the mixture onto Whatman 3MM chromatographic paper (Thomas et al. 1968). The paper was washed at 4°C, once with 10% trichloroacetic acid (TCA) for 30 minutes, once with 5% TCA for 30 minutes, and twice with 5% TCA at 18-20°C for 30 minutes. The treated paper was then washed with ethanol, rinsed with ether, and dried. After autoradiography, the spots were cut out and counted. One assay was performed in the absence of any kinase to show a specific binding (Figure 2, lane 1). To reduce background, 1% KH2PO4 and 1% Na4P2O7 (pyrophosphate) were included in all TCA solutions.
Fig. 1. A. Autoradiogram of proteins phosphorylated in the presence (lane 1) or absence (lane 2) of 1 mM spermine. The most prominent constitutively phosphorylated proteins are marked with a dash, the spermine-induced ones with an arrowhead.
B. Respective protein patterns after silver staining. Position of molecular mass standards are shown at the far right.
Figure 2 shows that these nuclear extracts contained an activity that was strongly induced (2- to 3-fold) by 2 mM spermine. Furthermore. we saw that heparin, a typical inhibitor of CKII, almost completely abolished any kinase activity, even in the presence of 2 mM spermine. These observations indicated that a CKII-like activity was active in the rice, nuclei. A third criterion establishing the CKII-like nature of the phosphorylation was the ability of GTP to substitute for ATP as a phosphoryl donor. However, when we used γ-GTP instead of γ-ATP for the spermine-induced phosphorylation of endogenous proteins, only the 23-kDa protein seemed to be phosphorylated well (data not shown). We do not know the nature of the kinase(s) that phosphorylate the other proteins. The fact that they too are clearly phosphorylated points towards the existence of an unknown kinase, inducible by spermine, but unable to use GTP. lt is also possible that the spermine-induced phosphorylation of the other proteins merely the result of a kinase cascade, with a spermine-inducible CKII which not only phosphorylates the 23-kDa target protein, but also one or more other kinases that are then activated and capable of phosphorylating the remaining "spermine-induced" proteins.
We are currently trying to unravel the existence for such a kinase cascade. Additional studies have shown that at least the 23-, 34-, and 55-kDa proteins bind DNA in vitro.
Fig. 2. Induction of kinase activity by spermine (panel A) and inhibition by heparin (panel B). The kinase assay was performed as described. The reaction was stopped after 30 minutes (panel A) and 15 minutes (panel B). Panel A: lane 1, background; lane 2, control; lanes 3, 4, and 5, induction by spermine at concentrations of 0. 1, 0.5, and 2 mM, respectively. Panel B: lane 1, background; lane 2, control; lane 3, induction by 2 mM spermine; lanes 4 and 5, inhibition by 2 μg/ml heparin in the absence and the presence of 2mM spermine, respectively.
The authors would like to thank Martine De Cock for her expert help in assembly and layout of this manuscript, and to the Rockefeller Foundation (RF #89066) and the Services of the Prime Minister (U.I.A.P. 120C01.87) for funding.
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