In a previous study, we reported the GDP-dependent phosphorylation of a 36 kD membrane protein, p36, in D. discoideum membranes prepared from starved (aggregation competent) cells (Anschutz et al., 1989). Here we show that p36 can be phosphorylated when membranes are supplied either ATP or GTP as the phosphate donor, but that a greater level of p36 phosphorylation is achieved with GTP. The rate of phosphorylation of p36, using either nucleotide triphosphate, is enhanced by GDP. This reflects a decrease in the apparent Km of the enzyme for the particular nucleotide triphosphate. p36 can also be phosphorylated in membranes prepared from vegetative cells. However, the ability of GDP to stimulate p36 phosphorylation is not observed in vegetative cell membranes. Competition experiments indicate that there are also developmental differences in the nucleotide triphosphate site(s) available to phosphorylate p36.
Increasing effort is directed toward elucidating the mechanisms by which guanine nucleotide-binding proteins regulate specific cellular processes. A common feature of this class of proteins is that GTP induces a transition from an inactive to an active conformation. The latter is limited by the hydrolysis of GTP and the coincident production of GDP. Here we provide evidence that guanine nucleotides may regulate biological processes by inducing the phosphorylation of specific proteins. In particular, we report a GDP-dependent phosphorylation of p36, a 36-kDa protein of Dictyostelium discoideum plasma membranes.Phosphorylation-dephosphorylation of key cellular components is a widely used mechanism for controlling biological processes. These modifications are catalyzed by protein kinases and phosphatases that recognize a diversity of substrates, such as hormone receptors, ion channels, enzymes, and structural proteins (1)(2)(3)(4). Protein kinases are themselves regulated by specific effector molecules. Frequently these effectors function as second messages for hormones, thus providing an important means of continuing the cascade of intracellular events that define the physiological response. Particular kinases are categorized with respect to their specific effectors-e.g., cAMP-dependent, cGMPdependent, Ca2 /calmodulin-dependent, or Ca2 /phospholipid-dependent protein kinase. We have observed that a protein kinase in Dictyostelium discoideum plasma membranes phosphorylates a 36-kDa protein (p36) also present in that subcellular fraction and does so in a GDP-dependent manner. Such an activity provides evidence for an unusual regulation of protein phosphorylation involving guanine nucleotides. MATERIALS AND METHODSStrains and Culture Conditions. Ax-2 amoebae (5) were grown in HL-5 medium (6). Cell differentiation to aggregation competence was induced by starving amoebae in 20 mM sodium phosphate (pH 6.5) (7) and was monitored morphologically (8).Membrane Phosphorylation. The plasma membraneenriched fraction was prepared from vegetative or starved cells and phosphorylated at 21°C using ['y-32P]ATP as described (9). Additions to the reaction mixture are designated in the figure legends. The reaction was terminated after the indicated times of incubation by the addition of SDS/PAGE sample buffer (10) and the products were analyzed by SDS/PAGE (10) and autoradiography. Where indicated, linear-range autoradiograms were analyzed using a Bio-Rad model 620 video densitometer and 1-D analyst software to determine peak areas.Other Procedures. Western blots were performed according to Mumby et al. (11) except that no detergents were added to the buffers. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin treatment of partially purified p36 was performed according to Fung and Nash (12 RESULTS AND DISCUSSIONIn the course of studies in this laboratory on phosphorylation mechanisms in D. discoideum (9, 13, 14), it was observed that the phosphorylation of a 36-kDa protein (p36) was enhanced by GDP. Fig. 1 sho...
We have examined the phosphorylation of the cyclic adenosine 3':5' monophosphate (cAMP) cell surface chemotactic receptor and a 36 kDa membrane-associated protein (p36) in Dictyostelium discoideum. The activity of CAR-kinase, the enzyme responsible for the phosphorylation of the cAMP receptor, was studied in plasma membrane preparations. It was found that, as in intact cells, the receptor was rapidly phosphorylated in membranes incubated with [gamma 32P] adenosine triphosphate (ATP) but only in the presence of cAMP. This phosphorylation was not observed in membranes prepared from cells which did not display significant cAMP binding activity. cAMP could induce receptor phosphorylation at low concentrations, while cyclic guanosine 3':5' monophosphate (cGMP) could elicit receptor phosphorylation only at high concentrations. Neither ConA, Ca2+, or guanine nucleotides had an effect on CAR-kinase. It was also observed that 2-deoxy cAMP but not dibutyryl cAMP induced receptor phosphorylation. The data suggest that the ligand occupied form of the cAMP receptor is required for CAR-kinase activity. Although the receptor is rapidly dephosphorylated in vivo, we were unable to observe its dephosphorylation in vitro. In contrast, p36 was rapidly dephosphorylated. Also, unlike the cAMP receptor, the phosphorylation of p36 was found to be regulated by the addition of guanine nucleotides. Guanosine diphosphate (GDP) enhanced the phosphorylation while guanosine triphosphate (GTP) decreased the radiolabeling of p36 indicating that GTP can compete with ATP for the nucleotide triphosphate binding site of p36 kinase. Thus was verified using radiolabeled GTP as the phosphate donor. Competition experiments with GTP gamma S, ATP, GTP, CTP, and uridine triphosphate (UTP) indicated that the phosphate donor site of p36 kinase is relatively non-specific.(ABSTRACT TRUNCATED AT 250 WORDS)
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