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...
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