GDP stimulates the phosphorylation of a 36-kDa membrane protein in Dictyostelium discoideum.

Abstract

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...

Evidence for allosteric regulation of succinyl-CoA synthetase

“…Phosphorylation was initiated by incubating samples with 10 nM [y-32P]GTP (1.0 Ci/,umol). Samples prepared in this manner could be monitored for p36 phosphorylation in the absence of any endogenous dephosphorylation, previously demonstrated in cruder preparations (Anschutz et al, 1989). To monitor dephosphorylation, the phosphorylation reaction was stopped by the addition of 30 mM EDTA and dialysed against buffer [50 mM Tris/HCl, 1 mM EDTA and 10 % (v/v) glycerol] to remove free radionucleotides.…”

“…Previous experiments from this laboratory (Anschutz et al, 1989; Um and Klein, 1991) have described the rapid phosphorylation and dephosphorylation of a 36 kDa protein (p36) in extracts of Dictyostelium. Phosphorylation of p36 was achieved preferentially using GTP, as opposed to ATP, as the phosphate donor, and was stimulated by the specific addition of relatively low concentrations of GDP.…”

Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation

“…It is also not clear to what extent this regulation is specific to Aform enzymes. In previous experiments examining the regulation of SCS phosphorylation in D. discoideum, we did not detect an increase in the level of protein phosphorylation upon the addition of high levels of either GDP or ADP (1,18). However, such experiments examined the enzyme released from mitochondria that were disrupted in the absence of detergent.…”