The Roc domain of GbpC is activated by the GbpC-RasGEF domain. A, the purified Roc-Cor fragment of GbpC was loaded with the fluorescent GDP analog mGDP, and the decay of fluorescence was followed over time in the presence of excess GDP. The stability of the protein was measured by incubating the mGDP-loaded protein in assay buffer without further additions. The intrinsic exchange activity was measured by incubating the mGDP-loaded protein with an excess of unlabeled GDP. The addition of the purified RasGEF domain of GbpC causes a dramatic increase in the exchange rate, meaning that GbpC uses its own RasGEF to activate the Roc domain. B, same assay as in A, but the Roc-Cor fragment of GbpC was replaced by several other GTPases. Intrinsic exchange rates and exchange rates in the presence of GbpC-RasGEF are shown. No differences were found upon the addition of the GbpC-RasGEF domain, confirming specificity of GbpC-RasGEF for its own Roc. All of the exchange assays were repeated at least twice on different days, yielding similar results. Dd, Dictyostelium discoideum; Hs, Homo sapiens.

Roc-mediated GTP-binding by GbpC is specific and is stimulated by cGMP. A and B, full-length GbpC-GFP (∼320 kDa) and GbpC-ΔcGMP-GFP, expressed in gbpC null cells, can be visualized by Western blotting. A truncated protein (∼140 kDa) is also visible, and its presence is developmentally regulated. Equal amounts of cells were taken at several time points during starvation. The cells were boiled in SDS loading buffer and resolved by SDS-PAGE, and GbpC-GFP expression was visualized with Western blotting, using anti-GFP antibodies. C, GbpC-GFP was expressed in gbpC null cells and pulled down from lysates with GTP-agarose beads in the presence of equal volumes of lysis buffer, cGMP, or GTP, respectively. Bound protein was visualized on a Western blot, using anti-GFP antibodies. D, GbpC and the GbpC-RocInactive mutant with an inactive Roc domain were pulled down with GTP-agarose. The results show very little binding of GbpC-RocInactive to GTP-agarose. The inputs are shown as a control, indicating equal expression levels of both constructs. The proteins were visualized on a Western blot, using anti-GFP antibodies. E, cGMP stimulates GTP binding in mutants with abolished kinase or LRR domains but does not stimulate GTP binding in a mutant that is unable to bind cGMP or in a mutant with a deletion of the catalytic part of the RasGEF domain. GFP-fused GbpC mutants were pulled down with GTP-agarose as in A and visualized with anti-GFP antibodies on a Western blot. Representatives of at least three independent experiments on different days are presented.

Model for the activation mechanism of GbpC. The essential core of GbpC consists of at least the LRR, GEF, Roc, and kinase domain. Inactivation or deletion of one of these domains abolishes all GbpC activity in vivo. cGMP is produced by sGC, and upon binding of cGMP to the cNBDs, GDP/GTP exchange on the Roc domain is stimulated by the RasGEF domain, which is exposed during cGMP binding. GTP binding to the Roc domain causes activation of the kinase domain, which is the output of the protein, analogous to other Roco proteins like LRRK1 and LRRK2. Abolished cGMP binding to GbpC does not fully inactivate its activity, and therefore a cGMP-independent activation mechanism also exists, hypothetically via a similar mechanism as the sGC protein.

Primer design, GbpC mutants and alignments. The gbpC open reading frame was amplified by PCR from cDNA in eight parts. The used primers consisted of the elements that are shown in A. The forward primer (5′-3′) contains a BglII or BamHI site for cloning in MB74 plasmids (in red), followed by a Kozak-sequence, a start codon (underlined), and a unique restriction site in the gbpC cDNA, which can be used for fusion of various parts. In the reverse primer (3′-5′), a unique site in the gbpC cDNA is followed by a SpeI or XbaI site for cloning in MB74-plasmids (in red) and a stop codon (underlined). B, overview of the GbpC domain structure and the constructs used in this study. The amino acid borders of eight parts of GbpC are indicated by numbers on the bar. The mutations and truncations of used constructs (left) and their corresponding names (right) are shown. The locations of point mutations are highlighted by asterisks. C, expression of GbpC mutants. The cytosols of AX3, gbpC null cells, and gbpC null cells expressing various GbpC-derived proteins were assayed for high affinity cGMP-binding sites, using 10 nm [³H]cGMP. The amount of binding sites is indicative for expression levels and protein stability and presented as a comparison with the amount of binding sites in AX3 cells. cGMP binding was determined in triplicate, and the assay was repeated at least once during the time course of this study to verify stable expression levels throughout the whole study. The average values ± S.D. are presented. D, alignment of the functionally important P-loop in the Roc domain of GbpC with a selection of other GTPases. The P-loop is underlined. A conserved lysine is indicated with an asterisk and was mutated to an asparagine to inactivate the nucleotide-binding abilities of the Roc domain. E, alignment of the first 31 amino acids of the kinase domain of GbpC with those of human LRRK1 and human LRRK2. Residues with dots are thought to be essential for ATP binding. A conserved lysine is indicated with an asterisk and was mutated to a tryptophan. F, alignment of part of the cyclic nucleotide-binding domains of GbpC and human protein kinase G. Underlined is the conserved FGE motif that is thought to be important for cGMP binding. The phenylalanines and glutamic acids of this motif (indicated with asterisks) were mutated to alanines in both cNBDs of GbpC to create a GbpC mutant that was unable to bind cGMP. G, alignment of part of the RasGEF domain of GbpC with human SOS1 and yeast CDC25. Underlined is a helical hairpin that is thought to be important for activity. A conserved phenylalanine is indicated with an asterisk and was mutated to an alanine. Dd, D. discoideum; Hs, H. sapiens.

Chemotaxis data of GbpC mutants. Seven-hour starved cells were analyzed in a small population assay and scored for their ability to chemotaxis toward drops with 10^-6 m cAMP in the presence of the PI3K inhibitor LY and the PLA₂ inhibitor p-bromophenacyl bromide. GbpC mutants were expressed in gbpC null cells and compared with the chemotaxis data of AX3 and gbpC null cells. The data presented are the means and standard error of the means of at least three independent measurements on different days. The data were analyzed by Student's t test; ^**, p < 0.001 versus GbpC and not significantly different from gbpC null at p > 0.05; ^*, significantly less than GbpC at p < 0.05 and significantly above gbpC null at p < 0.01.