Biochemical characterization of purified mammalian ARL13B protein indicates that it is an atypical GTPase and ARL3 guanine nucleotide exchange factor (GEF)

Abstract

Primary cilia play central roles in signaling during metazoan development. Several key regulators of ciliogenesis and ciliary signaling are mutated in humans, resulting in a number of ciliopathies, including Joubert syndrome (JS). ARL13B is a ciliary GTPase with at least three missense mutations identified in JS patients. ARL13B is a member of the ADP ribosylation factor family of regulatory GTPases, but is atypical in having a non-homologous, C-terminal domain of ∼20 kDa and at least one key residue difference in the consensus GTP-binding motifs. For these reasons, and to establish a solid biochemical basis on which to begin to model its actions in cells and animals, we developed preparations of purified, recombinant, murine Arl13b protein. We report results from assays for solution-based nucleotide binding, intrinsic and GTPase-activating protein-stimulated GTPase, and ARL3 guanine nucleotide exchange factor activities. Biochemical analyses of three human missense mutations found in JS and of two consensus GTPase motifs reinforce the atypical properties of this regulatory GTPase. We also discovered that murine Arl13b is a substrate for casein kinase 2, a contaminant in our preparation from human embryonic kidney cells. This activity, and the ability of casein kinase 2 to use GTP as a phosphate donor, may be a source of differences between our data and previously published results. These results provide a solid framework for further research into ARL13B on which to develop models for the actions of this clinically important cell regulator.

Keywords: ADP ribosylation factor (ARF); ARL13B; ARL3; GTPase; GTPase-activating protein (GAP); HEK cell expression; guanine nucleotide binding; guanine nucleotide exchange factor (GEF); intrinsic GTPase activity; kinetics.

Figure 1.

ARL13B is an atypical member of the ARF family of regulatory GTPases. Primary sequence alignment of murine MmArl13b, MmArl2, MmArf1, and MmHras is shown. Residues of ARL13B mutated and analyzed in our study are highlighted in red. Sites of phosphorylation identified in mouse ARL13B are underlined and in bold. The G1–G5 motifs are highlighted in green. The predicted coiled-coil and proline-rich regions are highlighted in gray and light blue, respectively. Residues involved in covalent modifications that promote membrane association are highlighted in yellow: N-terminal glycine for ARF1 myristoylation, C-terminal farnesylation for HRAS, and palmitoylation sites near the N terminus of ARL13B.

Figure 2.

Purified protein preparations of murine Arl13b. Murine GST-Arl13b was purified from HEK cells as described under “Experimental procedures.” The purified preparation (2 μg) is shown prior to (lane 2) or after overnight cleavage (at 4 °C) with TEV protease (lane 3). Gel filtration using a Sephadex S200 column resulted in removal of GST and TEV (lane 4). Protein standards are shown in lane 1, with sizes of each marked on the left.

Figure 3.

Filter trapping assay highlights the aberrant properties of murine Arl13b in this assay. The nitrocellulose filter trapping assay was performed as described under “Experimental procedures,” using either [³⁵S]GTPγS (A and C) or [³H]GDP (B) as ligands. Purified human ARL2 is included as a positive control for guanine nucleotide binding. Filled symbols show results with GST-Arl13b or Arl13b. A, GTPases (1 μm) were incubated in the presence of 10 μm [³⁵S]GTPγS at 30 °C, and time points were taken at the indicated times. Note that the amount of [³⁵S]GTPγS bound to ARL2 declines over time, as a result of protein instability. B, binding of [³H]GDP is shown for the same two proteins under the same conditions and again ARL2 binds rapidly but this is lost with time due to protein instability. In contrast, murine GST-ARL13B does not bind GDP. C, dissociation of the [³⁵S]GTPγS was monitored after pre-incubation as described in A by addition at t = 0 of excess (100 μm) unlabeled GTPγS. The experiments were performed in duplicates, with at least two different preparations of each protein. Each point shows the mean ± S.E.

Figure 4.

Murine Arl13b is a phosphoprotein that can be thiophosphorylated on Ser-328 by CK2 in standard in vitro assays for GTPγS binding. Left panels in each case show Coomassie Blue staining of the gel, and the right panel shows the results from autoradiography of the dried gel, revealing covalently bound [³⁵S]thiophosphate incorporation. The times of incubation at 30 °C are indicated at the top. A, indicated preparations of murine Arl13b (4 μm) were incubated in the radioligand binding assay using [³⁵S]GTPγS, but instead of filter trapping to detect bound nucleotide, the reactions were stopped with Laemmli SDS sample buffer, and proteins were resolved in denaturing SDS gels and stained with Coomassie Blue, prior to drying and exposure to film. B, murine and human GST-ARL13B (2 μg) were analyzed as described in A, in the absence or presence of the CK2 inhibitor (10 μm). C, commercially obtained and purified, recombinant CK2 increases the incorporation of thiophosphate into murine but not human GST-ARL13B (2 μm). The GTPases were incubated with [³⁵S]GTPγS for the times indicated at 30 °C with and without added CK2 (4 μm). Exposure times for films were the same within each panel but differed between panels. For example, C was exposed to film for a shorter time than in A, to minimize saturation of the signal.

Figure 5.

Solution-based binding of mant-Gpp(NH)p reveals that all ARL13B mutants, except K34A, bind with similar apparent affinities. The relative fluorescence intensity of different preparations of ARL13B were determined using mant-Gpp(NH)p in the binding assay, performed at 26 °C as described under “Experimental procedures.” Initial rates of binding were used to determine on-rates. After 25 min, excess (100 μm) unlabeled Gpp(NH)p was added to initiate monitoring of the rates of dissociation. A, binding and dissociation of mant-Gpp(NH)p to murine GST-Arl13b with (red circles) human GST-ARL13B (blue circles) are shown. Human ARL3 (green circles) is included as a positive control and GST alone (black circles) as a negative control. B, comparison of wild type to T35N and G75Q (red circles, black squares, and blue squares, respectively) reveal no significant differences in on- or off-rates. Murine GST-Arl13b (K34A) (green squares) was included as another negative control. C, three Joubert mutants GST-Arl13b (R79Q) (blue triangles), GST-Arl13b (Y86C) (green triangles), and GST-Arl13b (R200C) (black triangles) were analyzed in the same binding assay and are shown in comparison with the wild-type protein (red circles).

Figure 6.

All three Joubert mutations are devoid of ARL3 GEF activity. The ARL3 GEF assay was performed as described under “Experimental procedures.” This involved pre-incubation of purified ARL3 with [³H]GDP and then monitoring the rate of dissociation of the radioligand by addition of excess unlabeled GDP in the absence or presence of ARL13B preparations, as indicated above each graph. Time points were collected up to 15 min, and nucleotide remaining bound to ARL3 was determined by filter trapping. The amount of binding observed prior to addition of cold GDP and ARL13B was normalized to 100% to facilitate comparisons. Dissociation of [³H]GDP from ARL3 alone (no ARL13B added; filled circles) was determined in every assay performed but is only shown in A and B for clarity. The activity of each indicated ARL13B was determined for the apoprotein (no pre-incubation; filled squares) of after pre-loading of the ARL13B with GTPγS (100 μm; open triangles). A, we also show the results from pre-incubation of murine GST-Arl13b with 100 μm GDP (open squares). F, data for apo-GST-Arl13b (S328A) and apo-GST-Arl13b (S328D) are shown with asterisk and squares, respectively, and that for the same proteins pre-incubated with 100 μm GTPγS are shown with crosses and triangles, respectively. Each sample was assayed at least three times in duplicate using at least two different preparations of each protein. Bars represent one standard error, based upon at least six determinations.