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

Anna A Ivanova, Tamara Caspary, Nicholas T Seyfried, Duc M Duong, Andrew B West, Zhiyong Liu, Richard A Kahn

The Journal of biological chemistry 2017 Jun 30

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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. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Citation

Anna A Ivanova, Tamara Caspary, Nicholas T Seyfried, Duc M Duong, Andrew B West, Zhiyong Liu, Richard A Kahn. Biochemical characterization of purified mammalian ARL13B protein indicates that it is an atypical GTPase and ARL3 guanine nucleotide exchange factor (GEF). The Journal of biological chemistry. 2017 Jun 30;292(26):11091-11108