Abstract
The small GTPase Rab35 is a molecular switch for membrane trafficking that regulates a variety of cellular events. We previously showed that Rab35 promotes neurite outgrowth of nerve growth factor-stimulated PC12 cells through interaction with centaurin-β2 (also called ACAP2). Centaurin-β2 is the only Rab35-binding protein reported thus far that exclusively recognizes Rab35 and does not recognize any of the other 59 Rabs identified in mammals, but the molecular basis for the exclusive specificity of centaurin-β2 for Rab35 has remained completely unknown. In this study, we performed deletion and mutation analyses and succeeded in identifying the residues of Rab35 and centaurin-β2 that are crucial for formation of a Rab35·centaurin-β2 complex. We found that two threonine residues (Thr-76 and Thr-81) in the switch II region of Rab35 are responsible for binding centaurin-β2 and that the same residues are dispensable for Rab35 recognition by other Rab35-binding proteins. We also determined the minimal Rab35-binding site of centaurin-β2 and identified two asparagine residues (Asn-610 and Asn-691) in the Rab35-binding site as key residues for its specific Rab35 recognition. We further showed by knockdown-rescue approaches that neither a centaurin-β2 binding-deficient Rab35(T76S/T81A) mutant nor a Rab35 binding-deficient centaurin-β2(N610A/N691A) mutant supported neurite outgrowth of PC12 cells, thereby demonstrating the functional significance of the Rab35/centaurin-β2 interaction during neurite outgrowth of PC12 cells.
Highlights
Centaurin-2 recognizes Rab35 but not the other 59 Rabs
We focused on centaurin-2, an Arf6-GTPase-activating protein (GAP) [26] that is required for nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells [8, 14], and analyzed the exclusive specificity of the Rab35/centaurin-2 interaction with Rab35 by performing deletion and mutation analyses
Because Rab1A/B, Rab8A/B, Rab10, Rab13, Rab15, and Rab35 are phylogenetically similar and belong to the same large branch in the phylogenetic tree [39], the amino acid sequences of their switch II region are highly conserved. Careful inspection of their sequences, revealed two Thr residues, one at AA position 76 (Thr-76) and the other at AA position 81 (Thr-81), that are unique to Rab35 (Fig. 1A, arrowheads). To determine whether these two Thr residues are involved in the specific recognition of Rab35 by centaurin-2, we performed site-directed mutagenesis and generated a Rab35(T76S/T81A) mutant that carries a switch II region of Rab1A/B (Fig. 1A, arrowheads)
Summary
Centaurin-2 recognizes Rab but not the other 59 Rabs. Results: The T76S/T81A mutation in the switch II region of Rab impaired its centaurin-2 binding activity. Rab is one such Rab protein and has been shown to bind various candidate effector molecules, including MICAL-1 [7], MICAL-L1 [7, 9, 10], MICAL-cl [7], OCRL [7, 11], RUSC2 [12], Fascin1 [13], and centaurin-2 [8, 14], and to be involved in various cellular events, including cytokinesis [11, 15, 16], cell migration [17, 18], phagocytosis [19, 20], immunological synapse formation [21], myelination [22], and neurite outgrowth [10, 14, 23, 24], most likely through regulation of endocytic recycling [25]. We focused on centaurin-2 ( called ACAP2), an Arf6-GAP [26] that is required for nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells [8, 14], and analyzed the exclusive specificity of the Rab35/centaurin-2 interaction with Rab by performing deletion and mutation analyses. We discuss the utility of the Rab35(T76S/T81A) mutant as a tool to investigate the involvement of the Rab351⁄7centaurin-2 complex in Rab35-dependent cellular events
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