Abstract
Centaurin-alpha(1) is a phosphatidylinositol 3,4,5-trisphosphate binding protein as well as a GTPase activating protein (GAP) for the ADP-ribosylation factor (ARF) family of small GTPases. To further understand its cellular function, we screened a rat brain cDNA library using centaurin-alpha(1) as bait to identify centaurin-alpha(1) interacting proteins. The yeast two-hybrid screen identified a novel kinesin motor protein as a centaurin-alpha(1) binding partner. The motor protein, termed KIF13B, encoded by a single approximately 9.5-kb transcript, is widely expressed with high levels observed in brain and kidney. Yeast two-hybrid and GST pull-down assays showed that the interaction between centaurin-alpha(1) and KIF13B is direct and mediated by the GAP domain of centaurin-alpha(1) and the stalk domain of KIF13B. Centaurin-alpha(1) and KIF13B form a complex in vivo and the KIF13B interaction appears to be specific to centaurin-alpha(1) as other members of the ARF GAP family did not show any binding activity. We also show that KIF13B and centaurin-alpha(1) colocalize at the leading edges of the cell periphery whereas a deletion mutant of centaurin-alpha(1) that lacks the KIF13B binding site, failed to colocalize with KIF13B in vivo. Finally, we demonstrate that KIF13B binding suppresses the ARF6 GAP activity of centaurin-alpha(1) in intact cells. Together, our data suggest a mechanism where direct binding between centaurin-alpha(1) and KIF13B could concentrate centaurin-alpha(1) at the leading edges of cells, thus modulating ARF6 function.
Highlights
The ADP-ribosylation factor (ARF) are Ras-related small GTPases that regulate intracellular vesicle trafficking by shuttling between an inactive GDP-bound and an active GTP-bound form (Moss and Vaughan, 1998; Randazzo et al, 2000)
Interaction appears to be specific to centaurin-α1 as other members of the ARF GTPase activating protein (GAP) family did not show any binding activity
We show that KIF13B and centaurin-α1 colocalize at the leading edges of the cell periphery whereas a deletion mutant of centaurin-α1 that lacks the KIF13B binding site, failed to colocalize with KIF13B in vivo
Summary
The ARFs are Ras-related small GTPases that regulate intracellular vesicle trafficking by shuttling between an inactive GDP-bound and an active GTP-bound form (Moss and Vaughan, 1998; Randazzo et al, 2000). It has been shown recently that the cytohesin family of ARF GEFs is recruited to the plasma membrane in agonist-stimulated cells by binding phosphatidylinositol 3,4,5trisphosphate [PtdIns(3,4,5)P3], leading to the activation of ARF6 (Cullen and Venkateswarlu, 1999; Hawadle et al, 2002). This observation suggests a possible involvement of PtdIns(3,4,5)P3 in ARF6-associated cellular responses, a view further strengthened by the identification of ARF GAPs such as centaurin-α1 and ARAP3 (ARF GAP, Rho GAP, ankyrin repeat, PH protein 3) as PtdIns(3,4,5)P3 binding proteins (Hammonds-Odie et al, 1996; Krugmann et al, 2002).
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