Centaurin-alpha1 interacts directly with kinesin motor protein KIF13B.
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.
Venkateswarlu
Kanamarlapudi
K
Department of Pharmacology, School of Medical Sciences, The University of Bristol, University Walk, Bristol, BS8 1TD, UK. k.venkateswarlu@bristol.ac.uk
Hanada
Toshihiko
T
Chishti
Athar H
AH
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004285:Dogs; D020690:GTPase-Activating Proteins; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D051381:Rats; D020798:Two-Hybrid System Techniques
D000089982:ADP-Ribosylation Factor 6; C491062:Adap1 protein, rat; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D020409:Molecular Motor Proteins; D009419:Nerve Tissue Proteins; C504977:KIF13B protein, rat; D016547:Kinesins; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715349:Arf6 protein, rat