Venkateswarlu Kanamarlapudi, Hanada Toshihiko, Chishti Athar H
Department of Pharmacology, School of Medical Sciences, The University of Bristol, University Walk, Bristol, BS8 1TD, UK.
J Cell Sci. 2005 Jun 1;118(Pt 11):2471-84. doi: 10.1242/jcs.02369.
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.
CENTAURIN-α(1)是一种磷脂酰肌醇3,4,5-三磷酸结合蛋白，也是小GTP酶ADP-核糖基化因子(ARF)家族的GTP酶激活蛋白(GAP)。为了进一步了解其细胞功能，我们以CENTAURIN-α(1)为诱饵筛选大鼠脑cDNA文库，以鉴定与CENTAURIN-α(1)相互作用的蛋白。酵母双杂交筛选鉴定出一种新型驱动蛋白作为CENTAURIN-α(1)的结合伴侣。该驱动蛋白称为KIF13B，由一个约9.5 kb的单一转录本编码，广泛表达，在脑和肾中表达水平较高。酵母双杂交和GST下拉试验表明，CENTAURIN-α(1)与KIF13B之间的相互作用是直接的，由CENTAURIN-α(1)的GAP结构域和KIF13B的柄结构域介导。CENTAURIN-α(1)和KIF13B在体内形成复合物，KIF13B的相互作用似乎对CENTAURIN-α(1)具有特异性，因为ARF GAP家族的其他成员未显示任何结合活性。我们还表明，KIF13B和CENTAURIN-α(1)共定位于细胞周边的前沿，而缺乏KIF13B结合位点的CENTAURIN-α(1)缺失突变体在体内未能与KIF13B共定位。最后，我们证明KIF13B结合抑制完整细胞中CENTAURIN-α(1)的ARF6 GAP活性。总之，我们的数据提示了一种机制，即CENTAURIN-α(1)与KIF13B之间的直接结合可使CENTAURIN-α(1)集中在细胞前沿，从而调节ARF6功能。
