Centaurin-α1 interacts directly with kinesin motor protein KIF13B

Abstract

Centaurin-α1 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-α1 as bait to identify centaurin-α1 interacting proteins. The yeast two-hybrid screen identified a novel kinesin motor protein as a centaurin-α1 binding partner. The motor protein, termed KIF13B, encoded by a single ∼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-α1 and KIF13B is direct and mediated by the GAP domain of centaurin-α1 and the stalk domain of KIF13B. Centaurin-α1 and KIF13B form a complex in vivo and the KIF13B interaction appears to be specific to centaurin-α1 as other members of the ARF GAP family did not show any binding activity. We also 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. Finally, we demonstrate that KIF13B binding suppresses the ARF6 GAP activity of centaurin-α1 in intact cells. Together, our data suggest a mechanism where direct binding between centaurin-α1 and KIF13B could concentrate centaurin-α1 at the leading edges of cells, thus modulating ARF6 function.

The neuronal Arf GAP centaurin α1 modulates dendritic differentiation

“…The observation that Arf GAPs, such as centaurin ␣1 and ArfGEFs, such as EFA6 and ARNO are potentially regulated by PtdIns(3,4,5)P 3 , and found in dendritic and synaptic regions, lends support to the hypothesis that PI 3-kinase coordinately regulates GEFs and GAPs that control the Arf pathways required for dendritic development. In addition, centaurin ␣1 has multiple identified binding partners in addition to PtdIns(3,4,5)P 3 , including protein kinases and cytoskeletal proteins, which could provide input to centaurin ␣1 regulation from a variety of signaling pathways (Dubois et al, 2001;Dubois et al, 2003;Zemlickova et al, 2003;Thacker et al, 2004;Venkateswarlu et al, 2005;Striker et al, 2006). In turn, centaurin ␣1 may also provide cross-talk to other signaling pathways such as the Ras-MAP kinase pathway, which was shown to be diminished by reducing centaurin ␣1 expression (Hayashi et al, 2005).…”

“…Human Flag-centaurin ␣1 constructs were kindly provided by Kanamarlapudi Venkateswarlu (Venkateswarlu et al, 2005) and Muahamed Hawadle (Neural Development Unit, UCL Institute of Child Health, UK). eGFP vectors were obtained from Clontech.…”

PI-3-kinase-dependent membrane recruitment of centaurin-α2 is essential for its effect on ARF6-mediated actin cytoskeleton reorganisation

“…Centaurin-␣ 1 (also known as PIP3BP/p42 IP4 ) was originally identified as a PIP 3 or its inositol head group, inositol 1,3,4,5-tetrakisphosphate (IP 4 ), binding protein (Hammonds-Odie et al, 1996;Stricker et al, 1997;Tanaka et al, 1999). This protein acts as a PIP 3 -dependent ARF6 GAP that regulates GPCR internalisation and actin reorganisation (Lawrence et al, 2005;Venkateswarlu et al, 1999a;Venkateswarlu et al, 2004;Venkateswarlu et al, 2005). Centaurin-␣ 1 contains an Nterminal ARF GAP domain and two adjacent PH domains [one in the middle (N-PH) and the other at the C terminus (C-PH)] that are required for binding to PIP 3 (Venkateswarlu et al, 1999a).…”

RanBPM, a novel interaction partner of the brain‐specific protein p42IP4/centaurin α‐1

“…In mammalian (neuronal) cells, p42 IP4 regulates the actin cytoskeleton via ARFGAP-dependent and -independent mechanisms (Thacker et al 2004;Venkateswarlu et al 2004). It is involved in various signaling cascades, such as activation of the ERK mitogen-activated protein kinase cascade (Hayashi et al 2006), ARF6 signaling pathway (Venkateswarlu et al 2005) or transcription factor AP-1 activation cascade (Chanda et al 2003). In addition, p42 IP4 seems to be involved in neuronal diseases, such as Alzheimer disease, since we have found that besides the known intracellular localization, p42…”

“…p42 IP4 can shuttle between cytosol, nucleus and plasma membrane, depending on the cell stimulation situation . Several interaction partners of p42 IP4 have been identified, such as casein kinase I (Dubois et al 2001(Dubois et al , 2002, nucleolin , protein kinase C , several ARFs (Thacker et al 2004;Venkateswarlu et al 2004) GAKIN (Venkateswarlu et al 2005;Horiguchi et al 2006), the peptidase nardilysin (Stricker et al 2006) or F-actin (Thacker et al 2004). It has been shown that p42 IP4 is a component of the neuronal phosphatidyl inositol 3-kinase cascade and is involved in dendritic differentiation/synaptic plasticity and/or transport of PtdIns(3,4,5)P 3 -containing vesicles along axons to regulate neuronal cell polarity (Horiguchi et al 2006;Moore et al 2007).…”