Direct Functional Interaction of the Kinesin-13 Family Membrane Kinesin-like Protein 2A (Kif2A) and Arf GAP with GTP-binding Protein-like, Ankyrin Repeats and PH Domains1 (AGAP1)

Ruibai Luo,Pei-Wen Chen,Michael Wagenbach,Xiaoying Jian,Lisa Jenkins,Linda Wordeman,Paul A Randazzo

doi:10.1074/jbc.m116.732479

Ruibai Luo, Pei-Wen Chen + Show 5 more

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https://doi.org/10.1074/jbc.m116.732479

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Abstract

The molecular basis for control of the cytoskeleton by the Arf GTPase-activating protein AGAP1 has not been characterized. AGAP1 is composed of G-protein-like (GLD), pleckstrin homology (PH), Arf GAP, and ankyrin repeat domains. Kif2A was identified in screens for proteins that bind to AGAP1. The GLD and PH domains of AGAP1 bound the motor domain of Kif2A. Kif2A increased GAP activity of AGAP1, and a protein composed of the GLD and PH domains of AGAP1 increased ATPase activity of Kif2A. Knockdown (KD) of Kif2A or AGAP1 slowed cell migration and accelerated cell spreading. The effect of Kif2A KD on spreading could be rescued by expression of Kif2A-GFP or FLAG-AGAP1, but not by Kif2C-GFP. The effect of AGAP1 KD could be rescued by FLAG-AGAP1, but not by an AGAP1 mutant that did not bind Kif2A efficiently, ArfGAP1-HA or Kif2A-GFP. Taken together, the results support the hypothesis that the Kif2A·AGAP1 complex contributes to control of cytoskeleton remodeling involved in cell movement.

Highlights

The ADP-ribosylation factor (Arf)3 GTPase-activating proteins (GAPs) are a family of structurally diverse proteins with the common function of catalyzing the hydrolysis of GTP bound to Arf, thereby converting Arf1⁄7GTP to Arf1⁄7GDP (1–3)
We found that the GLD and pleckstrin homology (PH) domains of AGAP1 are critical for regulated GAP activity (25)
As an initial test of the possibility that there are other binding partners that bind to the GLDPH domains of AGAP1 to regulate catalytic and biological activity, we screened for binding partners using a proteomic approach

Summary

Introduction

The ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs) are a family of structurally diverse proteins with the common function of catalyzing the hydrolysis of GTP bound to Arf, thereby converting Arf1⁄7GTP to Arf1⁄7GDP (1–3). The five Arf isoforms in humans regulate membrane traffic and actin remodeling (4). Site-specific targeting, regulation, and effector function of Arf GAPs remain relatively unexplored. Like other Arf GAPs, the AGAPs studied to date affect membrane trafficking and actin cytoskeleton. The related protein AGAP2 binds to ␤-arrestin, which affects Erk signaling (11), and to focal adhesion kinase, which controls focal adhesions and, presumably, cell migration (12), a plausible function for AGAP1 as well given its effect on both actin cytoskeleton and membrane traffic (8). The closely related Kif2C diffuses along the microtubule lattice independent of ATP hydrolysis. It binds the end of the microtubules where ATP hydrolysis drives the removal of tubulin dimers (16).

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