Arf GAPs and molecular motors

Abstract

Arf GTPase-activating proteins (Arf GAPs) were first identified as regulators of the small GTP-binding proteins ADP-ribosylation factors (Arfs). The Arf GAPs are a large family of proteins in metazoans, outnumbering the Arfs that they regulate. The members of the Arf GAP family have complex domain structures and some have been implicated in particular cellular functions, such as cell migration, or with particular pathologies, such as tumor invasion and metastasis. The specific effects of Arfs sometimes depend on the Arf GAP involved in their regulation. These observations have led to speculation that the Arf GAPs themselves may affect cellular activities in capacities beyond the regulation of Arfs. Recently, 2 Arf GAPs, ASAP1 and AGAP1, have been found to bind directly to and influence the activity of myosins and kinesins, motor proteins associated with filamentous actin and microtubules, respectively. The Arf GAP-motor protein interaction is critical for cellular behaviors involving the actin cytoskeleton and microtubules, such as cell migration and other cell movements. Arfs, then, may function with molecular motors through Arf GAPs to regulate microtubule and actin remodeling.

Keywords: ADP-ribosylation factor GTPase-activating protein; ADP-ribosylation factors; AGAP1; ASAP1; Arf; Arf GAP; Kif2A; NM2A; kinesin-13; nonmuscle myosin 2A.

Figure 1.

Domain structures of Arf GAPs and associated motor proteins. (A) Domain structures of the human Arf GAP subfamilies are depicted. Abbreviations are: ALPS, ArfGAP1 lipid-packing sensor; ArfGAP, ArfGAP domain; ANK, ankyrin repeat; BAR, Bin/Amphiphysin/Rvs; BoCCS, binding of coatomer, cargo and SNARE; CALM, CALM binding domain; CB, clathrin-box; CC, coiled-coil; FG repeats, multiple copies of the XXFG motif; GLD, GTP-binding protein-like domain; GRM, Glo3 regulatory motif; PBS, Paxillin binding site; PH, pleckstrin homology domain; Pro(PxxP)3, cluster of 3 Proline-rich (PxxP) motifs; Pro(D/ELPPKP)8, 8 tandem Proline-rich (D/ELPPKP) motifs; RA, Ras association motif; RhoGAP, RhoGAP domain; SAM, sterile α-motif; SH3, Src homology 3 domain; SHD, Spa-homology domain. Adapted from Kahn et al. 2009. (B) Domain structure of Nonmuscle myosin 2A. NM2A is composed of 2 heavy chains, 2 essential light chains (ELCs) and 2 regulatory light chains (RLCs). Each heavy chain contains a motor head domain, a neck region, a coiled-coil rod and a non-helical tail (NHT). (C) Conformations of NM2. D. Domain structure of Kif2A.

Figure 2.

Model for ASAP1 function through NM2A to control maturation of stress fibers and associated integrin adhesions. (A) Hypothetical model for assembly of contractile stress fibers. Transverse arcs are assembled by endwise joining of NM2 and α-actinin cross-linked F-actin. The dorsal stress fibers emanating from integrin adhesions attach to arcs to form a continuous contractile stress fiber networks. The network contracts and flow toward the cell center. Ventral stress fibers are generated from transverse arcs located between 2 dorsal stress fibers. (B) ASAP1 couples assembly of actomyosin stress fibers to maturation of FAs. 1. Integrins are delivered to the cell surface and make contact with the extracellular matrix resulting in their activation. 2. Activated integrins recruit several proteins such as talin and paxillin, which recruit additional proteins including Crk and FAK. 3. ASAP1 is recruited to the nascent adhesionsvia binding of its proline rich domain to Crk and binding of its SH3 domain to FAK. 4. ASAP1 associates with PIP2 and Arf•GTP that are enriched in the forming adhesions. 5. PIP2 and Arf•GTP-bound ASAP1 interacts with NM2A (note that the roles of PIP2 and Arf in NM2A association are purely speculative, see text), stabilizing association of NM2A with actin filaments at the junction between dorsal stress fibers and transverse arcs. 6. Contractility of actomyosin stress fibers drives enlargement and maturation of the integrin adhesion.

Figure 3.

Model for ASAP1 interaction with NM2A regulated by PI(4,5)P2 and Arf. In this model, PI(4,5)P2 (PIP2 in figure) binding to the PH domain of ASAP1 facilitates Arf•GTP binding to ASAP1. ASAP1 undergoes a conformational change allowing interaction with NM2A. Subsequent hydrolysis of Arf•GTP induces further conformational change in ASAP1 that promotes NM2A activity and returns ASAP1 to a conformation capable for another round of NM2A binding. NM2 state 1 and state 2 represent inactive and active NM2 respectively. The transition of NM2 state 1 to state 2 could be from 10S to 6S form, or single 6S to 6S bipolar filaments, or unphosphorylated to phosphorylated Thr18/Ser19 of RLC.

Figure 4.

Hypothesized mechanisms for control of enzymatic activity of AGAP1 by PI(4,5)P2 and Kif2A. 1. Inactive form of AGAP1. We have identified charged patches in the GLD and insert in the PH domain important for activation. The drawing shows a speculated salt bridge, but its existence has not been tested. 2. PI(4,5)P2 (PIP2 in figure) binding to PH domain. The effects of Kif2A are dependent on PI(4,5)P2 binding to the PH domain leading us to speculate that PI(4,5)P2 binding results in a domain rearrangement favoring productive interaction with Kif2A. 3. Kif2A binding to the GLD and PH domains. Kif2A binding was found to increase GAP activity, leading us to speculate that Kif2A causes further domain rearrangement that optimizes AGAP1 binding to the substrate Arf1•GTP. 4. Catalytically active PI(4,5)P2·Kif2A·AGAP1 complex. Based on work with ASAP1, another Arf GAP that contains a PH domain, we speculate that the catalytic site comprises both the Arf GAP and PH domains which are optimally oriented for binding Arf1•GTP and hydrolyzing GTP with the PI(4,5)P2- and Kif2A-induced domain rearrangements.

Figure 5.

AGAP1 association with Kif2A may affect microtubule dependent FA dynamics. (A) Docking of microtubules with FAs results in disassembly of FAs through multiple mechanisms, including delivery of an Arf exchange factor, Brag2 (transported when associated with MAP4K4), which controls endocytosis of integrins, delivery of NBR1, which controls autophagy of FA plaque proteins (illustrated as yellow geometric shapes) and delivery of metalloproteinase (MMP), which hydrolyzes the extracellular part of the FA. Microtubule contact limits FAK activity. (B) AGAP1-Kif2A may increase the depolymerization of microtubules, preventing their association with FAs and consequent delivery of molecules that drive disassembly. The reduced microtubule contact results in increased FAK activity, indicated by the bold green “FAK,” by mechanisms that may involve AGAP1 directly as described in the text. As a result, the FA would be stabilized. N, nucleus.

Figure 6.

ASAP1 and AGAP1 cooperate to stabilize FAs. In this speculative model, ASAP1 association with NM2A stabilizes NM2A association with actin filaments at the contact sites between FAs and actomyosin stress fibers, which stabilizes FAs. At the same time, AGAP1 functions through Kif2A to destabilize microtubules, preventing the contact of microtubules with FAs, contributing to stabilization of the FAs by preventing endocytosis of integrins that accompanies FA turnover. N, nucleus.