Partial Characterization of an Interaction between Kinesin Associated Protein 3 (Kap3) of Kinesin-2 and the Actin Cytoskeleton

Research Article

Austin Biochem.2016; 1(1): 1003.

Partial Characterization of an Interaction between Kinesin Associated Protein 3 (Kap3) of Kinesin-2 and the Actin Cytoskeleton

Tenney A, Price M, Macatangay J, Hatt D, Kline T and Berezuk MA*

Department of Biology and Chemistry, Azusa Pacific University, USA

*Corresponding author: Berezuk MA, Department of Biology and Chemistry, Azusa Pacific University, 675 E Foothill Blvd, Azusa, CA 91702, USA

Received: September 29, 2016; Accepted: November 10, 2016; Published: November 14, 2016


Kinesin-2 is a plus-end directed microtubule based motor involved in the transport of diverse intracellular cargoes. It has been specifically shown to function in systems that help define sub cellular structures and cell morphology in general. The non-motor accessory subunit of kinesin-2, KAP3, is proposed to act as a multifunctional linker between the motor subunits and an assortment of target cargoes. Human KAP3 contains an array of eleven armadillo repeats, a well characterized motif common to various protein-protein interactions. Previous work showed that native kinesin-2 and bacterially expressed KAP3 armadillo repeat bearing fragments can co-cycle with actin filaments in vitro. However, these studies left the putative KAP3-actin interaction poorly characterized and did not address what in vivo significance, if any, a KAP3- actin interaction might have. Here we report that KAP3 associates with actin polymers exclusively through its central core of armadillo repeats. Conversely, KAP3 appears incapable of binding to monomeric, globular actin in solution. Co-localization of over expressed GFP-tagged full length KAP3 with phalloidin stained actin filaments in cell culture substantiates an in vivo consequence to our biochemical description. This work helps to refine our understanding of the KAP3-actin interaction in vitro and expands it to a cellular context. Thus, providing further insight into how KAP3 facilitates kinesin-2 transport activities, specifically those related to the formation and maintenance of specific cellular processes and cell shape.

Keywords: Kinesin-2; Actin; Cytoskeleton


Kinesins are a class of predominantly plus-end directed, microtubule-based motors capable of transforming the energy generated from ATP hydrolysis into mechanical force for intracellular transport [1-6]. While all kinesins share nearly identical motor domains, their non-motor components and accessory subunits are quite varied [4,7-11]. Kinesin-2 is one of the plus-end directed members of the kinesin super family and is essential for metazoan life [9,12]. It is unique amongst kinesin motors in that it consists of two configurations of heterologous motor subunits (kinesin-2 A/B or A/C) and a non-motor, accessory protein, KAP3 [13-15]. The kinesin-2 holoenzyme has been shown to function in the intracellular transport of organelles, viruses and RNAs as well as having roles in the regulation of cell polarity, cell-cell adhesion, mitosis, neurite extension, intraflagellar transport and cilium assembly/maintenance [9,16-18]. However, the exact mechanisms of these activities and how kinesin-2 associates with the myriad of cargoes involved is poorly understood. Most studies implicate the KAP3 subunit of kinesin-2 as the likely mediator of target molecule/organelle recognition and attachment to the motor subunits [2,19-29]. While promising, these reports have only identified a small proportion of the potentially large number of cargo associating molecules present for KAP3 and kinesin-2 to bind within the cell.

Structurally, KAP3 contains a central grouping of armadillo repeats [8,14,30], a common protein interaction motif often found in proteins that associate with the actin cytoskeleton [31-36]. Previous work using His-tagged KAP3 fragments identified an association between the armadillo repeats of KAP3 and actin [27]. This novel finding provided us with a model for kinesin-2 recognizing a specific cargo in the context of a known functional interaction [22,37]. Here we report studies which confirm that the armadillo repeat regions of KAP3 are capable of co-cycling with actin filaments in vitro. However, we are unable to detect any direct interaction between these same fragments and globular actin. To address the cellular significance of the observed biochemical interaction, we cloned a Green Fluorescent Protein (GFP) fusion construct of full length human KAP3 into a mammalian expression vector. Transient transfection and overexpression in COS-7 cell culture coupled with fluorescence staining of actin filaments revealed specific co-localization between GFP-KAP3 and filamentous actin. Our continued characterization of this novel interaction furthers our understanding of how these distinct cytoskeletal systems can communicate and potentially regulate one another in performing a variety of cellular functions.

Materials and Methods

His-tagged protein expression and purification

KAP3 fragment constructs cloned into the His-tagged pRSET B bacterially expression vector [27] were a generous gift from T.A. Schroer. The coding region for KAP3 included in each clone corresponds to the N-terminus [KAP3 N; a.a. 1-161], armadillo repeats 1-3 (KAP3 A1-3; a.a 162-333), 4-8 (KAP3 A4-8; aa. 334-534), 9-11 (KAP3 A9-11; aa. 535 -662) and the C terminus (KAP3 C; aa. 663-772) of human KAP3 (Figure 1). Plasmids were transformed into Rosetta (DE3) competent cells (EMD Millipore, Billerica, MA). Cultures were grown and protein expression induced with 0.1 μM IPTG for 16 hours at 37 C. Cells were lysed by sonication and sterile filtrate loaded onto a HiTrap™ Chelating HP column (GE Healthcare, Marlborough, MA) for binding (binding buffer; 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM imidazole) and elution (0 to 100% gradient of elution buffer; 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 500 mM imidazole) using an AKTA®purifier FPLC system (GE Healthcare, Marlborough, MA).