Induction of lamellipodia by Kalirin does not require its guanine nucleotide exchange factor activity

Abstract

Guanine nucleotide exchange factor (GEF) domains of the Dbl family occur in a variety of proteins that include multiple protein–protein and protein–lipid interaction domains. We used an epithelial-derived cell line to investigate the mechanisms by which the two GEF domains of Kalirin, a neuronal Rho GEF, influence morphology. As expected, Kal-GEF1, an efficient GEF for Rac1 and RhoG, induced the formation of lamellipodia resembling those induced by active Rac1. Although Kal-GEF1 activated Rac and Pak, its ability to induce formation of lamellipodia was not blocked by dominant negative Rho GTPases or by catalytically inactive Pak. Consistent with this, a catalytically inactive mutant of Kal-GEF1 induced formation of lamellipodia and activated Pak. Active Pak was required for the GEF-activity independent effect of Kal-GEF1 and the lamellipodia produced were filled with ribs of filamentous actin. Kal-GEF1 and a GEF-dead mutant co-immunoprecipitated with Pak. The interaction of Kal-GEF1 with Pak is indirect and requires the regulatory protein binding domain of Pak. Filamin A, which is known to interact with and activate Pak, binds to both catalytically active and inactive Kal-GEF1, providing a link by which catalytically inactive Kal-GEF1 can activate Pak and induce lamellipodia. Together, our results indicate that Kal-GEF1 induces lamellipodia through activation of Pak, where GEF activity is not required. GEF-activity-independent effects on downstream targets may be a general property of RhoGEFs.

Introduction

Living organisms perform a variety of functions that require rapid rearrangement of their cytoskeleton. Actin-rich protrusions called filopodia and flattened sheets with ruffles called lamellipodia are just two of a number of structures important for cell structure and motility. RhoGTPases play a central role in modulation of these structures and several proteins and pathways are emerging as important components regulating and maintaining these structures. RhoGEFs are the upstream activators of RhoGTPase, and several RhoGEFs link receptors to downstream effects upon cell morphology and cell signaling.

Spatial precision and timing are critical to RhoGEF function. Among the Dbl family of GDP/GTP exchange factors (GEFs) for small GTP binding proteins of the Rho subfamily, Kalirin and Trio and their Drosophila and C. elegans paralogs (dTrio and Unc-73, respectively) are distinct, having dual RhoGEF domains [1], [2]. The only other protein with two Dbl domains is DdracGAP [3] (by SMART analysis). Kalirin isoforms with single or dual GEF domains are generated through the tissue-specific and developmentally regulated use of several promoters and splice sites with Kalirin 12 being the longest Kalirin isoform (Fig. 1A) [4], [5], [6]. While genetic screens in worms and flies have consistently yielded mutations in the first GEF domain of these Kalirin paralogs, the fact that these proteins contain two GEF domains is likely critical to their function, allowing the coordinated control of multiple RhoGTPases [1], [7].

The Kalirin GEF domains bind to RhoGTPases and the isolated GEF1 domain catalyzes nucleotide exchange on Rac1 and RhoG [8], [9], [10]. Kal-GEF1 is dominant in superior cervical ganglion cultures, as exogenous Kal-GEF1 produces initiation of new axons and Kal-GEF2 is without effect on axon initiation [10]. Likewise, Kal7, an isoform containing GEF1, but lacking GEF2, plays a role in Ephrin-induced spine formation in cortical neurons that requires its GEF activity [11]. In contrast, expression of exogenous Kal-GEF2 in cortical neurons causes axons to lengthen in a RhoA-dependent manner while expression of Kal-GEF1 causes the Rac1-dependent retraction of processes [12]. In this example, Kal-GEF2 dominates the morphological response, suggesting that the opposing morphological activities of the two GEF domains are controlled. Thus, the response of cells to Kalirin will be dependent upon the RhoGTPase-effector systems and Kalirin binding proteins present in each type of cell. In order to compare the activities of the two Kalirin GEFs, we have chosen a hEK-293 cell variant because it allows consistent scoring of morphological responses and because its high transfection efficiency allows evaluation of the mechanism underlying the morphological responses.

Our experiments indicate that Kal-GEF1 is an active GEF, but has GEF-independent activities that may be just as important as its ability to activate RhoGTPases. While the catalytic activity of most GEFs is clearly essential for their effects on cell morphology and transformation (e.g., [13]), GEF-independent activities of RhoGEFs are not unprecedented. For example, an αPIX mutant devoid of exchange factor activity still activates Pak, and the RhoGEF Vav plays a role in the maturation of myeloid cells that does not require its GEF activity [14], [15]. GEFs may act through non-catalytic interactions with RhoGTPases. RhoGEFs such as Lfc, Ost/Dbs, and Kal-GEF2 bind to selected GTPases without catalyzing nucleotide exchange [12], [16].

Members of the Kalirin/Trio family are also known to have GEF-activity-independent effects. Trio-GEF1 binds Filamin A, a protein involved in cross-linking actin filaments [17], and Trio-GEF1-mediated membrane ruffling in fibroblasts requires Filamin A, but not the catalytic activity of Trio [18]. Tara, another actin binding protein, also interacts with Trio-GEF1, although the relationship of this interaction to exchange activity is unknown [19]. Trio-GEF1 mutants lacking GEF activity activate JNK kinase by an unknown mechanism [20]. Worms expressing an Unc-73 mutant devoid of GEF activity toward Rac show a pathfinding error that lacks complete penetrance [21]. While this is attributed to parallel pathways, another explanation is GEF-independent effects of Unc-73 [21]. Finally, dTrio-GEF2 does not activate any of the six Drosophila RhoGTPases [7], suggesting that it functions in a manner independent of its GEF activity, or may require accessory factors for activity. In this study, we found that Kalirin GEF1 can induce lamellipodia independent of its GEF activity.