Elsevier

Cellular Signalling

Volume 26, Issue 1, January 2014, Pages 9-18
Cellular Signalling

RACK1 promotes neurite outgrowth by scaffolding AGAP2 to FAK

https://doi.org/10.1016/j.cellsig.2013.08.036Get rights and content

Highlights

  • RACK1 functions as a signalling switch and integrates distinct signalling pathways.

  • RACK1 is required for the regulation of FAK activity.

  • Differentiation signals promote an interaction between RACK1 and FAK.

  • We have identified AGAP2 as a novel RACK1-interacting protein.

  • RACK1 scaffolds AGAP2 to FAK to regulate FAK activity.

Abstract

RACK1 binds proteins in a constitutive or transient manner and supports signal transmission by engaging in diverse and distinct signalling pathways. The emerging theme is that RACK1 functions as a signalling switch, recruiting proteins to form distinct molecular complexes. In focal adhesions, RACK1 is required for the regulation of FAK activity and for integrating a wide array of cellular signalling events including the integration of growth factor and adhesion signalling pathways. FAK is required for cell adhesion and migration and has a well-established role in neurite outgrowth and in the developing nervous system. However, the mechanism by which FAK activity is regulated in neurons remains unknown. Using neuronal cell lines, we determined that differentiation of these cells promotes an interaction between the scaffolding protein RACK1 and FAK. Disruption of the RACK1/FAK interaction leads to decreased neurite outgrowth suggesting a role for the interaction in neurite extension. We hypothesised that RACK1 recruits proteins to FAK, to regulate FAK activity in neuronal cells. To address this, we immunoprecipitated RACK1 from rat hippocampus and searched for interacting proteins by mass spectrometry. We identified AGAP2 as a novel RACK1-interacting protein. Having confirmed the RACK1–AGAP2 interaction biochemically, we show RACK1–AGAP2 to localise together in the growth cone of differentiated cells, and confirm that these proteins are in complex with FAK. This complex is disrupted when RACK1 expression is suppressed using siRNA or when mutants of RACK1 that do not interact with FAK are expressed in cells. Similarly, suppression of AGAP2 using siRNA leads to increased phosphorylation of FAK and increased cell adhesion resulting in decreased neurite outgrowth. Our results suggest that RACK1 scaffolds AGAP2 to FAK to regulate FAK activity and cell adhesion during the differentiation process.

Introduction

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has an important role in cell migration. At the leading edge of the cell, FAK is recruited early as new focal adhesion complexes are established. At the trailing edge of the cell, focal adhesion complexes are dismantled which facilitates cell migration [1]. These events must be precisely coordinated for cell migration to occur. The dynamic process of focal adhesion assembly and disassembly is tightly regulated and relies heavily on crosstalk between growth factor and adhesion signalling pathways.

FAK is a ubiquitously expressed protein with high levels reported in the brain, in particular, in the specific brain regions of the cortex and the hippocampus [2], [3]. FAK has a well-established role in neurite outgrowth [4], [5], [6], an important process that is central to neuronal migration and differentiation. The process involves the initiation of neurite protrusion and the extension of an axon or dendrite from the neuronal cell body. Growth cones, which are a common feature of neurite outgrowth, are highly motile structures found at the end of neurites and are composed of actin-rich structures called filopodia and lamellipodia [7]. They have a crucial role in neural development as they act in a similar way to the leading edge of a migrating cell [8]. Growth cones possess cell surface receptors for neurite path determination (or pathfinding) and contain large amounts of signalling and cytoskeletal proteins important for motor activity (reviewed by Vitriol and Zheng [9]). During cell migration, this dynamic area of the cell can respond to both attractive and repulsive environmental cues. FAK is particularly enriched in the growth cone [8] and FAK has a key role to play in mediating the signalling downstream of both attractive and repulsive cues (reviewed by Cheng and Poo [10], Chacón and Fazzari [11]).

Many reports have indicated that FAK activity is elevated during the invasion of gliomas, glioblastomas and astrocytomas [12], [13]. Therefore, the regulation of FAK activity in neuronal cells is critical. However, the signalling processes regulating FAK activity in the brain are poorly understood. Most of what we understand about FAK is derived from studies in fibroblasts and epithelial cells. Although FAK displays elements of autoinhibition [14], [15], the central theme is that FAK activity and function is regulated by a series of binding partners such as kinases, phosphatases and a series of adaptor proteins. RACK1 has emerged as an important regulator of FAK activity and focal adhesion assembly [16], [17], [18]. RACK1 is a scaffolding protein with 7 WD repeats in the central pathways regulating cell adhesion and cell migration [16], [19], [20], [21], [22], [23], [24]. It is now well established that RACK1 functions as a convergence point for both growth factor and adhesion signalling [25]. RACK1 is also highly expressed in the brain cortex and hippocampus [26] and has previously been implicated in the process of neurite outgrowth [27], [28]. Kiely et al. [16] were the first to report an interaction between RACK1 and FAK [16] and mapped the interaction site to Y52 in WD2 of RACK1. We showed that cells expressing the FAK binding mutant of RACK1 are significantly impaired in adhesion, growth, and foci formation [16]. It is now well established that the RACK1/FAK complex, in conjunction with PDE4D5, controls cell spreading and directional sensing to help establish cell polarity [17], [18]. Taken together, these findings suggest that a FAK/RACK1 interaction is essential for focal adhesion formation and in the regulation of signalling events required to initiate cell migration.

The objective of this study was to investigate a role for RACK1 in the regulation of FAK activity in neuronal cells. Our hypothesis was that RACK1 is required to precisely integrate a wide array of signalling events leading to FAK activation during cell migration. We predicted that RACK1 provides both spatial organization and specificity of signalling proteins for FAK activation in migrating neurones by positioning them in close proximity to their substrate FAK. Having confirmed the interaction between RACK1 and FAK in rat hippocampus, we used cell models to determine that the RACK1–FAK interaction is regulated by differentiation signals and is required for neurite extension. To identify proteins that may be scaffolded by RACK1 to regulate FAK activity, we employed mass spectrometry analysis to identify a series of RACK1 interacting proteins in rat hippocampus. We identified several novel RACK1-interacting proteins in our screen and here we report how AGAP2 is scaffolded by RACK1 to FAK in response to differentiation signals to regulate neurite outgrowth.

Section snippets

Reagents and antibodies

Recombinant NGF was purchased from R&D Systems Europe Ltd. (Abington, UK). Mouse anti-RACK1 and anti-FAK antibodies were purchased from BD Transduction Laboratories (Heidelberg, Germany). The rabbit anti-FAK and mouse anti-PIKE antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-HA antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). The anti-actin monoclonal antibody was from Sigma-Aldrich (Wicklow, IE). The anti-GFP antibody was from Abcam (Cambridge,

RACK1 interacts with FAK in rat hippocampus and in differentiated PC12 cells

We have previously shown that RACK1 regulates FAK phosphorylation in response to IGF-I and adhesion signalling [16]. It is now known that the RACK1/FAK complex, in conjunction with PDE4D5 controls cell spreading and directional sensing to help establish cell polarity [17], [18]. In the brain, RACK1 has a well-established role in regulating the signalling cascades downstream of NMDA receptors but a role for RACK1 in regulating FAK activity in the brain is poorly understood. We asked whether

Discussion

In this study, we demonstrate the involvement of the RACK1/FAK interaction in neurite outgrowth using a series of quantitative biochemical approaches and morphological analysis. Furthermore, we identify AGAP2 as a novel RACK1 interacting protein in the brain. We provide strong evidence indicating that RACK1 recruits AGAP2 to FAK during neurite outgrowth and we confirm that the complex between RACK1, AGAP2 and FAK is a key regulator of the neurite outgrowth process.

During neurite outgrowth

Acknowledgements

This work was supported, in whole by grants received from the Health Research Board of Ireland (HRB); Grant HRA/2009/188 (to P.K.). We are grateful to our colleagues in the Laboratory of Cellular and Molecular Biology for helpful discussions and critical review.

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