A Complex between FAK, RACK1, and PDE4D5 Controls Spreading Initiation and Cancer Cell Polarity

Abstract

A fundamental question in cell biology concerns how cells respond to their environment by polarizing after sensing directional cues. This requires the differential localization of protein complexes in cells, and it is important to identify and understand how these complexes function. Here we describe a novel "direction-sensing" pathway that links the integrin effector focal adhesion kinase (FAK), the molecular scaffold protein RACK1, and activity of one of its client proteins, PDE4D5, a cAMP-degrading phosphodiesterase. The complex is recruited to nascent adhesions and promotes cell polarity. We identify FAK FERM domain residues whose mutation impairs RACK1 binding. When re-expressed in cancer cells in which endogenous fak is deleted by Cre-lox-mediated recombination, the RACK1-binding-impaired FAK mutant protein does not support formation of nascent actin adhesion structures as cells spread. These cancer cells, like FAK-deficient cells, cannot undergo directional responses, including wound-induced polarization or chemotactic invasion into three-dimensional matrix gels. We show that RACK1 serves as the molecular bridge linking FAK to the recruitment of PDE4D5. FAK/RACK1/PDE4D5 is a novel 'direction-sensing' complex that acts to recruit specific components of the cAMP second-messenger system to nascent integrin adhesions and to the leading edge of polarizing cells.

Signaling of the direction-sensing FAK/RACK1/PDE4D5 complex to the small GTPase Rap1

“…Using Squamous Cell Carcinoma (SCC) cells, in which FAK could be genetically deleted by tamoxifen treatment, we found that FAK is required for both cell spreading and RACK1 localization to nascent adhesions close to the cell-substratum interface as imaged by Total Internal Reflection Fluorescence microscopy (TIRF). 4 Small patches of FAK/RACK1 co-localization are also evident at nascent adhesive structures in fully spread cells. Disruption of this interaction through expression cells first make contact locally with the ExtraCellular Matrix (ECM; reviewed in ref.…”

“…25 Nascent lamellipodia, which originate at the tips of SICs, do not form in FAK-deficient cells or in cells in which FAK cannot be tyrosine phosphorylated after integrin engagement. 4,25 Thus, FAK links integrin signaling directly to the actin assembly machinery, and we have implicated the FAK FERM domain in this process. 4,25 In more recent work, we identified that the FAK FERM domain, specifically residues E139 and D140, which is required for direct binding of FAK to RACK1.…”

“…4,25 Thus, FAK links integrin signaling directly to the actin assembly machinery, and we have implicated the FAK FERM domain in this process. 4,25 In more recent work, we identified that the FAK FERM domain, specifically residues E139 and D140, which is required for direct binding of FAK to RACK1. Unlike the previously identified complex between FAK and Arp3, where binding is regulated by integrin induced FAK-Y397 phosphorylation, the FAK/ RACK1 complex does not change under these conditions.…”

“…In particular, FAK is needed for wound-induced polarization of SCC cells and chemotactic invasion through 3D matrix gels. 4 We believe this represents a novel "directionsensing complex" that is essential for the formation of nascent adhesion structures which permit cells to sense and polarize towards chemotactic factors. As the FAK/ RACK1 complex was also evident in fully spread cells, we used time-lapse microscopy to monitor RACK1 position in live cells.…”

“…24 FAK is present and co-localizes with RACK1, at nascent protrusive structures that form as Mouse Embryo Fibroblasts (MEFs) spread on ECM components. 4,25 FAK is a pivotal signal integrator operating at focal adhesions (its biological functions are depicted in fig. 1a) and is required for cell spreading, optimal integrin-dependent cell migration and integrin-induced signaling that promotes proliferation and survival.…”