ReviewαPIX and βPIX and their role in focal adhesion formation
Introduction
α and βPIX belong to a family of proteins acting as guanine nucleotide exchange factors (GEFs) towards Rho GTPases. These are small GTP-binding proteins that function as binary switches and cycle between an active GTP-bound and an inactive GDP-bound form (van Aelst and D’Souza-Schorey, 1997). GEF proteins activate Rho GTPases by catalyzing the exchange of bound GDP for GTP (Schmidt and Hall, 2002), whereas the GTPase-activating proteins (GAPs) accelerate the intrinsic GTPase activity which leads to inactivation of Rho proteins (Bernards and Settleman, 2004). Of the Rho GTPase family members, RhoA, Rac1, and Cdc42 have been extensively studied. They are best known for their important roles in regulating signal transduction pathways linked to the actin cytoskeleton. In addition, they influence a variety of diverse biological processes, namely cell polarity, gene transcription, cell cycle progression, microtubule dynamics, and vesicular transport pathways (Etienne-Manneville and Hall, 2002).
Regulation of actin dynamics is the best-characterized function of Rho GTPases. In tissue culture studies using fibroblast cells, activation of Rho increases cell contractility and leads to the formation of focal adhesions and actin stress fibers (Hotchin and Hall, 1995; Ridley and Hall, 1992), whereas Cdc42 and Rac activation propagates the formation of filopodia, lamellipodia, and peripheral membrane ruffles as well as focal contacts/complexes (Nobes and Hall, 1995; Ridley et al., 1992). Focal adhesions as well as focal complexes are protein complexes that link the extracellular matrix (ECM) to the actin cytoskeleton via heterodimeric transmembrane receptors, the integrins (Burridge and Chrzanowska-Wodnicka, 1996; Schoenwaelder and Burridge, 1999). The turnover of focal adhesions is highly coordinated and important for adhesion-dependent processes, such as cell migration and cell spreading (Raftopoulou and Hall, 2004; Ridley et al., 2003). Upon activation of integrins via ECM engagement, regulation of the actin cytoskeletal dynamics occurs primarily via the Rho family of small GTPases (Hotchin and Hall, 1995; Schoenwaelder and Burridge, 1999). The molecular mechanisms implicated in ECM- and growth factor-mediated activation of Rho GTPases are beginning to emerge, and it is not surprising that the regulated action of GEF proteins, such as α and βPIX, seems to be highly important for these processes. Here, we will summarize recent findings on α and βPIX, two closely homologous proteins, and their role in the formation of focal adhesions. To distinguish between different functions of α and βPIX, we refer to the respective name in the text. In case the data most likely apply to both α and βPIX, we only mention ‘PIX’ in the text.
Section snippets
Domain structure and function of PIX proteins
In 1997, a novel src homology 3 (SH3) domain-containing mouse protein, p85SPR, was characterized. In A431 cells, p85SPR showed a dispersed distribution in the cytoplasm; however, it was clearly enriched in a punctate pattern at the cell periphery and co-localized with the focal adhesion protein paxillin (Oh et al., 1997). One year later, the orthologous rat and human proteins, named βPIX or Cool-1, as well as another human variant, αPIX or Cool-2, were characterized in more detail (Bagrodia et
The PIX-PAK-GIT complex
Various independent experiments showed that αPIX strongly stimulates PAK activity and it has been assumed that this results from its ability to act as a GEF for Rac1 and Cdc42 (Bagrodia et al., 1999; Daniels et al., 1999; Feng et al., 2002; Ku et al., 2001; Li et al., 2003). Moreover, PIX was also found to be important for PAK recruitment and localization to focal complexes and focal adhesions. It has been proposed that activated Cdc42 tightly binds to PAK and concomitantly to PIX, thereby
The αPIX-β-parvin interaction
Recently, a novel αPIX-binding partner, β-parvin, was identified in a yeast two-hybrid screen using αPIX as bait protein (Rosenberger et al., 2003). The interaction was confirmed by co-immunoprecipitation and GST pull-down and is mediated by both the N-terminal CH domain and the C-terminal coiled-coil domain of αPIX, suggesting that either αPIX homo- or αPIX-βPIX heterodimers bind to β-parvin through the CH domains. αPIX co-localized with β-parvin and integrin-linked kinase (ILK), respectively,
The αPIX-calpain 4 connection
Another αPIX-binding protein, calpain 4, was identified in the same yeast two-hybrid screen that yielded β-parvin (Rosenberger et al., 2005). Calpain 4 is the small subunit of calpain proteases; these are Ca2+-dependent cysteine proteases, with a large number of substrates including several proteins involved in adhesion and motility, such as talin, α-actinin, paxillin, vinculin, or β-integrins (Glading et al., 2002; Sato and Kawashima, 2001). μ- and m-calpain are the two major forms of calpain
Conclusions
The common theme for α and βPIX is their involvement in integrin-dependent signaling processes at cell adhesion sites. The available data point to a role of PIX in both focal adhesion assembly as well as disassembly. PIX proteins, in particular βPIX, as part of the multiprotein complex p95-APP1/GIT-PAK-paxillin are responsible to recycle important focal adhesion components as well as part of the machinery required for Rac-mediated actin reorganization to the membrane of migrating cells. During
Acknowledgements
We are grateful to Hans-Jürgen Kreienkamp for critically reading the manuscript. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB444).
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