Review
αPIX and βPIX and their role in focal adhesion formation

https://doi.org/10.1016/j.ejcb.2005.10.007Get rights and content

Abstract

α and βPIX belong to the group of guanine nucleotide exchange factors (GEFs) that mediate activation of members of the Rho GTPase family, in particular Rac1 and Cdc42, by stimulating the exchange of GDP for GTP. Rho family proteins are well known as regulators of the actin cytoskeleton and have been implicated in the formation of various types of focal adhesion structures. However, the function of GEF proteins during focal adhesion formation is only beginning to emerge. Here, we highlight the recent findings on α and βPIX and their involvement in integrin-dependent signaling and suggest models for the role of PIX proteins during focal adhesion turnover.

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).

References (69)

  • A. Huttenlocher et al.

    Regulation of cell migration by the calcium-dependent protease calpain

    J. Biol. Chem.

    (1997)
  • T. Kim et al.

    Molecular cloning and characterization of a novel mouse betaPix isoform

    Mol. Cells

    (2001)
  • S. Kim et al.

    Molecular cloning of neuronally expressed mouse betaPix isoforms

    Biochem. Biophys. Res. Commun.

    (2000)
  • S. Kim et al.

    Leucine zipper-mediated homodimerization of the p21-activated kinase-interacting factor, beta Pix. Implication for a role in cytoskeletal reorganization

    J. Biol. Chem.

    (2001)
  • S. Kulkarni et al.

    Calpain mediates integrin-induced signaling at a point upstream of Rho family members

    J. Biol. Chem.

    (1999)
  • Z. Li et al.

    Directional sensing requires G beta gamma-mediated PAK1 and PIX alpha-dependent activation of Cdc42

    Cell

    (2003)
  • S. Linder et al.

    Podosomes: adhesion hot-spots of invasive cells

    Trends Cell Biol.

    (2003)
  • E. Manser et al.

    PAK kinases are directly coupled to the PIX family of nucleotide exchange factors

    Mol. Cell

    (1998)
  • Z. Nie et al.

    Arf and its many interactors

    Curr. Opin. Cell Biol.

    (2003)
  • S.N. Nikolopoulos et al.

    Molecular dissection of actopaxin-integrin-linked kinase-Paxillin interactions and their role in subcellular localization

    J. Biol. Chem.

    (2002)
  • C.D. Nobes et al.

    Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia

    Cell

    (1995)
  • W.K. Oh et al.

    Cloning of a SH3 domain-containing proline-rich protein, p85SPR, and its localization in focal adhesion

    Biochem. Biophys. Res. Commun.

    (1997)
  • R.T. Premont et al.

    The GIT family of ADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2 through alternative splicing

    J. Biol. Chem.

    (2000)
  • R.T. Premont et al.

    The GIT/PIX complex: an oligomeric assembly of GIT family ARF GTPase-activating proteins and PIX family Rac1/Cdc42 guanine nucleotide exchange factors

    Cell. Signal.

    (2004)
  • M. Raftopoulou et al.

    Cell migration: Rho GTPases lead the way

    Dev. Biol.

    (2004)
  • S. Rhee et al.

    BetaPix-b(L), a novel isoform of betaPix, is generated by alternative translation

    Biochem. Biophys. Res. Commun.

    (2004)
  • A.J. Ridley et al.

    The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors

    Cell

    (1992)
  • A.J. Ridley et al.

    The small GTP-binding protein rac regulates growth factor-induced membrane ruffling

    Cell

    (1992)
  • G. Rosenberger et al.

    AlphaPIX associates with calpain 4, the small subunit of calpain, and has a dual role in integrin-mediated cell spreading

    J. Biol. Chem.

    (2005)
  • S.M. Schoenwaelder et al.

    Bidirectional signaling between the cytoskeleton and integrins

    Curr. Opin. Cell Biol.

    (1999)
  • N. Vitale et al.

    GIT proteins, a novel family of phosphatidylinositol 3,4,5-trisphosphate-stimulated GTPase-activating proteins for ARF6

    J. Biol. Chem.

    (2000)
  • Y. Zhang et al.

    Distinct roles of two structurally closely related focal adhesion proteins, alpha-parvins and beta-parvins, in regulation of cell morphology and survival

    J. Biol. Chem.

    (2004)
  • A. Bhatt et al.

    Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain

    J. Cell Sci.

    (2002)
  • K. Bialkowska et al.

    Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active

    J. Cell Biol.

    (2000)
  • Cited by (72)

    • KCC2 regulates dendritic spine development

      2020, Neuronal Chloride Transporters in Health and Disease
    • Context-Specific Mechanisms of Cell Polarity Regulation

      2018, Journal of Molecular Biology
      Citation Excerpt :

      It has been suggested that SCRIB regulates cell orientation and promotes directed migration by interacting with Rho GTPases [75]. Indeed, SCRIB interacts with the guanine nucleotide exchange factor (GEF) βPIX, which plays a critical role in regulating activity of Rho GTPases, CDC42 and RAC1 (Fig. 2b) (reviewed in Ref. [76]). In addition, SCRIB and βPIX are required for a robust localization and activation of CDC42 at the leading edge [77,78].

    • Binding of the extreme carboxyl-terminus of PAK-interacting exchange factor β (βPIX) to myosin 18A (MYO18A) is required for epithelial cell migration

      2014, Biochimica et Biophysica Acta - Molecular Cell Research
      Citation Excerpt :

      Fourth, MYO18A is linked to βPIX/Rac1 axis regulation in epithelial cells. βPIX is known to be responsible for recycling critical focal adhesion components required for Rac1-mediated actin reorganization to the membranes of migrating cells [15,18–20]. In the current work, we observed that Rac1 activity in MYO18Aα-silenced M1-7 cells was significantly lower than that in V1 control cells, indicating a specific role for MYO18Aα in Rac1-mediated cytoskeletal organization (rearrangement) or related cellular events, which may provide insight into the regulation of adhesion and migration by the βPIX/Rac1 axis in epithelial cells.

    • The genetics and pathogenesis of CAKUT

      2023, Nature Reviews Nephrology
    View all citing articles on Scopus
    View full text