parvin: Biological Overview | References
Gene name - parvin
Cytological map position - 18E5-18F1
Function - cytoskeletal regulator
Keywords - modulates integrin connection to the cytoskeleton, IPP complex member that maintains the integrin-actin link at embryonic muscle attachment sites, affects the organization of actin cytoskeleton in both wing and eye epithelia
Symbol - parvin
FlyBase ID: FBgn0052528
Genetic map position - chrX:19640090-19642491
Classification - Calponin homology domain (actin-binding domain)
Cellular location - cytoplasmic
|Recent literature||Vakaloglou, K. M., Chrysanthis, G. and Zervas, C. G. (2016). IPP complex reinforces adhesion by relaying tension-dependent signals to inhibit integrin turnover. Cell Rep 14: 2668-2682. PubMed ID: 26972014
Cytoskeleton-mediated forces regulate the assembly and function of integrin adhesions; however, the underlying mechanisms remain unclear. The tripartite IPP complex, comprising ILK, Parvin, and PINCH, mediates the integrin-actin link at Drosophila embryo muscle attachment sites (MASs). This study demonstrate a developmentally earlier function for the IPP complex: to reinforce integrin-extracellular matrix (ECM) adhesion in response to tension. In IPP-complex mutants, the integrin-ECM linkage at MASs breaks in response to intense muscle contractility. Mechanistically, the IPP complex is required to relay force-elicited signals that decelerate integrin turnover at the plasma membrane so that the integrin immobile fraction is adequate to withstand tension. Epistasis analysis shows that alleviation of muscle contractility, downregulation of endocytosis, and enhanced integrin binding to the ECM are sufficient to restore integrin-ECM adhesion and maintain integrin-adhesome organization in IPP-complex mutants. These findings reveal a role for the IPP complex as an essential mechanosensitive regulatory switch of integrin turnover in vivo.
Integrin-linked kinase (ILK), PINCH (Steamer Duck in Drosophila) and Parvin constitute the tripartite IPP complex that maintains the integrin-actin link at embryonic muscle attachment sites (MASs) in Drosophila. This study shows that parvin null mutants in Drosophila exhibit defects in muscle adhesion, similar to ILK and PINCH mutants. Furthermore, the identical muscle phenotype of the triple mutant, which for the first time in any organism removed the entire IPP-complex function, genetically demonstrated that Parvin, ILK and PINCH function synergistically. This is consistent with the tight localization of the tripartite complex at sites of integrin adhesion, namely MASs in the developing embryo and focal-contact-like structures in the wing epithelium. Parvin contains tandem unconventional calponin-homology (CH) domains separated by a linker sequence, and a less-well conserved N-terminal region. In vivo structure-function analysis revealed that all the domains are essential for Parvin function, whereas recruitment at integrin adhesion sites is mediated by two localization signals: one located within the CH2 domain as previously reported, and a second novel signal within the CH1 domain. Interestingly, this site is masked by the linker region between the two CH domains, suggesting a regulatory mechanism to control Parvin localization. Finally, whereas in muscles only ILK controls the stability and localization of both PINCH and parvin, in the wing epithelium the three proteins mutually depend on each other. Thus molecular differences exist in the assembly properties of IPP complex in specific tissues during development, where differential modulation of the integrin connection to the cytoskeleton is required (Vakaloglou, 2012).
During animal development cells assemble into tissues and establish specific adhesion sites with the surrounding extracellular matrix (ECM). ECM enables the stable attachment of cells and their separation in distinct layers permitting tissue morphogenesis. The integrin family of transmembrane proteins mediate physical contact of cells with the ECM. This connection requires direct binding of the extracellular domain of both the α- and the β-subunit of integrin to their ECM ligands, whereas their small cytoplasmic tails bind a network of proteins to form the integrin adhesome and mediate the link to the actin cytoskeleton (Geiger, 2011). The molecular composition of the integrin adhesome network is diverse, highly dynamic and largely determined by the cell type and the physical strength required by the local developmental microenvironment (Vakaloglou, 2012).
The tripartite IPP complex containing integrin-linked kinase (ILK), PINCH and parvin is central to the integrin adhesome network (Wickstrom, 2010; Wu, 2001). In invertebrates, the IPP-complex components are encoded by single genes, in contrast to the two PINCH genes and three parvin genes (α-, β- and 7gamma;-parvin) in mammals. The early evolutionary appearance of ancestral IPP-complex components suggest that the IPPb complex may have been one of the first molecular machines used by integrins (Sebe-Pedros, 2010; Vakaloglou, 2012 and references therein).
ILK plays a central role in the formation of the IPP complex. It contains five tandem ankyrin repeats (ANKRs) followed by a kinase-like domain, and interacts with the β1 and β3 integrin cytoplasmic tails). The ANKRs of ILK bind to PINCH, an adaptor protein containing five LIM domains, whereas the kinase-like domain of ILK binds to parvin, which in turn binds to actin (Legate, 2006). Thus, a simple model of IPP-complex function envisages either direct or indirect anchoring of ILK to integrins, with parvin mediating the link to actin. This model is supported by studies showing linear assembly of IPP complex in invertebrates. In Caenorhabditis elegans, ILK is required for both parvin and PINCH subcellular localization but not the reverse, and in Drosophila ILK is similarly required for PINCH stability and recruitment, although parvin has not been tested (Vakaloglou, 2012).
In mammalian cells, genetic elimination of one component of the IPP complex results in a significant decrease of the other two members, although some compensation has been observed for parvins and PINCH (Wickstrom, 2011). Moreover, distinct IPP complexes are formed within cells containing different parvin and PINCH members, resulting in different functional properties (Wu, 2004). However, it is unknown whether removing all IPP components enhances the defects of single deletions, and thus whether all components function together or have other individual functions (Vakaloglou, 2012).
The parvin family was initially identified by sequence similarity to α-actinin. Recent detailed analysis of parvin CH2-domain interactions with the ILK kinase domain and the leucine aspartate (LD)-rich repeats of paxillin suggested that parvin contributes to the assembly of focal adhesions in mammalian cells (Fukuda, 2009; Lorenz, 2008; Nikolopoulos, 2000; Olski, 2001; Wang, 2008). Genetic ablation of α-parvin in mice uncovered its function in vascular morphogenesis as a negative regulator of RhoA/ROCK signaling (Lange, 2009; Montanez, 2009). However, due to functional compensation by β-parvin, earlier developmental functions of the parvin family were hard to study, possibly explaining why deletion of α-parvin does not phenocopy defects in early embryo development observed with genetic elimination of ILK or PINCH-1. Expression of, γ-parvin is restricted to the hematopoietic system, where it is not essential (Vakaloglou, 2012).
Previously found that ILK is required to link the actin cytoskeleton to the integrin-containing junctions at embryonic muscle attachment sites (MASs) in Drosophila, and PINCH was subsequently shown to be required for the same process. To date, there have been no genetic studies of the single Drosophila parvin gene, resulting in a currently incomplete characterization of IPP-complex function in flies. This paper reports a detailed functional characterization of parvin and the IPP complex in Drosophila development by generating a series of specific mutants and analyzing their effects on protein function and localization, in both the MASs of developing embryos and the wing epithelium (Vakaloglou, 2012).
This study has established the essential role of parvin in integrin-mediated adhesion and has demonstrated that parvin participates together with ILK and PINCH in the maintenance of the integrin-actin link in vivo. These conclusions are supported by: (1) the identical muscle phenotype of parvin mutants to mutants of ILK, PINCH and the triple mutant; (2) colocalization of all three proteins at sites of integrin adhesion; (3) the fact that ILK is necessary and sufficient for parvin stability and recruitment to MASs; (4) the suppression of dominant negative effects of overexpressed parvin within muscle by ILK; and (5) interdependence of all three IPP-complex components for their stability and subcellular localization at the basal side of wing epithelia, unlike the central role of ILK in IPP-complex assembly at the embryonic MASs. The results suggest that depending on the cell context, discrete molecular events coordinate the assembly of adhesion complexes into functional attachment structures (see Genetic hierarchy of integrin-actin linker complex assembly at MASs and in wing epithelium) (Vakaloglou, 2012).
parvin mutants were generated to complete the functional characterization of the entire IPP complex in Drosophila development. parvin null mutants shared similar phenotypic abnormalities with ILK and PINCH mutants as well as the integrin hypomorphic mutations described previously, including muscle defects due to actin filament retraction from the muscle ends in the late embryo. The muscle defects and lethality associated with parvin mutants was completely rescued by moderate expression of a wild-type parvin transgene mainly in somatic muscles and tendon cells, indicating that parvin has essential functions in these tissues. This study generated the first in vivo triple null mutant in an animal for all proteins that comprise the IPP complex. The identical loss-of-function phenotype at muscles in the triple versus single mutants strongly suggests that these three proteins work together as a complex by reinforcing the integrin-actin link at MASs in the Drosophila embryo. Although loss of parvin function at the muscles causes defective adhesion, high levels of parvin is also detrimental for the developing organism, suggesting that unlike overexpression of other IPP-complex members, the amount and subcellular localization of parvin needs to be tightly regulated (Vakaloglou, 2012).
parvin recruitment at MASs appears to depend exclusively on its interaction with ILK. In the absence of ILK, endogenous parvin protein levels are drastically reduced as previously reported (Lin, 2003; Wu, 2004). Although endogenous parvin protein levels also decrease in the absence of PINCH, most likely due to moderate reduction of the parvin mRNA levels, this can be completely rescued by coexpression of ILK. Previously, it was shown that removal of PINCH does not affect the levels of a genomic ILK-GFP transgene. However, its effect on endogenous ILK levels remains undetermined due to the lack of a specific antibody against Drosophila ILK. Therefore, even a slight reduction of endogenous ILK levels in pinch mutants can not be excluded, providing an alternative explanation for both the moderate reduction of parvin and its complete restoration upon ILK overexpression. Overexpressed parvin in muscle cells remains in the cytoplasm, suggesting that the number of available binding sites for parvin at MASs is limited. However, simultaneous overexpression of parvin and ILK results in accumulation of parvin at MASs, indicating that ILK is necessary and sufficient for subcellular localization of parvin. High levels of cytoplasmic uncomplexed parvin in muscle cells induce dominant lethality, which is reversed upon coexpression of ILK. Previous studies in cells have shown that an imbalance in the amount of α- and β-parvin complexed with ILK induces apoptosis. Taken together these results suggest that the stoichiometry of ILK and parvin is crucial. Indeed, in Drosophila embryos and S2R+ cells, all components of the IPP complex are co-purified in stoichiometric amounts (Kadrmas, 2004; Vakaloglou, 2012 and references therein).
Parvin is known to be recruited to sites of integrin adhesion by direct interaction of its CH2 domain with the kinase domain of ILK (Fukuda, 2009). The current study also showed that the CH2 domain of Drosophila parvin is sufficient for localization to MASs. In addition, a novel ability of the parvin CH1 domain to localize at integrin junctions was uncovered. The close correlation between the levels of ILK and the stability and localization of the CH1 domain strongly suggest their direct interaction. Based on the similar negative effect of the E356K mutation of ILK on the recruitment of each CH domain, it is predicted that they bind to the same site within the kinase domain of ILK, although with different affinity. An alternative explanation that is favored less is the existence of a factor-X that recruits the CH1 domain at MASs, whereas ILK mediates factor-X recruitment. One candidate could be the fly homolog of αPIX, dPix, which is localized at MASs and binds to the CH1 domain of β-parvin in mammalian cells (Vakaloglou, 2012).
The data suggests that the CH1 domain localization signal is masked by the linker region. Although no direct biochemical evidence was provided supporting such an intramolecular interaction, a similar interaction has been reported in filamin, another CH-domain-containing protein. Given the strong possibility that both CH domains of parvin interact with ILK, the linker might function as a regulatory module ensuring that only high affinity interactions between parvin and ILK occur, thus controlling recruitment of parvin at MASs. The functional significance of parvin CH1-domain interaction with ILK requires further investigation involving the identification of specific mutations within the CH1 domain that could disrupt the binding on ILK (Vakaloglou, 2012).
Proteins containing tandem CH domains possess the ability to bind to filamentous actin (Gimona, 2002). Mammalian parvins show a differential ability to bind to F-actin. Although human α-parvin binds to actin filaments, β-parvin does not, despite 73% sequence identity. In mammalian cells, both α and β-parvin are largely associated with focal adhesions rather than the actin cytoskeletal filaments (Nikolopoulos, 2000; Olski, 2001; Yamaji, 2001). In the living fly embryo, it was found that parvin-GFP expressed from its own promoter was similarly not colocalized with F-actin in the muscle cells, but instead accumulated heavily at MASs. By contrast, the two CH domains of Short stop colocalized with filamentous actin. These findings reflect differences in affinity for actin as supported by in vitro dissociation constants (ShotCH 0.022μM and α-parvin 8μM). Thus, within muscles parvin does not appear to have sufficient actin binding activity to co-distribute with actin filaments. Given that several proteins are known to bind to both CH domains in regions overlapping the putative actin binding sites, the role of parvin as an actin-binding protein In vivo is questionable. Hence, interaction of the CH domains with F-actin appears to be either less favored or to occur only in a highly regulated manner at sites of integrin adhesion (Vakaloglou, 2012).
Studies of mammalian parvins have suggested possible biochemical functions for Drosophila parvin. To gain further insight into the mechanisms by which parvin functions in integrin-mediated adhesion, the functional requirement of each protein domain was assessed. It was demonstrated that both the CH1 and CH2 domains are essential to strengthen the integrin-actin link and their importance coincides with their capacity to mediate an interaction with ILK that is responsible for their individual localization. Moreover, both CH domains are required in the same molecule. Hence, it is likely that these parvin domains link ILK and other proteins together to maintain the integrin-actin link. This study also found that the less well conserved N-terminal region of parvin is not essential for integrin-mediated adhesion at MASs, similar to pat-6 N-terminal domain in C. elegans. However, later in development the N-terminus becomes indispensable, suggesting a different set of essential molecular interactions for parvin depending on the developmental context (Vakaloglou, 2012).
In mammalian cells all three proteins of the IPP complex are mutually dependent for their stability and recruitment at sites of integrin adhesion. However, genetic studies both in C. elegans and Drosophila have shown that in muscle cells ILK behaves as a master component for complex assembly. Several studies have pointed out that the two well established models for stable adhesion in Drosophila, MASs and the basal side of the wing epithelium, exhibit molecular differences in assembly properties of the integrin adhesion complex, as well as differential sensitivity in genetically induced perturbations. In agreement with these studies, this study showed that in the wing epithelium the hierarchy of genetic interactions among the IPP-complex members differs and that, like in mammalian cells, the three proteins are interdependent for their stability and subcellular localization. This finding highlights the existence of diverse molecular strategies capable of mediating distinct adhesion properties in the developing organism and therefore underlines the importance of in vivo analysis of the same molecular machine of integrin adhesion junctions in various morphogenetic processes (Vakaloglou, 2012).
Parvin is a putative F-actin binding protein important for integrin-mediated cell adhesion. This study used overexpression of Drosophila Parvin to uncover its functions in different tissues in vivo. Parvin overexpression caused major defects reminiscent of metastatic cancer cells in developing epithelia, including apoptosis, alterations in cell shape, basal extrusion and invasion. These defects were closely correlated with abnormalities in the organization of F-actin at the basal epithelial surface and of integrin-matrix adhesion sites. In wing epithelium, overexpressed Parvin triggered increased Rho1 protein levels, predominantly at the basal side, whereas in the developing eye it caused a rough eye phenotype and severely disrupted F-actin filaments at the retina floor of pigment cells. Genes were identified that suppressed these Parvin-induced dominant effects, depending on the cell type. Co-expression of both ILK and the apoptosis inhibitor DIAP1 blocked Parvin-induced lethality and apoptosis and partially ameliorated cell delamination in epithelia, but did not rescue the elevated Rho1 levels, the abnormal organization of F-actin in the wing and the assembly of integrin-matrix adhesion sites. The rough eye phenotype was suppressed by coexpression of either PTEN or Wech, or by knock-down of Xrp1. Two main conclusions can be drawn from these studies: (1) high levels of cytoplasmic Parvin are toxic in epithelial cells; (2) Parvin in a dose dependent manner affects the organization of actin cytoskeleton in both wing and eye epithelia, independently of its role as a structural component of the ILK-PINCH-Parvin complex that mediates the integrin-actin link. Thus, distinct genetic interactions of Parvin occur in different cell types and second site modifier screens are required to uncover such genetic circuits (Chountala, 2012).
Parvin proteins are highly conserved and participate in the assembly and function of the integrin adhesome. This study employed the UAS/Gal4 system to investigate additional functions of Parvin upon overexpression in a tissue specific manner and to identify novel genetic interactions in the wing and the eye. Drosophila Parvin promotes apoptosis when overexpressed in vivo, similar to mammalian β-Parvin in HeLa cells. Expression of β-Parvin in breast cancer cells was recently shown to inhibit tumor progression and cell proliferation suggesting that this study of the cellular and molecular changes associated with Parvin overexpression in Drosophila may be relevant to cancer pathology. At the cellular level it was demonstrated that overexpressed Parvin induced alterations in the organization of the actin cytoskeleton, disruption of cell-matrix adhesion, cell invasion and cell delamination. Mechanistically, it was shown that overexpressed Parvin causes JNK activation and enhanced MMP1 levels. A functional link between Parvin and subcellular distribution of Rho1 was revealed. Interestingly, it was shown that these Parvin-induced signaling effects are not dependent on its interaction with ILK (Chountala, 2012).
Among the three counterparts of the ILK/PINCH/Parvin-complex, only overexpression of full-length Parvin induced ectopic apoptosis and excessive lethality in the larval and pupae developmental stages. Nevertheless, in the wing imaginal discs overexpression of other components of the integrin adhesome such as tensin and paxillin also results in apoptosis and lethality, including activation of the JNK pathway and modulation of Rho1 activity, respectively. Overexpression of Parvin increases Rho1 protein levels predominantly at the basal side of the wing epithelium, although loss of Parvin did not cause a reciprocal reduction of Rho1 levels. Given the previous reports that mammalian Parvins interact with two regulators of the small GPTases family, the GEF α pix and the CdGAP respectively, one hypothesis would be that high levels of Parvin sequester these factors and interfere with their interaction with Rho1. As a consequence, Rho1 is released from the apicolateral side where normally is enriched. The elevated Rho1 levels in the basal compartment of the epithelium could explain the formation of ectopic actin accumulation in accordance with previous studies. As already described Rho1 is able to induce JNK-dependent apoptosis and F-actin organization defects in the wing epithelia cells. Therefore, it is plausible that the elevated JNK activity observed upon Parvin overexpression is caused by aberrant elevation of Rho1 basaly. Taking these findings together, it is proposed that Parvin-induced cellular defects in the wing epithelia are mediated by increased levels of Rho1, however, a putative role of additional unidentified factors that are activated downstream of Parvin independently of Rho1 cannot be ruled out (Chountala, 2012).
It was recently showed that coexpression of ILK together with Parvin-GFP in the mesoderm is sufficient to completely rescue Parvin-induced lethality and control Parvin subcellular localization (Vakaloglou, 2012), suggesting that coupling of Parvin to ILK could have a protective effect in epithelia viability. Rescue experiments were performed to investigate whether Parvin function in the wing epithelium is mechanistically linked to its interaction with ILK, by coexpressing Parvin with ILK. Expression of ILK alone did not completely rescue the dominant effects of Parvin overexpression in the developing wing epithelia, had a mild suppressive effect on the rough eye phenotype and did not change the subcellular distribution of Parvin-GFP in the wing epithelial cells. Both the JNK activity and the increase in Rho1 protein levels were also not affected by ILK coexpression. Even when high levels of ILK are present, the putative interaction of Parvin with GTPase regulators is not disturbed and the imbalance of Rho1 subcellular distribution is maintained. That is not unexpected given that both αpix and CdGAP interact with the N-terminus region of Parvin, whereas ILK binds on the C-terminus. These findings demonstrate that the functional interplay between Parvin and ILK depends on the cell context and that Parvin interacts with other proteins and perform additional roles. In addition to functioning as a structural element of the integrin-actin link, it also acts as a dosage dependent modulator of actin cytoskeleton organization and cell homeostasis in the developing epithelia, via modulating the subcellular distribution of Rho1 (Chountala, 2012).
Because overexpression of Parvin caused extensive apoptosis in the wing epithelium, to mechanistically uncouple the Parvin-induced cellular defects from Parvin-induced apoptosis, rescue experiments were performed by coexpressing Parvin and DIAP1, which blocks apoptosis by inhibiting both the initiator caspase DRONC and the effector caspases DriCe and Dcp-1. DIAP1 alone did not efficiently suppress the cellular defects of Parvin in the wing. Both ILK and DIAP1 had to be coexpressed to completely rescue the lethality, presumably by coupling the reduction of excessive cytoplasmic Parvin by ILK and the inhibition of DRONC-mediated apoptosis by DIAP1. Coexpression of ILK and DIAP1 rescue both cell apoptosis and cell extrusion in the wing poutch cells, but not in the hinge and notum. These findings were not entirely unexpected, given previous documentation of regional differences within the wing imaginal disc regarding the differential requirement of actin regulators for epithelial integrity. However, consistent with these results from ILK rescue experiments, coexpression of DIAP1 or both ILK and DIAP1 did not ameliorate either the irregular organization of F-actin or the disorganized integrin-matrix adhesion sites and did not change the elevated levels of Rho1 in the basal side of the wing epithelium. These results demonstrate that the Parvin-induced cellular defects are not a simple consequence of apoptosis, but rather a distinct feature of Parvin function (Chountala, 2012).
Overexpression of Parvin in the eye generates a rough eye phenotype. At the cellular level the basal actin cytoskeleton in the eye retina is severely disrupted, suggesting that a cause of the abnormal eye development could be initiated by abnormalities in the cell shape of pigment cells, as in the case of the wing epithelium. Because the Parvin-induced eye phenotype was sensitive to the copy number of Parvin transgenes and to temperature, a modifier screen was performed to uncover novel genetic interactors. It was found that elevated levels of Wech and PTEN antagonize the Parvin-induced dominant effects in the developing eye and completely suppress the rough eye phenotype, whereas high levels of ILK has only minimal suppression activity (Chountala, 2012).
Wech is an ILK binding protein and it is not clear why it could suppress Parvin-induced dominant defects at elevated levels rather than ILK itself, which directly binds to Parvin and rescues lethality completely in the mesoderm (Vakaloglou, 2012) and significantly in enGal4 expressing cells. The lack of data regarding Wech function in the eye, preclude further analysis at this point. The second surprising result of this study was the ability of high levels of PTEN to suppress the rough eye phenotype induced by Parvin overexpression. UAS::PTEN overexpression under GMRGal4 has been reported to induce a rough-eye phenotype by inhibiting cell-cycle progression in proliferating cells and inducing apoptosis in a cell-context dependent manner. In the current experiments expression of the same UAS::PTEN lines obtained from two different donors did not result in eye roughening. One possible explanation could be the use of longGMRGal4 in these experiments, because previous studies drove expression of UAS::PTEN with GMRGal4. In addition, previous reports suggested that expression by longGMRGal4 driver in the developing eye follows a more strict pattern in the photoreceptor cells. Taken these data and previous reports together, it is speculated that Parvin and PTEN have antagonistic functions within the eye epithelium and coexpression of both proteins counterbalance their induced dominant effects upon overexpression. Currently insufficient data is available to point a specific pathway that could be modified by Parvin and PTEN and leads to rough eye phenotype. However, the recent report that Parvin is associated with PKB (Kimura, 2010) together with previous data suggesting that Parvin may facilitates the recruitment of PKB at plasma membrane Fukuda, 2009, suggests that Parvin could antagonized the negative effects of PTEN on PKB activation by reducing PIP3 levels (Chountala, 2012).
The third suppressor gene found was Xrp1. Xrp1 contains an AT-hook motif that is found in nuclear proteins with DNA binding activity. Currently, sufficient information is available to speculate on putative functional interaction between Parvin-induced signaling and nuclear activity. However, previous studies on Xrp1 point on its role as a p53-dependent negative regulator of cell proliferation following genotoxic stress. Among the genes that enhanced the Parvin-induced rough eye were all of the integrin subunits known to be expressed in the eye, including αPS1, αPS2 and βPS, the cytoskeletal regulators ZASP52 and the transgelin homolog encoded by CG14996 of unknown function (Chountala, 2012).
In conclusion, the findings revealed novel cell context-dependent roles for Parvin in the whole organism. Besides its known function as a structural component of the IPP-complex that mediate the integrin-actin link, it was demonstrated that Parvin can also affect cell-matrix adhesion, organization of actin cytoskeleton and cell homeostasis, by regulating Rho1 and JNK levels in an ILK-independent manner. These findings are relevant to situations where cell homeostasis is altered ranging from the physiological renewal of tissues to cancer pathology. In addition, the modifier genetic screen revealed novel interactors that affect Parvin function in a living organism. These in vivo data provide the first insight into genetic circuits influenced by Parvin and offer a framework for additional detailed studies to elucidate how these genetic networks interact (Chountala, 2012).
Integrin-linked kinase (ILK) is an essential component of a multiprotein complex that links actin to the plasma membrane. This study has used a genetic approach to examine the molecular interactions that are essential for the assembly of this ILK-containing complex at Drosophila muscle attachment sites (MASs). Downstream of integrins, talin plays a decisive role in the recruitment of three proteins: ILK, Lim domain protein PINCH (steamer duck) and paxillin. The accumulation of ILK at MASs appears to follow an amplification mechanism, suggesting that numerous binding sites are generated by minimal levels of the upstream integrin and talin effectors. This property suggests that ILK functions as an essential hub in the assembly of its partner proteins at sites of integrin adhesion. PINCH stability, and its subcellular localization at MASs, depends upon ILK function, but ILK stability and localization is not dependent upon PINCH. An in vivo structure-function analysis of ILK demonstrated that each ILK domain has sufficient information for its independent recruitment at embryonic MASs, whereas at later developmental stages only the kinase domain was effectively recruited. These data strengthen the view that the ILK complex is assembled sequentially at sites of integrin adhesion by employing multiple molecular interactions, which collectively stabilize the integrin-actin link (Zervas, 2011).
In multicellular organisms, tissue integrity depends upon the stable association of cells with the extracellular matrix (ECM). This requires a link between matrix ligands and the cytoskeleton, which is primarily mediated by the integrin family of surface receptors. Each integrin is composed of one α and one β subunit, and because these subunits have very short cytoplasmic tails it has been postulated that other scaffolding proteins assist integrins in the recruitment of downstream components that mediate the link to actin. The cytoskeletal molecules involved in this link, and the hierarchy of their interactions, are currently a subject of intense investigation, and genetic approaches in model organisms have provided novel insights into the function of these proteins. One such example is integrin-linked kinase (ILK), a protein initially identified by its ability to bind directly to the cytoplasmic tail of the β1 integrin subunit. ILK is a modular protein containing five tandem ankyrin repeats (ANKRs), a putative phosphoinositide-binding site and a kinase-like domain. ILK is required to maintain the molecular link between integrins and the sarcomeric actin filaments in Drosophila embryonic muscles and to assemble the link between integrins and the contractile apparatus of developing Caenorhabditis elegans muscles. Knockout of the single mouse gene showed that ILK is required for diverse developmental processes, with its earliest function being in epithelial polarization and normal actin distribution of the developing epiblast. In all three organisms, certain site-directed mutations within the kinase domain of ILK that should eliminate kinase activity did not show any defects, suggesting that the essential role of ILK is not the phosphorylation of target proteins. Instead, ILK binds to multiple proteins, suggesting that its primary function is as an adaptor. The recently published structure of the kinase domain of ILK supports the view that it is a binding adaptor rather than a kinase. Thus, genetic and structural data indicate that ILK is a pseudokinase. However, a clear picture of the essential adaptor function of ILK in assisting integrin function has yet to be developed (Zervas, 2011).
The large number of proteins that bind ILK suggests it is a central scaffolding molecule for the assembly of a protein complex. Interacting proteins have been identified by yeast two-hybrid screening and biochemical methods. Among these proteins are: PINCH, which contains five LIM domains and binds to the ANKRs of ILK through its first LIM domain; paxillin, which has four LIM domains and five leucine-rich motifs (LD) and binds to the ILK kinase domain (Nikolopoulos, 2001); and parvin, which has two calponin homology domains and binds to the ILK kinase domain. Both the ANKRs and kinase domains are essential for recruitment of ILK at focal adhesions in vertebrate cells in culture. Even before its association with integrins, ILK is in a cytoplasmic complex with PINCH and parvin and a fourth protein, Ras suppressor protein 1 (Rsu1), which binds the fifth PINCH LIM domain. So far the model of functional coordination between ILK, PINCH and parvin has not been fully supported by genetic studies. ILK is required for parvin (Pat-6) recruitment to sites of integrin adhesion in C. elegans but not the reverse, and PINCH is not required for ILK recruitment in either Drosophila or C. elegans (Zervas, 2011 and references therein).
This study combined genetic analysis and structure-function approaches to examine the mutual interactions of ILK and PINCH, as well as their interactions with talin and paxillin. Low levels of integrins were found to be sufficient to recruit substantial levels of ILK to the major sites of integrin adhesion in the embryo and larva, namely the muscle attachment sites (MASs), suggesting an amplification mechanism. Complete removal of talin resulted in the loss of ILK, PINCH and paxillin from MASs, in agreement with its crucial contribution to integrin function. Neither ILK nor PINCH was required for paxillin recruitment. Unexpectedly, PINCH stability and recruitment required both domains of ILK (i.e., PINCH-binding ANKRs and kinase domains). In embryos each ILK domain was recruited to the MASs, but recruitment of the isolated ANKRs was lost during development, indicating a change in mechanism. Finally, the function of a number of conserved ILK residues that have been shown to be important for interactions with other proteins were tested, but they were found to be dispensable. Collectively, the results indicate that ILK functions upstream of PINCH in the muscle and that the function of ILK is executed by simultaneous interactions of both the kinase and ANKR domains (Zervas, 2011).
In cell culture models, focal adhesions are the prototype form of sites of integrin adhesions. Several lines of evidence suggest that the molecular assembly of focal adhesions is a multistep process, where different cytoplasmic proteins enter the adhesion sites in a defined sequential order. This study provides genetic evidence that supports a hierarchical model for formation of integrin adhesions, using the Drosophila embryonic MASs as a model system (Zervas, 2011).
This study reexamined whether integrins are essential for the localization of ILK to MASs. A previous study found that ILK was recruited independently of integrins. This conclusion has now been corrected: complete elimination of integrins resulted in loss of ILK recruitment. This result, therefore, reveals that the small amount of maternally provided integrin recruits unexpectedly high levels of ILK. This surprising finding has two important implications: 1) the recruitment of ILK and maintenance of cell-matrix adhesion have different requirements for the amount of integrin (low levels of integrin are sufficient to recruit ILK, but high levels are needed for functional adhesion), 2) the amount of ILK recruited by integrins is not set by the quantity of integrin at MASs, suggesting that there is an amplification mechanism so that a single integrin can recruit multiple ILK molecules (although the experiments do not reveal the relative number of molecules). A similar, but less extreme, lack of correlation between the levels of integrin and an intracellular integrin-associated protein at MASs has been found for talin. This apparent amplification could be explained by a multidocking scaffolding protein that is able to recruit, either directly or indirectly, several ILK molecules. Alternatively, ILK could have the ability to employ a variety of additional interactions with different binding partners in the integrin-containing junctions, so that even if integrins or other components are much reduced, multiple ILK molecules are recruited. The mechanism of the amplification is unknown, with a number of the integrin-associated proteins being potential components; the kindlin proteins are particular good candidates, because they bind both integrins and ILK (Karaköse, 2010), and Wech has been shown to bind both talin and ILK and to contribute to ILK recruitment (Löer, 2008; Zervas, 2011 and references therein).
Talin binding to integrins is one of the first molecular events, and is a prerequisite, in the formation of the integrin-cytoskeleton link. Therefore, whether talin is required to recruit components of the ILK-containing complex at sites of integrin adhesion was examined. As with integrins, it was essential to remove both the maternal and zygotic production of talin to reveal defects in ILK, PINCH and paxillin recruitment. Consequently, these results show an important function for talin in recruiting the ILK-containing complex. A similar role for talin in ILK recruitment at myotendinous junctions in mice has been reported. Currently there are no data to support a direct association between components of the ILK complex and talin, but Wech binds to talin and ILK suggesting that it could be the key linker between the two proteins (Löer, 2008), at least in muscles. Therefore, a model is favored whereby talin, in concert with integrins, recruits Wech and additional proteins (see Comparison of ILK recruitment mechanisms). These proteins then in turn recruit ILK and paxillin, and ILK indirectly recruits PINCH (Zervas, 2011).
In vertebrate cells ILK and PINCH have mutually dependent functions. Their association is required to protect each other from a proteasome-dependent degradation and to facilitate each other's localization to focal adhesions. The direct interaction between ILK and PINCH has also been confirmed in Drosophila, and PINCH-mediated stabilization of ILK in mice has been confirmed genetically. In Drosophila, it was found that neither ILK protein stability nor its recruitment to MASs was affected in the absence of PINCH. This is in agreement with the persistent subcortical localization of ILK in the pinch-1-knockout mouse embryoid bodies, as well as in unc-97 mutants in C. elegans. By contrast, loss of ILK function reduced PINCH levels in Drosophila embryos. The instability of PINCH in the absence of ILK was overcome by overexpressing PINCH but, under these conditions, PINCH still was not localized to MASs. The isolated ILK ANKRs did localize to MASs in the absence of endogenous ILK, but were not able to recruit PINCH. Thus, rather than ILK recruiting PINCH by binding of its ANKRs to the LIM1 domain of PINCH, it appears to do so indirectly. This finding raises two important points. First, the interaction between the ILK ANKRs and LIM1 of PINCH might not be always maintained at sites of integrin adhesion. The finding that LIM1 is crucial for recruitment, but apparently not through interaction with ILK, suggests alternative interactions might be required. This is consistent with the observation that high levels of PINCH-GFP can be recruited to MASs, even when only the endogenous levels of ILK are present. Second, recent work in mammalian cells provided strong evidence that the primary function of PINCH was the recruitment of ILK through the ANKRs-LIM1 interaction (Stanchi, 2009). Notably, the recruitment of tensin and ILK at focal adhesions in cells lacking PINCH could be rescued by a direct fusion of the ILK kinase domain to integrin. However, the result suggests that, in Drosophila muscles, the reverse is true, and a primary function of ILK might be the stabilization and recruitment of PINCH (Zervas, 2011).
Finally, whether ILK and PINCH influence the subcellular localization and protein stability of paxillin was examined. Previous studies have demonstrated an interaction between the kinase domain of mammalian ILK and paxillin, and further suggested that this interaction controls ILK localization at focal adhesions. These studies identified key residues in ILK that contribute to paxillin interaction and ILK recruitment, such that ILK mutations at E359 and T384 impaired recruitment of ILK to focal adhesions. However, the ILK E359K mutation does not impair its ability to rescue the null phenotype or alter its recruitment in flies or mice. This study showed that mutating T384 in Drosophila ILK, equivalent to part of the paxillin-binding region in human ILK, also failed to impair the biological activity or recruitment of ILK, and neither ILK nor PINCH were required for paxillin recruitment. Thus, in MASs, the stability of paxillin and its subcellular localization does not depend on the ILK-PINCH complex (Zervas, 2011).
This study has examined the functions of the two main domains that constitute ILK (i.e. the ANKRs and the kinase domain), as well as specific binding sites that have been proposed to mediate interactions with phosphoinositides or paxillin and have been implicated in the regulation of the protein. From this work, it became evident that deletion of either the ANKRs or the kinase-like domain eliminates the essential functions of ILK in the developing organism. These two domains are required in the same molecule to execute the essential function of ILK. The proposed phosphoinositide-binding motif is dispensable for embryonic development but is required in the epithelial tissues of the wing, and therefore does contribute to ILK function. This suggests that the highest levels of ILK function are required in the wing. This is consistent with the finding that elimination of tensin only causes a wing-blister phenotype, even though it is also concentrated at MASs. By contrast, hypomorphic mutations in the gene encoding the αPS2 integrin subunit were found to cause muscle defects but not cause wing blisters, indicating that integrin adhesive sites are differentially sensitive to loss of different components of the integrin adhesion complex. The residue F436, which is located at the far end of the C-terminal region, was found to be crucial in vertebrate cells, but its mutation only mildly affected recruitment of the overexpressed protein and was dispensable for ILK function. It is possible that the F436A mutation reduces the binding affinity of ILK with Wech (Zervas, 2011).
Both the isolated ANKRs and the kinase domain are recruited to MASs, in contrast with the recruitment to focal adhesions seen in mammalian cells. This suggests that recruitment is more robust at MASs, so impaired ILK recruitment can still be visualized. It was surprising to find that the recruitment of the ANKRs of ILK did not involve an interaction with PINCH, as removing PINCH did not impair recruitment. Given that there is only one Drosophila pinch gene, in contrast with two in mammals, the ANKRs must be binding to another type of protein. One alternative candidate is Mlp84B, a protein that contains five LIM domains and is localized at MASs in an integrin-independent manner; it is known that the related zebrafish protein CSRP3 binds ILK (Zervas, 2011).
In summary, the results have demonstrated that, at the robust integrin adhesive sites that form in the developing muscles, ILK recruitment is much less sensitive to perturbation compared with recruitment in focal adhesions in cells in culture. The results show that integrins and talin are essential for the recruitment of ILK, and ILK in turn is essential for the recruitment of PINCH. How much of the function of ILK is mediated by PINCH, and whether it also functions independently through its interaction with parvin, will be the focus of future research (Zervas, 2011).
Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. PINCH (Steamer duck), a protein required for integrin-dependent cell adhesion and actin-membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, RSU-1, a regulator of Ras signaling in mammalian cells, has been identified as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding Drosophila RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. These results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development (Kadrmas, 2004).
To determine if PINCH contributes to DC, its localization was examined in stage 14 embryos. PINCH and ß-PS integrin colocalize in both the LE and the amnioserosa, consistent with PINCH's established role as an integrin effector. The amnioserosa is an extraembryonic tissue present on the dorsal surface of the embryo. Since it has been established that coordinated signaling between the amnioserosa and migrating epithelium is key to formation of LE focal complexes, PINCH could exert an effect in the LE epithelium, the amnioserosa, or both tissues. stck homozygous mutant embryos rescued with a PINCH:GFP transgene under the control of the endogenous PINCH promoter display PINCH-GFP at the LE of the advancing epithelial sheets. Within the LE, PINCH is precisely localized to areas of active phosphotyrosine signaling at triangular nodes corresponding to apical adherens junctions (Kadrmas, 2004).
Zygotic stck mutants proceed normally through DC with complete lethality arising at the embryo-to-larval transition. When maternal PINCH contribution is eliminated, only 12% of cuticles have wild-type morphology. Dorsal puckers and dorsal holes characteristic of aberrant DC are observed at a 36% and 23% frequency, respectively, indicating that maternally inherited PINCH is a key contributor to the process of DC. Moreover, in the absence of maternal PINCH, epithelial defects are observed in ventral patterning and head involution, indicating that PINCH may have additional functions in the developing embryo. Cuticles from embryos lacking both maternal and zygotic PINCH have the same array of phenotypes (Kadrmas, 2004).
PINCH is composed of five LIM domains, each of which could serve as a protein-binding interface. The SH2-SH3 adaptor protein, Nck2, has been reported to interact with mammalian PINCH. This association is intriguing because the Drosophila homologue of Nck2, Dreadlocks, interacts directly with Misshapen (Msn), a MAP4K in the JNK signaling cascade. As with other components of the JNK pathway, null mutations in msn result in embryonic lethality due to failure of DC. Although no direct binding of PINCH to Dreadlocks was observed in Drosophila, this study uncovered a link between PINCH's role in DC and the JNK cascade by testing for genetic interaction between stck and msn. Reduction of PINCH protein levels by introduction of a single copy of the loss-of-function allele, stck18, into the msn102 homozygous null background allows partial restoration of DC, suggesting that PINCH functions as a negative regulator of JNK signaling (Kadrmas, 2004).
Puckered (Puc) is a JNK phosphatase whose expression is up-regulated in response to JNK activation, setting up a negative feedback loop. During DC, JNK-regulated expression of a Puc-LacZ fusion reporter is restricted to the LE cells. In embryos lacking maternal PINCH, expression of the Puc-LacZ fusion protein is disorganized and present in an expanded number of cells, including those beyond the LE border. This phenotype is similar to Puc-LacZ expression observed in puc loss-of-function mutants and further supports a role for PINCH in the negative regulation of the JNK cascade (Kadrmas, 2004).
Thorax closure is a post-embryonic developmental process with features common to DC, including migration of epithelial sheets and a dependence on JNK signaling. Within the wing disc, cells of the stalk region are functionally similar to LE cells during DC. These cells comprise the eventual fusion site for adjacent imaginal discs and are active in JNK signaling. Spatially restricted JNK signaling in the stalk of wing disc can be visualized via a Puc-LacZ reporter, and PINCH expression overlaps with Puc-LacZ in this area of active JNK signaling. Therefore, as in DC, PINCH is properly positioned to act as a regulator of the JNK cascade (Kadrmas, 2004).
Although msn null mutations are embryonic lethal due to DC failure, flies homozygous for the hypomorphic allele msn3349 are semi-viable and a large proportion of the eclosing adults have thorax closure defects. These observations underscore the similarities between thorax closure and DC. In a stck18 heterozygous background, a greater percentage of msn3349 homozygotes are able to eclose, supporting the hypothesis that PINCH is a negative regulator of the JNK pathway in both dorsal and thorax closure (Kadrmas, 2004).
Drosophila PINCH was purified in complex with its binding partners using tandem affinity purification (TAP)-tagged PINCH (TAP-PINCH). stck homozygous mutant embryos rescued with a TAP:PINCH transgene driven by the endogenous stck promoter to wild-type levels afford material for purification of soluble, cytoplasmic TAP-PINCH complexes in the absence of endogenous PINCH protein. Three partners that copurified stoichiometrically with TAP-PINCH from embryos, as well as in complex with TAP-PINCH from cultured Drosophila S2R+ cells, were identified by mass spectrometric analysis. Consistent with what is observed in mammalian cells, ILK copurified with PINCH. The Drosophila homologue of the parvin/actopaxin family of proteins, CG32528, is also present in PINCH protein complexes. Parvin is known to bind ILK and actin in mammalian systems, but the isolated Parvin/ILK/PINCH complexes are the first to be described in Drosophila. Additionally, a novel 31-kD protein was identified as Drosophila CG9031. The CG9031 protein is 55% identical and 74% similar to human RSU-1, a leucine-rich repeat containing protein first identified as a suppressor of cell transformation by v-Ras and subsequently implicated in regulation of MAP kinase signaling, specifically the JNK and ERK cascades, when overexpressed in cultured cells. Despite its potent ability to act as a tumor suppressor, little is known about the mechanism of action of RSU-1. Its partnership with the PINCH protein allows placement of RSU-1 in a molecular pathway that is linked to integrins (Kadrmas, 2004).
To assess the specificity and nature of the interaction between PINCH and RSU-1, domain-mapping studies were performed in cell culture and in yeast two-hybrid assays. Drosophila RSU-1 copurifies with full-length His-tagged PINCH, but not with a truncated His-tagged PINCH containing only LIM13, confirming the specificity of the interaction and suggesting LIM4 and/or 5 is the site of binding. ILK, which binds LIM1 of PINCH, copurifies with both full-length and the truncated LIM13 version of His-tagged PINCH, serving as a positive control. Both PINCH and ILK are copurified with His-tagged RSU-1. Moreover, endogenous PINCH and RSU-1 can be coimmunoprecipitated. The site of RSU-1 binding to PINCH was further mapped using yeast two-hybrid analysis. Only cells expressing LIM5 bait/RSU-1 prey activated all three reporters, indicating LIM5 is the site of RSU-1 binding. Consistent with the view that they interact in vivo, PINCH:GFP and RSU-1 are prominently colocalized at integrin-rich muscle attachment sites in Drosophila embryos (Kadrmas, 2004).
Drosophila RSU-1, which displays seven leucine-rich repeats with high sequence similarity to small GTPase regulators, is encoded by the CG9031 locus. A P-element insertion allele was characterized that disrupts the RSU-1 coding sequence. Flies homozygous for this mutation within CG9031 are viable and fertile, and lack RSU-1 protein as indicated by Western analysis with multiple anti-RSU-1 antibodies. PINCH and RSU-1 are both expressed in larval wing discs and similar to stck wing clones, the mutation within CG9031 produces flies with wing blisters at 60% penetrance. These data are consistent with PINCH and RSU-1 acting in concert to support integrin-dependent adhesion. The CG9031 gene was named icarus (ics) after the son of Daedalus who had unstable wings (Kadrmas, 2004).
Although elimination of RSU-1 function does not result in dorsal or thorax closure defects, the role of RSU-1 in these processes was evaluated by testing for genetic interactions between ics and msn. Similar to what occurs with reduction of stck dosage, homozygous mutation of ics suppresses DC defects observed in msn102 mutant embryos. Absence of RSU-1 also increases eclosure rates of msn3349 hypomorphs and completely suppresses the thorax defects present in msn3349 animals, suggesting that like PINCH, RSU-1 can function as a negative regulator of JNK signaling. To confirm that the suppression of msn DC defects by ics mutation is mediated by the JNK signaling cascade, RSU-1 was eliminated in basket (bsk) embryos that lack zygotic JNK, the terminal kinase in this cascade. Homozygous ics mutation suppresses the DC defects of bsk1 mutants, confirming that ics loss-of-function mutations affect DC by influencing the JNK cascade. Moreover, wing discs isolated from ics mutants display a 30% increase in active phospho-JNK relative to wild type, providing direct biochemical confirmation that RSU-1 influences JNK activation state in vivo. Although no localized accumulation of RSU-1 during DC was detected, RSU-1 is readily detected by Western analysis in stage 13 embryos that are undergoing DC. Thus, the temporal pattern of RSU-1 expression is consistent with genetic results that highlight its role in regulation of JNK-dependent morphogenesis (Kadrmas, 2004).
Analysis of PINCH and RSU-1 levels in wild-type versus stck or ics mutant embryos provided insight into the physiological significance of their association. In embryos mutant for both maternal and zygotic stck, RSU-1 is dramatically reduced relative to wild-type levels. Likewise, in ics embryos, PINCH levels are also decreased. These observations suggest that PINCH and RSU-1 are reciprocally dependent on each other for maximal expression and/or stability. The mechanism for coordinate regulation of RSU-1 and PINCH remains to be determined. Because the phenotypes associated with loss of RSU-1 represent a subset of stck phenotypes, the processes disturbed in ics mutants may be exquisitely sensitive to PINCH levels. Alternately, RSU-1 may have functions that are independent of its role in PINCH stabilization (Kadrmas, 2004).
The data are consistent with a model in which PINCH could modulate JNK signaling in two distinct ways. (1) PINCH is present at areas where JNK is active and antagonizes JNK signaling. This behavior is reminiscent of Drosophila Puc, a phosphatase regulator of the JNK cascade that establishes a negative feedback loop. PINCH has no intrinsic catalytic activity, but might recruit proteins that could alter the availability or activity of JNK signaling components. Like Puc, PINCH expression is up-regulated in response to constitutive JNK signaling. Availability of RSU-1 at these sites of active JNK signaling could independently regulate JNK signaling or modulate the effects of PINCH on JNK through regulation of PINCH stability. (2) PINCH and RSU-1 are required for integrin-dependent adhesion. PINCH links integrins to the actin cytoskeleton via ILK and Parvin, and these connections could influence both integrin-dependent adhesion and signaling. Integrin signaling, through a variety of tyrosine kinases and Rac, stimulates the JNK cascade; therefore, PINCH may also exert an influence on JNK signaling via integrin. The current findings illustrate that the cellular concentration of PINCH affects the level of RSU-1 and vice versa. Thus, modulation of the ratio of RSU-1 to PINCH could provide a mechanism to regulate JNK signaling during DC and thorax closure in Drosophila. It is hypothesized that PINCH/RSU-1 complexes fine-tune and integrate the JNK and integrin signaling cascades required during morphogenesis, highlighting the potential role of integrin-associated apical junctional complexes as signal coordination points for epithelial morphogenesis (Kadrmas, 2004).
Search PubMed for articles about Drosophila Parvin
Chountala, M., Vakaloglou, K. M. and Zervas, C. G. (2012). Parvin overexpression uncovers tissue-specific genetic pathways and disrupts F-actin to induce apoptosis in the developing epithelia in Drosophila. PLoS One 7: e47355. PubMed ID: 23077599
Fukuda, K., Gupta, S., Chen, K., Wu, C. and Qin, J. (2009). The pseudoactive site of ILK is essential for its binding to alpha-Parvin and localization to focal adhesions. Mol Cell 36: 819-830. PubMed ID: 20005845
Geiger, B. and Yamada, K. M. (2011). Molecular architecture and function of matrix adhesions. Cold Spring Harb Perspect Biol 3. PubMed ID: 21441590
Gimona, M., Djinovic-Carugo, K., Kranewitter, W. J. and Winder, S. J. (2002). Functional plasticity of CH domains. FEBS Lett 513: 98-106. PubMed ID: 11911887
Kadrmas, J. L., Smith, M. A., Clark, K. A., Pronovost, S. M., Muster, N., Yates, J. R. and Beckerle, M. C. (2004). The integrin effector PINCH regulates JNK activity and epithelial migration in concert with Ras suppressor 1. J Cell Biol 167: 1019-1024. PubMed ID: 15596544
Karaköse, E., Schiller, H. and Fässler, R. (2010). The kindlins at a glance. J. Cell Sci. 123: 2353-2356. PubMed Citation: 20592181
Kimura, M., Murakami, T., Kizaka-Kondoh, S., Itoh, M., Yamamoto, K., Hojo, Y., Takano, M., Kario, K., Shimada, K. and Kobayashi, E. (2010). Functional molecular imaging of ILK-mediated Akt/PKB signaling cascades and the associated role of beta-parvin. J Cell Sci 123: 747-755. PubMed ID: 20164304
Lange, A., Wickstrom, S. A., Jakobson, M., Zent, R., Sainio, K. and Fassler, R. (2009). Integrin-linked kinase is an adaptor with essential functions during mouse development. Nature 461: 1002-1006. PubMed ID: 19829382
Legate, K. R., Montanez, E., Kudlacek, O. and Fassler, R. (2006). ILK, PINCH and parvin: the tIPP of integrin signalling. Nat Rev Mol Cell Biol 7: 20-31. PubMed ID: 16493410
Lin, X., Qadota, H., Moerman, D. G. and Williams, B. D. (2003). C. elegans PAT-6/actopaxin plays a critical role in the assembly of integrin adhesion complexes in vivo. Curr Biol 13: 922-932. PubMed ID: 12781130
Löer, B., et al. (2008). The NHL-domain protein Wech is crucial for the integrin-cytoskeleton link. Nat. Cell Biol. 10(4): 422-8. PubMed Citation: 18327251
Lorenz, S., Vakonakis, I., Lowe, E. D., Campbell, I. D., Noble, M. E. and Hoellerer, M. K. (2008). Structural analysis of the interactions between paxillin LD motifs and alpha-parvin. Structure 16: 1521-1531. PubMed ID: 18940607
Montanez, E., Wickstrom, S. A., Altstatter, J., Chu, H. and Fassler, R. (2009). Alpha-parvin controls vascular mural cell recruitment to vessel wall by regulating RhoA/ROCK signalling. EMBO J 28: 3132-3144. PubMed ID: 19798050
Nikolopoulos, S. N. and Turner, C. E. (2000). Actopaxin, a new focal adhesion protein that binds paxillin LD motifs and actin and regulates cell adhesion. J Cell Biol 151: 1435-1448. PubMed ID: 11134073
Olski, T. M., Noegel, A. A. and Korenbaum, E. (2001). Parvin, a 42 kDa focal adhesion protein, related to the alpha-actinin superfamily. J Cell Sci 114: 525-538. PubMed ID: 11171322
Sebe-Pedros, A., Roger, A. J., Lang, F. B., King, N. and Ruiz-Trillo, I. (2010). Ancient origin of the integrin-mediated adhesion and signaling machinery. Proc Natl Acad Sci U S A 107: 10142-10147. PubMed ID: 20479219
Stanchi, F., et al. (2005). Consequences of loss of PINCH2 expression in mice. J. Cell Sci. 118(Pt 24): 5899-910. 16317048
Vakaloglou, K. M., Chountala, M. and Zervas, C. G. (2012). Functional analysis of parvin and different modes of IPP-complex assembly at integrin sites during Drosophila development. J Cell Sci 125: 3221-3232. PubMed ID: 22454516
Wang, X., Fukuda, K., Byeon, I. J., Velyvis, A., Wu, C., Gronenborn, A. and Qin, J. (2008). The structure of alpha-parvin CH2-paxillin LD1 complex reveals a novel modular recognition for focal adhesion assembly. J Biol Chem 283: 21113-21119. PubMed ID: 18508764
Wickstrom, S. A., Lange, A., Montanez, E. and Fassler, R. (2010). The ILK/PINCH/parvin complex: the kinase is dead, long live the pseudokinase! EMBO J 29: 281-291. PubMed ID: 20033063
Wickstrom, S. A., Radovanac, K. and Fassler, R. (2011). Genetic analyses of integrin signaling. Cold Spring Harb Perspect Biol 3. PubMed ID: 21421914
Wu, C. and Dedhar, S. (2001). Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. J Cell Biol 155: 505-510. PubMed ID: 11696562
Yamaji, S., Suzuki, A., Kanamori, H., Mishima, W., Yoshimi, R., Takasaki, H., Takabayashi, M., Fujimaki, K., Fujisawa, S., Ohno, S. and Ishigatsubo, Y. (2004). Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction. J Cell Biol 165: 539-551. PubMed ID: 15159419
Zervas, C. G., et al. (2011). A central multifunctional role of integrin-linked kinase at muscle attachment sites. J. Cell Sci. 124(Pt 8): 1316-27. PubMed ID: 21444757
date revised: 10 April 2013
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