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Cell Adh Migr. 2009 Apr-Jun; 3(2): 191–194.
PMCID: PMC2679884

Regulatory models of RhoA suppression by dematin, a cytoskeletal adaptor protein


Cell motility, adhesion and actin cytoskeletal rearrangements occur upon integrin-engagement to the extracellular matrix and activation of the small family of Rho GTPases, RhoA, Rac1 and Cdc42. The activity of the GTPases is regulated through associations with guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and guanine dissociation inhibitors (GDIs). Recent studies have demonstrated a critical role for actin-binding proteins, such as ezrin, radixin and moesin (ERM), in modulating the activity of small GTPases through their direct associations with GEFs, GAPs and GDI's. Dematin, an actin binding and bundling phospho-protein was first identified and characterized from the erythrocyte membrane, and has recently been implicated in regulating cell motility, adhesion and morphology by suppressing RhoA activation in mouse embryonic fibroblasts. Although the precise mechanism of RhoA suppression by dematin is unclear, several plausible and hypothetical models can be invoked. Dematin may bind and inhibit GEF activity, form an inactive complex with GDI-RhoA-GDP, or enhance GAP function. Dematin is the first actin-binding protein identified from the erythrocyte membrane that participates in GTPase signaling, and its broad expression suggests a conserved function in multiple tissues.

Key words: dematin, RhoA, actin, GTPase, signaling

Cell adhesion and motility are mediated through activation of integrin receptors and the family of small Rho GTPases.1 Engagement of integrin receptors to the extracellular matrix leads to the activation of multiple kinase pathways (i.e., FAK, Src), inducing the assembly of the focal adhesion complex and actin/myosin contraction. Furthermore, activation of the receptor tyrosine kinases (i.e., insulin receptor) or G-protein coupled receptors (i.e., LPA receptor), leads to downstream signaling events that also trigger multiple kinase pathways that regulate the protrusive and contractile actin/myosin dynamics. These adhesion-dependent or receptor-driven signaling cascades ultimately result in the activation of the small family of Rho GTPases: Cdc42, Rac1 and RhoA, key regulators of actin cytoskeleton assembly. The activation of these GTPases induces lamellipodia (Rac1), filopodia (Cdc42), actin stress fiber formation (RhoA) and focal adhesion complex formation (Rac1 and RhoA). Nascent focal adhesion complex formation within the lamellipodia is a result of Rac1 activation;2 however, mature focal adhesions and actin cytoskeletal rearrangements are a direct consequence of RhoA activation.3

The regulation of RhoA activity occurs through its interactions with guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and guanine dissociation inhibitors (GDIs) (reviewed in refs. 46). The regulatory intricacies that govern RhoA association with GEFs, GAPs and GDIs remain poorly understood. However, it is now well accepted that actin-binding proteins participate and play a significant role in regulating the functional activity of RhoA GTPase through their direct association with RhoGEFs, GDI's and RhoGAPs (Table 1). Dematin, an actin binding protein, has been recently identified as a novel suppressor of RhoA activation; however, the precise mechanism of this function remains unknown.7

Table 1
List of actin-binding proteins that are known to directly bind to GEFs, GAPs or GDIs and regulate RhoA activation

Dematin, previously known as erythrocyte membrane protein band 4.9, is a member of the villin family of headpiece-containing actin-binding proteins. It contains a c-terminal actin-binding domain, and an N-terminal “core-domain” of unknown function.8,9 Dematin was first isolated and characterized from the mature erythrocyte membrane,10,11 where it functions to maintain erythrocyte shape and membrane structural integrity via a novel linkage at the actin-spectrin junctional complex through glucose transporter-1 (GLUT1) in a species specific manner.12 Despite its wide-spread expression, relatively little is known about the biological function of this actin-binding protein in non-erythroid cells. Previous studies have shown that the human dematin gene (EPB4.9) maps to 8p21.1,9 a chromosomal region that is frequently deleted in prostate cancers. Interestingly, it was demonstrated that a sub-set of metastatic prostate tumors show a loss of heterozygosity of the dematin gene. Furthermore, it was demonstrated that in PC-3 cells, a prostate cancer cell line, the overexpression of the dematin gene was able to revert the oncogenic morphology (cell rounding) to a normal prostate epithelial morphology (microvillar and cytoplasmic extensions), thus suggesting a possible role for dematin in modulating these cellular processes.13 To determine the in vivo function of dematin, a dematin headpiece-null mouse (HPKO) model was generated in our laboratory, lacking the c-terminal actin binding headpiece domain. Consequently, the HPKO model expresses a truncated variant of dematin containing the N-terminal “core-domain.” Hematological analysis of the HPKO erythrocytes revealed evidence of membrane fragility, spherocytosis and mild hemolytic anemia.14 Since the loss of the dematin actin-binding headpiece resulted in morphological defects in the erythrocyte, we extended these studies to investigate if these defects would manifest in non-erythroid cells. Isolated mouse embryonic fibroblasts from HPKO mice display abnormal cell morphology, motility and adhesion, presumably resulting from RhoA hyperactivation and subsequent phosphorylation of downstream signaling molecules, such as focal adhesion kinase (FAK) and myosin light chain (MLC).7 These data suggest that dematin acts upstream of RhoA perhaps by associating with one of the known regulators of RhoA activation: GEFs, GAPs and GDIs (Fig. 1).

Figure 1
Hypothetical models of dematin mediated regulation of RhoA signaling. (A) Dematin has been shown to bind the DH domain of RasGRF2, but does not modulate Rac1 or Ras activation through RasGRF2. In several yeast-2-hybrid RasGRF2 clones, an insert from the ...

Previous evidence has shown that dematin binds to calcium activated Ras-guanine nucleotide-releasing factor 2 (RasGRF2).15 RasGRF2 is a bifunctional guanine nucleotide exchange factor (GEF) that can modulate the activation of Ras through its Cdc25 domain and Rac1 through its DH-PH domains (Dbl and Plekstrin homology domains).16 Although dematin binds to the DH domain of RasGRF2, dematin was unable to modulate the activation of Rac1 or Ras. Moreover, the yeast-2 hybrid results revealed that several of the isolated RasGRF2 clones contained an insert from the GEFD2 domain of Trio, a RhoA GEF.17 It is therefore plausible, that in vivo, dematin associates with Trio, and inhibits RhoA activation, similar to TRIPalpha, the first known inhibitor of a RhoA GEF, which specifically blocks the Trio GEFD2-exchange activity of RhoA.18 The significance of the postulated in vivo dematin interactions with Ras-GRF2 and TrioRhoGEF has not been established, but taken together; this model may provide a mechanistic link between dematin and RhoA (Fig. 1).

RhoGAPs catalyze the hydrolysis of the active GTP-bound state of RhoA to the inactive GDP-bound form through intrinsic GTPase activity. Although there is no indication that dematin binds to a RhoGAP, it is possible that dematin behaves similarly to actopaxin19 and VRP1,20 actin-binding proteins that provide spatial and temporal regulation of RhoGAP function, and consequently RhoA inhibition. In addition to the regulation of RhoA through GEFs and GAPs, the actin-binding proteins, ezrin, radixin and moesin (ERMs) are known to sequester the guanine dissociation inhibitor, GDI, from RhoGDP.21 The tethering of GDI to the actin cytoskeleton reduces GDI activity, resulting in an increase in RhoA activation. Furthermore, recent studies have shown that PKA phosphorylation of GDI results in an increase in the association between GDI and RhoA-GDP, thus resulting in a decrease in RhoA activity.22 Interestingly, PKA phosphorylates and inhibits dematin's actin-bundling activity by inducing a conformational change in the dematin actin-binding headpiece domain.10,23 It is possible that in the absence of PKA, dematin robustly interacts with GDI resulting in a stronger and tighter linkage to the actin cytoskeleton; thus in turn resulting in an increase in RhoA activation. Phosphorylation of dematin by PKA may result in the release of GDI from dematin and the actin cytoskeleton and causing subsequent suppression of RhoA activity. It is also possible that dematin retains inactive Rho-GDP in the cytosol, through an association with RhoGDI and the actin cytoskeleton. RhoA activation would occur when the dematin-RhoGDI-RhoA-GDP complex disassociates from the cytoskeleton via intracellular signaling events (Fig. 1).

In addition to the aforementioned mechanisms of RhoA regulation through GEFs, GAPs and GDIs, it is also possible that dematin participates in the signaling cascade several steps upstream of RhoA activation. Dematin's interaction with GLUT1,12 and with the scaffolding protein 14-3-3ζ may provide alternative models to investigate the mechanism of dematin-mediated suppression of RhoA. Since dematin interacts with GLUT1, it is possible that dematin mediates GLUT1 trafficking to the plasma membrane. In the absence of dematin, GLUT1 trafficking may be altered, thus resulting in abnormal glucose uptake. Metabolic defects have significant effects on intracellular signaling, which manifest itself in a variety of phenotypes, such as altered cell morphology, motility and adhesion.25 Proteomic analysis, as well as seven consensus 14-3-3 binding motifs, suggests that dematin may interact in vivo with the scaffolding protein, 14-3-3ζ.24 Recent evidence has shown that PI3-Kinase/Akt activation induces the association of an ankyrin repeat domain-containing protein, KANK, with 14-3-3ζ, which in turn results in RhoA activation.26 The mechanism by which KANK negatively regulates 14-3-3ζ-activation of RhoA is unknown. However, it has been reported that the RhoGEF, AKAP-Lbc, is inhibited by anchoring PKA to 14-3-3ζ.27 It is thus possible that dematin exists in a similar complex to suppress RhoA activation.

The unexpected finding that dematin functions as a suppressor of RhoA activity has its significance as being the first protein isolated from the erythrocyte that has been functionally linked to a small GTPase and regulates its activity. There is a significant amount of RhoA in the human erythrocytes,28 and it is possible that other cytoskeletal components of the erythrocyte membrane are also able to modulate small Rho-GTPases in vivo. Recent evidence has implicated the small GTPase, Rac1 and Rac2 in modulating the deformability of the erythrocyte membrane29 and Rac GTPases together with mDia2 regulate enucleation in mammalian erythroblasts.30 Although the precise mechanism of these processes is not yet clear, it raises the possibility that the erythrocyte membrane yet again serves as a paradigm for elucidating fundamental biochemical processes beyond the field of red cell biology. Future studies on the dematin-RhoA signaling pathway will be directed toward elucidating the mechanism by which dematin is able to suppress RhoA activation in relevant cell types.


guanine nucleotide exchange factor
GTPase activating protein
guanine dissociation inhibitor
dematin headpiece-null knockout mouse


Previously published online as a Cell Adhesion & Migration E-publication:


1. Hotchin NA, Hall A. Regulation of the actin cytoskeleton, integrins and cell growth by the Rho family of small GTPases. Cancer Surv. 1996;27:311–322. [PubMed]
2. Rottner K, Hall A, Small JV. Interplay between Rac and Rho in the control of substrate contact dynamics. Curr Biol. 1999;9:640–648. [PubMed]
3. Chrzanowska-Wodnicka M, Burridge K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol. 1996;133:1403–1415. [PMC free article] [PubMed]
4. Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 2005;6:167–180. [PubMed]
5. Moon SY, Zheng Y. Rho GTPase-activating proteins in cell regulation. Trends Cell Biol. 2003;13:13–22. [PubMed]
6. DerMardirossian C, Bokoch GM. GDIs: central regulatory molecules in Rho GTPase activation. Trends Cell Biol. 2005;15:356–363. [PubMed]
7. Mohseni M, Chishti AH. The headpiece domain of dematin regulates cell shape, motility and wound healing by modulating RhoA activation. Mol Cell Biol. 2008;28:4712–4718. [PMC free article] [PubMed]
8. Rana AP, Ruff P, Maalouf GJ, Speicher DW, Chishti AH. Cloning of human erythroid dematin reveals another member of the villin family. Proc Natl Acad Sci USA. 1993;90:6651–6655. [PubMed]
9. Azim AC, Knoll JH, Beggs AH, Chishti AH. Isoform cloning, actin binding and chromosomal localization of human erythroid dematin, a member of the villin superfamily. J Biol Chem. 1995;270:17407–17413. [PubMed]
10. Chishti AH, Levin A, Branton D. Abolition of actin-bundling by phosphorylation of human erythrocyte protein 4.9. Nature. 1988;334:718–721. [PubMed]
11. Chishti AH, Faquin W, Wu CC, Branton D. Purification of erythrocyte dematin (protein 4.9) reveals an endogenous protein kinase that modulates actin-bundling activity. J Biol Chem. 1989;264:8985–8989. [PubMed]
12. Khan AA, Hanada T, Mohseni M, Jeong JJ, Zeng L, Gaetani M, et al. Dematin and adducin provide a novel link between the spectrin cytoskeleton and human erythrocyte membrane by directly interacting with glucose transporter-1. J Biol Chem. 2008;283:14600–14609. [PMC free article] [PubMed]
13. Lutchman M, Pack S, Kim AC, Azim A, Emmert-Buck M, van Huffel C, et al. Loss of heterozygosity on 8p in prostate cancer implicates a role for dematin in tumor progression. Cancer Genet Cytogenet. 1999;115:65–69. [PubMed]
14. Khanna R, Chang SH, Andrabi S, Azam M, Kim A, Rivera A, et al. Headpiece domain of dematin is required for the stability of the erythrocyte membrane. Proc Natl Acad Sci USA. 2002;99:6637–6642. [PubMed]
15. Lutchman M, Kim AC, Cheng L, Whitehead IP, Oh SS, Hanspal M, et al. Dematin interacts with the Ras-guanine nucleotide exchange factor Ras-GRF2 and modulates mitogenactivated protein kinase pathways. Eur J Biochem. 2002;269:638–649. [PubMed]
16. Fam NP, Fan WT, Wang Z, Zhang LJ, Chen H, Moran MF. Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras. Mol Cell Biol. 1997;17:1396–1406. [PMC free article] [PubMed]
17. Debant A, Serra-Pagès C, Seipel K, O'Brien S, Tang M, Park SH, et al. The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains. Proc Natl Acad Sci USA. 1996;93:5466–5471. [PubMed]
18. Schmidt S, Diriong S, Méry J, Fabbrizio E, Debant A. Identification of the first Rho-GEF inhibitor, TRIPalpha, which targets the RhoA-specific GEF domain of Trio. FEBS Lett. 2002;523:35–42. [PubMed]
19. LaLonde DP, Grubinger M, Lamarche-Vane N, Turner CE. CdGAP associates with actopaxin to regulate integrin-dependent changes in cell morphology and motility. Curr Biol. 2006;16:1375–1385. [PubMed]
20. Roumanie O, Peypouquet MF, Bonneu M, Thoraval D, Doignon F, Crouzet M. Evidence for the genetic interaction between the actin-binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae. Mol Microbiol. 2000;36:1403–1414. [PubMed]
21. Takahashi K, Sasaki T, Mammoto A, Takaishi K, Kameyama T, Tsukita S, et al. Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J Biol Chem. 1997;272:23371–23375. [PubMed]
22. Qiao J, Huang F, Lum H. PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction. Am J Physiol Lung Cell Mol Physiol. 2003;284:972–980. [PubMed]
23. Jiang ZG, McKnight CJ. A phosphorylation-induced conformation change in dematin headpiece. Structure. 2006;14:379–387. [PubMed]
24. Angrand PO, Segura I, Völkel P, Ghidelli S, Terry R, Brajenovic M, et al. Transgenic mouse proteomics identifies new 14-3-3-associated proteins involved in cytoskeletal rearrangements and cell signaling. Mol Cell Proteomics. 2006;5:2211–2227. [PubMed]
25. Howell SL, Tyhurst M. The cytoskeleton and insulin secretion. Diabetes Metab Rev. 1986;2:107–123. [PubMed]
26. Kakinuma N, Roy BC, Zhu Y, Wang Y, Kiyama R. Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K-Akt signaling. J Cell Biol. 2008;181:537–549. [PMC free article] [PubMed]
27. Diviani D, Abuin L, Cotecchia S, Pansier L. Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex. EMBO J. 2004;23:2811–2820. [PubMed]
28. Boukharov AA, Cohen CM. Guanine nucleotide-dependent translocation of RhoA from cytosol to high affinity membrane binding sites in human erythrocytes. Biochem J. 1998;330:1391–1398. [PubMed]
29. Kalfa TA, Pushkaran S, Mohandas N, Hartwig JH, Fowler VM, Johnson JF, et al. Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton. Blood. 2006;108:3637–3645. [PubMed]
30. Ji P, Jayapal SR, Lodish HF. Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2. Nat Cell Biol. 2008;10:314–321. [PubMed]
31. Takahashi K, Sasaki T, Mammoto A, Hotta I, Takaishi K, Imamura H, et al. Interaction of radixin with Rho small G protein GDP/GTP exchange protein Dbl. Oncogene. 1998;16:3279–3284. [PubMed]
32. Seipel K, O'Brien SP, Iannotti E, Medley QG, Streuli M. Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization. J Cell Sci. 2001;114:389–399. [PubMed]
33. Ryan XP, Alldritt J, Svenningsson P, Allen PB, Wu GY, Nairn AC, et al. The Rho-specific GEF Lfc interacts with neurabin and spinophilin to regulate dendritic spine morphology. Neuron. 2005;47:85–100. [PubMed]
34. Maeda M, Matsui T, Imamura M, Tsukita S, Tsukita S. Expression level, subcellular distribution and rho-GDI binding affinity of merlin in comparison with Ezrin/Radixin/Moesin proteins. Oncogene. 1999;18:4788–4797. [PubMed]

Les articles de Cell Adhesion & Migration ont été offerts à titre gracieux par Taylor & Francis.