Ubiquitination and the Regulation of Rho Family Pathways Rho News Rho Publications Rho Research Tools OCT 2012 Upcoming Meetings Neuroscience 2012 New Orleans, LA, USA Booth # 512 Oct. 13-17, 2012 ASCB 2012 San Francisco, CA, USA Booth # 901 Dec. 15-19, 2012 Cytoskeleton Products Actin Proteins Activation Assays Antibodies ECM Proteins ELISA Kits G-LISA ® Kits Pull-down Assays Motor Proteins Small G-Proteins Tubulin & FtsZ Proteins Contact Us P: 1 (303) 322.2254 F: 1 (303) 322.2257 E: [email protected]W: cytoskeleton.com Distributors www.cytoskeleton.com/distributors/ www.cytoskeleton.com CYTOSKELETON NEWS NEWS FROM CYTOSKELETON INC. this issue Ubiquitination and the Regulation of Rho Family Pathways Rho Ubiquitination Publications Rho Family Research Tools The mammalian proteome is esmated to contain approximately 1,000,000 unique proteins. This level of complexity is derived from a relavely simple genome (approx. 25,000 genes), a transcriptome which increases the potenal protein footprint to 100,000, and protein post-translaonal modificaons (PTMs) which account for a further order of magnitude increase in proteome complexity and an almost limitless potenal for funconal diversity 1-3 . One of the over 200 disnct PTMs described in the literature is ubiquinaon (a.k.a. ubiquitylaon) 4 . Ubiquin (Ub) and ubiquin-like proteins (Ubls, e.g., SUMO, Nedd) are a group of approximately 15 proteins with a molecular weight of approx. 8 kDa. During the ubiquinaon process, one or more of these proteins are conjugated via acvang (E1), conjugang (E2), and ligang (E3) enzymes to lysines of target proteins 5,6 . To read more about ubiquinaon, see these excellent reviews 7-9 . The classic funcon of ubiquinaon is to target proteins for proteosomal or lysosomal degradaon as well as regulang spao-temporal cell signaling events 5,7 . An emerging funcon of ubiquinaon is the acvaon of proteins via the creaon of unique protein:protein interacons 10 . Like most PTMs, ubiquinaon is reversible, catalyzed by ubiquin-specific proteases (a.k.a. deubiquinang enzymes) 11 . The reversible nature of ubiquinaon further enhances the potenal of this PTM to dynamically regulate the funcon of proteins, including Rho family GTPases. The Emerging Roles of Ub and Ubl PTMs in Rho Family Protein Regulaon Rho GTPases act as molecular switches and their physiological acvity is dependent upon proper cellular localizaon 12 and the binding of GTP to achieve the acve, signal propagang conformaon. Conversely, the inacve, GDP-bound form is generally unable to bind the effectors necessary for downstream signal transducon 12 . Cycling between GTP/GDP is regulated by GTPase acvang proteins (GAPs), GTPase exchange factors (GEFs) and guanine-nucleode dissociaon inhibitors (GDIs). Together these proteins regulate acvaon (GEFs), inacvaon (GAPs), and cytosolic stabilizaon (GDIs) of Rho GTPases 12 . Recently, it has become apparent that this relavely simple paradigm is only the p of the iceberg in Rho GTPase regulaon as GDP-bound Rho proteins can also propagate signal transducon pathways 13 . Moreover, ubiquinaon goes beyond the simple house-keeping task of tagging Rho proteins for degradaon to also regulang the spao-temporal dynamics of Rho GTPase acvity, including an alternave way to terminate GTPase acvity 7 (see Table 1). While a complete understanding of the role of ubiquinaon in the regulaon of Rho signaling pathways is lacking, some general concepts are emerging: 1) Ub and Ubl modificaons can regulate spao- temporal modulaon of Rho pathways. Several studies have shown that the RhoA ubiquin ligase, Smurf1, can be recruited to specific cellular locaons where it regulates the localized degradaon of Rho which can regulate cell shape, molity, polarity, and epithelial to mesenchymal transion (EMT) 14-17 . 2) Ub and Ubl modificaons do not always result in protein degradaon; for example, the SUMOylaon of Rac1 results in maintenance of the acve, GTP-bound state, possibly through increased binding to a GEF or decreased binding to a GAP 18 . 3) Ub and Ubl-modified proteins tend to represent a small percentage of any given GTPase populaon. However, they appear to be more selecve for the acve GTP-bound form of the protein, and in this way have a large effect on temporal signal transducon 19-20 . 4) Ub or Ubl conjugaon may be a major regulator of atypical Rho family proteins (e.g., RhoBTB2) that do not hydrolyze GTP and hence remain constuvely acve 21 . The scope of ubiquin regulaon for atypical Rho GTPases is currently under invesgaon 22-24 with this modulaon offering at least a paral explanaon for how Rho proteins are regulated even in the absence of a classical GTP/GDP switch mechanism.
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Ubiquitination and the Regulation of Rho Family Pathways
CYTOSKELETON NEWSN E W S F R O M C Y T O S K E L E T O N I N C .
this issue
Ubiquitination and the Regulation of Rho Family Pathways Rho Ubiquitination Publications
Rho Family Research Tools
The mammalian proteome is estimated to contain approximately 1,000,000 unique proteins. This level of complexity is derived from a relatively simple genome (approx. 25,000 genes), a transcriptome which increases the potential protein footprint to 100,000, and protein post-translational modifications (PTMs) which account for a further order of magnitude increase in proteome complexity and an almost limitless potential for functional diversity1-3. One of the over 200 distinct PTMs described in the literature is ubiquitination (a.k.a. ubiquitylation)4. Ubiquitin (Ub) and ubiquitin-like proteins (Ubls, e.g., SUMO, Nedd) are a group of approximately 15 proteins with a molecular weight of approx. 8 kDa. During the ubiquitination process, one or more of these proteins are conjugated via activating (E1), conjugating (E2), and ligating (E3) enzymes to lysines of target proteins5,6. To read more about ubiquitination, see these excellent reviews7-9.
The classic function of ubiquitination is to target proteins for proteosomal or lysosomal degradation as well as regulating spatio-temporal cell signaling events5,7. An emerging function of ubiquitination is the activation of proteins via the creation of unique protein:protein interactions10. Like most PTMs, ubiquitination is reversible, catalyzed by ubiquitin-specific proteases (a.k.a. deubiquitinating enzymes)11. The reversible nature of ubiquitination further enhances the potential of this PTM to dynamically regulate the function of proteins, including Rho family GTPases.
The Emerging Roles of Ub and Ubl PTMs in Rho Family Protein Regulation
Rho GTPases act as molecular switches and their physiological activity is dependent upon proper cellular localization12 and the binding of GTP to achieve the active, signal propagating conformation. Conversely, the inactive, GDP-bound form is generally unable to bind the effectors necessary for downstream signal transduction12. Cycling between GTP/GDP is regulated by GTPase activating proteins (GAPs), GTPase exchange factors
(GEFs) and guanine-nucleotide dissociation inhibitors (GDIs). Together these proteins regulate activation (GEFs), inactivation (GAPs), and cytosolic stabilization (GDIs) of Rho GTPases12. Recently, it has become apparent that this relatively simple paradigm is only the tip of the iceberg in Rho GTPase regulation as GDP-bound Rho proteins can also propagate signal transduction pathways13. Moreover, ubiquitination goes beyond the simple house-keeping task of tagging Rho proteins for degradation to also regulating the spatio-temporal dynamics of Rho GTPase activity, including an alternative way to terminate GTPase activity7 (see Table 1). While a complete understanding of the role of ubiquitination in the regulation of Rho signaling pathways is lacking, some general concepts are emerging:
1) Ub and Ubl modifications can regulate spatio-temporal modulation of Rho pathways. Several studies have shown that the RhoA ubiquitin ligase, Smurf1, can be recruited to specific cellular locations where it regulates the localized degradation of Rho which can regulate cell shape, motility, polarity, and epithelial to mesenchymal transition (EMT)14-17.
2) Ub and Ubl modifications do not always result in protein degradation; for example, the SUMOylation of Rac1 results in maintenance of the active, GTP-bound state, possibly through increased binding to a GEF or decreased binding to a GAP18.
3) Ub and Ubl-modified proteins tend to represent a small percentage of any given GTPase population. However, they appear to be more selective for the active GTP-bound form of the protein, and in this way have a large effect on temporal signal transduction19-20.
4) Ub or Ubl conjugation may be a major regulator of atypical Rho family proteins (e.g., RhoBTB2) that do not hydrolyze GTP and hence remain constitutively active21. The scope of ubiquitin regulation for atypical Rho GTPases is currently under investigation22-24 with this modulation offering at least a partial explanation for how Rho proteins are regulated even in the absence of a classical GTP/GDP switch mechanism.
Rho Family Small G-protein ToolsContinued from Page 1Small G-protein Activation Assays Method Cat. # AmountCdc42 G-LISA® Activation Assay, colorimetric G-LISA® BK127 96 assays
Rac1 His Protein, wild-type >90% RC01-A 1 x 100 µg
RhoA His Protein, constitutively-active (Q63L) >90% R6301-A 1 x 10 µg
RhoA His Protein, wild-type >80% RH01-A 1 x 100 µg
Small G-protein Antibodies Host/Type Species Reactivity
Cat. # Amount
Cdc42 Specific AntibodyHuman Cdc42 Peptide
Mouse/mAb Hu, Ms, Rt, other extracts
ACD03-AACD03-B
1 x 100 µg3 x 100 µg
Rac1 Specific AntibodyHuman C-terminal Peptide
Mouse/mAb Hu, Ms, Rt, other extracts
ARC03-AARC03-B
2 x 50 µg6 x 50 µg
RhoA Specific AntibodyHuman RhoA Peptide
Mouse/mAb Hu, Ms, Rt, other extracts
ARH03-AARH03-B
1 x 100 µg3 x 100 µg
References
5) Ubiquitination appears to be an important PTM in regulating the activity of many Rho GTPase modulators; for example, RhoGDI25, the Rho GEF Pbl, and the Cdc42 GEF hPEM-2 have all been demonstrated to be targets for ubiquitination25-27.
In conclusion, it is clear that comprehensive analyses of Rho GTPase regulation by ubiquitination should greatly enhance our understanding of Rho signal transduction pathways in health and disease.
Table 1: Examples of Ubiquitination Involvement in the Regulation of Rho Pathways
w w w . c y t o s k e l e t o n . c o m
Actin ProductsRHO FAMILY PRODUCTS
1. International human genome sequencing consortium. 2004. Nature. 431:931-945.