Ubiquitination and the Regulation of Rho Family Pathways

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Ubiquitination and the Regulation of Rho Family Pathways Rho Ubiquitination Publications

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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

Cdc42 Pull-down Activation Assay Biochem Kit™ Pull-down BK034BK034-S

50 assays20 assays

Rac1,2,3 G-LISA® Activation Assay, colorimetric G-LISA® BK125 96 assays

Rac1 G-LISA® Activation Assay, colorimetric G-LISA® BK128 96 assays

Rac1 Pull-down Activation Assay Biochem Kit™ Pull-down BK035BK035-S

50 assays20 assays

RhoA / Rac1 / Cdc42 Activation Assay Combo Kit Pull-down BK030 3 x 10 assays

RhoA G-LISA® Activation Assay, colorimetric G-LISA® BK124 96 assays

RhoA G-LISA® Activation Assay, luminescence G-LISA® BK121 96 assays

RhoA Pull-down Activation Assay Biochem Kit™ Pull-down BK036BK036-S

80 assays20 assays

Rhotekin-RBD and PAK-PBD Purity Cat. # AmountPAK-PBD ProteinBinds specifically to active (GTP-bound) Cdc42 and Rac

>80% PAK01-APAK01-B

1 x 250 µg4 x 250 µg

PAK-PBD BeadsBinds specifically to active (GTP-bound) Cdc42 and Rac

>80% PAK02-APAK02-B

1 x 500 µg4 x 500 µg

Rhotekin-RBD ProteinBinds specifically to active (GTP-bound) Rho

>90% RT01-ART01-B

1 x 500 µg3 x 500 µg

Rhotekin-RBD BeadsBinds specifically to active (GTP-bound) Rho

>85% RT02-ART02-B

2 x 2 mg6 x 2 mg

G-protein Modulator Cell Entry Mechanism

Protein Modulation

Cat. # Amount

Rho Activator IIDeamidation of Rho Gln-63

Cell permeable

Direct CN03-ACN03-B

3 x 20 µg9 x 20 µg

Rho Inhibitor IADP ribosylation of Rho Asn-41

Cell permeable

Direct CT04-ACT04-B

1 x 20 µg5 x 20 µg

Rho/Rac/Cdc42 Activator IDeamidation of Rho Gln-63 & Rac/Cdc42 Gln-61

Cell permeable

Direct CN04-ACN04-B

3 x 20 µg9 x 20 µg

Rho Pathway Inhibitor I Rho kinase (ROCK) inhibitor Y-27632

Cell permeable

Direct CN06-ACN06-B

5 x 10 units20 x 10 units

Rho Activator ISHP-2 phosphatase-mediated Rho activation

Cell permeable

Indirect CN01-ACN01-B

5 x 10 units20 x 10 units

Rac/Cdc42 Activator IIEGF receptor-mediated Rac/Cdc42 activation

Receptor mediated

Indirect CN02-ACN02-B

5 x 10 units20 x 10 units

Purified G-proteins Purity Cat. # AmountCdc42 His Protein, constitutively-active (Q61L) >70% C6101-A 1 x 10 µg

Cdc42 GST Protein, dominant-negative (T17N) >90% C17G01-A 1 x 25 µg

Cdc42 GST Protein, wild-type >90% CDG01-A 8 x 25 µg

Cdc42 His Protein, wild-type >90% CD01-A 1 x 100 µg

Rac1 His Protein, constitutively-active (Q61L) >90% R6101-A 1 x 10 µg

Rac1 GST Protein, dominant-negative (T17N) >90% R17G01-A 1 x 25 µg

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

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Actin ProductsRHO FAMILY PRODUCTS

1. International human genome sequencing consortium. 2004. Nature. 431:931-945.

2. Jensen, O.N. 2004. Curr. Opin. Chem. Biol. 8:33-41.

3. Ayoubi, T.A. et al. 1996. FASEB J. 10:453-460.

4. Walsh, C. 2006. Posttranslational modification of proteins: Expanding nature’s inventory. Roberts & Co. Greenwood Village, CO. 490 p.

5. Grabbe, C. et al. 2011. Nat. Rev. Mol. Cell. Biol. 12:295-307.

6. Deshaies, R.J. & Joazeiro, C.A. 2009. Ann. Rev. Biochem. 78: 399-434.

7. Schaefer, A. et al. 2012. Biochem. J. 442:13-25.

8. Varshavsky, A. 2012. Annu. Rev. Biochem. 81:167-176.

9. Yamaguchi, H. et al. 2012. Front. Oncol. 2:15.

10. Lomeli, H. & Vazquez. 2011. Cell Mol. Life Sci. 68:4045-4064.

11. Faesen, A.C. et al. 2012. Biochem. Soc. Trans. 40:539-545.

12. Ridley, A.2012. Meth. Mol. Biol. 827: 3-12.

13. Neel, N.F. et al. 2007. J. Cell Sci. 120:1559-1571.

14. Bryan, B. et al. 2005. FEBS Lett. 597:1015-1019.

15. Wang, H.R. et al. 2003. Science. 302:1775-1779

16. Sahai, E. et al. 2007. J. Cell Biol. 176:35-42.

17. Ozdamar, B. et al. 2005. Science. 307:1603-1609.

18. Castillo-Lluva, S. et al. 2010. Nat. Cell Biol. 12:1078-1085.

19. Boyer, L. et al. 2006. Mol. Biol. Cell. 17:2489-2497.

20. Rolli-Derkinderen, M. et al. 2005. Circ. Res. 96:1152-1160.

21. Chardin, P. 2006. Nat. Rev. Mol. Cell. Biol. 7:54-62.

22. Wilkins, A. et al. 2004. Genes Dev. 18: 856-861.

23. Berthold, J. et al. 2008. Exp. Cell Res. 314:3453-3465.

24. Goh, L.L. & Manser, E. 2012. J. Biol. Chem. 287:31311-31320.

25. Su, L. et al. 2006. J. Immunol. 177:7559-7566.

26. Reiter, L.T. et al. 2006. Hum. Mol. Genet. 15:2825-2835.

27. Yamaguchi, K. et al. 2008. Biol. Chem. 389:405-413.

28. Oberoi, T.K. et al. 2011. EMBO J. 31:14-28.

Target Protein

Modification Cellular Function Reference

RhoA Poly-ubiquitination Regulation of cell protrusions 15

RhoA Poly-ubiquitination Regulation of neurite outgrowth 14

RhoA Poly-ubiquitination Regulation of tight junction dissolution during EMT

17

Rac1 SUMOylation Required for maintenance of GTP bound Rac1 and optimal cell migration

18

Rac1 Poly-ubiquitination Control of cell migration plasticity through Rac1-GTP and Rac1-GDP degradation

28

RhoBTB2 Poly-ubiquitination Required to maintain physiological levels of RhoBTB2

22