Tài liệu Báo cáo khoa học: Activated Rac1, but not the tumorigenic variant Rac1b, is ubiquitinated

Activated Rac1, but not the tumorigenic variant Rac1b, is

ubiquitinated on Lys 147 through a JNK-regulated process

Orane Visvikis1,2, Patrick Lore` s1,2, Laurent Boyer3

, Pierre Chardin4

, Emmanuel Lemichez3

and Ge´rard Gacon1,2

1 Institut Cochin, Universite´ Paris Descartes, CNRS (UMR8104), Paris, France

2 INSERM, U567, Paris, France

3 Faculte´ de Me´decine, INSERM U627, Nice, France

4 Institut de Pharmacologie du CNRS, Sophia Antipolis, France

In all eukaryotic cells, Rho GTPases (Rho, Rac,

Cdc42) control basic cellular functions, including actin

cytoskeleton organization, vesicular trafficking, tran￾scriptional regulation and cell cycle progression, and

are therefore major intracellular regulators of cell

growth and division, and cell migration [1,2]. Consis￾tent with their key role in cell signaling, dysregulation

of Rho-dependent pathways is being found to be caus￾ally involved in the pathophysiology of a growing

number of human diseases [3]. Hence, mutations in

genes encoding Rho regulators are responsible for

human developmental defects and, particularly, several

Keywords

JNK; Rac1b; Rho GTPases; ubiquitination

Correspondence

G. Gacon, Institut Cochin, De´partement

Ge´ne´tique et De´ veloppement, 24 rue du

Faubourg Saint-Jacques, 75014 Paris,

France

Fax: +33 1 44 41 24 41

Tel: +33 1 44 41 24 70

E-mail: [email protected]

(Received 29 August 2007, revised 18

November 2007, accepted 27 November

2007)

doi:10.1111/j.1742-4658.2007.06209.x

Ubiquitination and proteasomal degradation have recently emerged as an

additional level of regulation of activated forms of Rho GTPases. To char￾acterize this novel regulatory pathway and to gain insight into its biological

significance, we studied the ubiquitination of two constitutively activated

forms of Rac1, i.e. the mutationally activated Rac1L61, and the tumori￾genic splice variant Rac1b, which is defective for several downstream

signaling pathways, including JNK activation. Whereas Rac1L61 under￾goes polyubiquitination and subsequent proteasomal degradation in

HEK293 cells, Rac1b is poorly ubiquitinated and appears to be much more

resistant to proteasomal degradation than Rac1L61. Mutational analysis of

all lysine residues in Rac1 revealed that the major target site for Rac1

ubiquitination is Lys147, a solvent-accessible residue that has a similar con￾formation in Rac1b. Like Rac1L61, Rac1b was found to be largely associ￾ated with plasma membrane, a known prerequisite for Rac1 ubiquitination.

Interestingly, Rac1b ubiquitination could be stimulated by coexpression of

Rac1L61, suggesting positive regulation of Rac1 ubiquitination by Rac1

downstream signaling. Indeed, ubiquitination of Rac1L61 is critically

dependent on JNK activation. In conclusion: (a) Rac1b appears to be more

stable than Rac1L61 with regard to the ubiquitin–proteasome system, and

this may be of importance for the expression and tumorigenic capacity of

Rac1b; and (b) ubiquitination of activated Rac1 occurs through a JNK￾activated process, which may explain the defective ubiquitination of Rac1b.

The JNK-dependent activation of Rac1 ubiquitination would create a regu￾latory loop allowing the cell to counteract excessive activation of Rac1

GTPase.

Abbreviations

CNF1, cytotoxic necrotizing factor 1; EMT, epithelial–mesenchymal transition; GST, glutathione S-transferase; HA, hemagglutinin;

UbR48, ubiquitin mutant lacking Lys48; UbR63, ubiquitin mutant lacking Lys63; UPS, ubiquitin–proteasome system.

386 FEBS Journal 275 (2008) 386–396 ª 2007 The Authors Journal compilation ª 2007 FEBS