Regulation of ubiquitin-binding proteins by monoubiquitination

Presented by: Wouter Lokers, Cherina Fleming, Myrthe BraamSupervised by: Ger Strous

Daniela Hoeller, Nicola Crosetto, Blagoy Blagoev, Camilla Raiborg, Ritva Tikkanen, Sebastian Wagner, Katarzyna Kowanetz, Rainer Breitling, Matthias Mann, Harald Stenmark & Ivan Dikic

Introduction Sts 1 and 2

Suppressors of T-cell receptor signalling Activate EGF via ubiquitin ligase Cbl Bind to ubiquitinated receptor via UBA

domain

Interfere with EGFR endocytosis and degradation

Both proteins can be monoubiquitinated

Research question

What is the functional role and mechanism of this monoubiquitination?

Is Sts1 and 2 ubiquitination dependant of UBA domains?

Sts1 and Sts2 WT binds to HA-Ub When the UBA is mutated no binding to binding to

ubiquitin takes place

UBA domains are required for monoubiquitination of Sts1 and 2

Which lysine is monoubiquitinated in Sts proteins in vivo?

Localisation of minor (blue) and major (red) ubiquitination sites of Sts2

Mutation of Lys202 to Arg impaired Sts2 monoubiquitination9

Lys202 is the main monoubiquitination site of Sts2 in vivo

Which consequences does mono-ubiquitination has for Sts binding?

Monoubiquitinated Sts is located in a different band than wt Sts

Monoubiquitinated Sts does not interact with exogenous ubiquitin

Monoubiquitination of Sts proteins abolishes binding to exogenous ubiquitin

Which consequences does mono-ubiquitination has for Sts binding?

Several permanently mono-ubiquitinated proteins are created

All ubiquitin chimerae are impaired in inter-action with exogenous ubiquitin

Monoubiquitination of UIM- and UBA-containing proteins neutralizes their ubiquitin-binding capacities

Do monoubiquitin and UBD have an intermoleculair interaction?

Dimerization-deficient Sts1-ΔPGM and its ubiquitin-chimera are subjected to chemical cross-linking

No detectable dimerization of Sts1-ΔPGM-ubiquitinNo biochemical evidence for intermolecular interactions

between UBA of monomeric Sts1-ΔPGM and ubiquitin of another Sts1-ΔPGM molecule

Do monoubiquitin and UBD have an intramoleculair interaction?

FRET-technology: FRET donor CFP attached to C-terminus and acceptor citrine to amino terminus of Sts2-ΔPGM

Intramolecular interactions between monoubiquitin and UBA of Sts1-ΔPGM occur and are sufficient to block ubiquitin-binding ability of Sts1 and 2

What is the functional importance of monoubiquitination of Sts?

Monoubiquitination of Sts2 inhibits its capacity to block ligand-induced degradation of EGFRs

Expression of Sts2 caused stabilization/accumulation of EGFR-GFP

Overexpression of Sts2-ubi-quitin caused significantly decreased EGFR levels

Results (monoubiquitination site)

UBA domains are required for monoubiquitination of Sts1 and 2

Lys202 is the main monoubiquitination site of Sts2 in vivo

Results (monoubiquitin binding)

Monoubiquitination of Sts proteins abolishes binding to exogenous ubiquitin

Monoubiquitination of UIM- and UBA-containing proteins neutralizes their ubiquitin-binding capacities

Results (monoubiquitin

interactions)

No intermolecular interactions between UBA of monomeric Sts1-ΔPGM and ubiquitin of another Sts1-ΔPGM molecule

Intramolecular interactions between monoubiquitin and UBA of Sts1-ΔPGM occur and are sufficient to block ubiquitin-binding ability of Sts1 and 2.

Results(monoubiquitin function)

Monoubiquitination of Sts2 inhibits its capacity to block ligand-induced degradation of EGFRs

Monoubiquitination of UBD-containing proteins triggers intramolecular interactions with the UBDs, thereby preventing them from binding to ubiquitinated targets

Proposed model