Regulation of protein function by monoubiquitination

Research project

The monoubiquitin (monoUb)-network is emerging as a major regulator of protein function beyond its well-established role in protein degradation. While initial observations, in various labs, project a physiological relevance of monoubiquitination comparable to that of phosphorylation, the characterization of the system has only just begun. We are trying to integrate a wide proteomic approach to monoUb as a signaling device with high-resolution studies of how monoUb can modulate the function of endocytic/signaling proteins. Presently three major levels of study are being pursued in the lab:

Fig1: The ubiquination process [+zoom]

Elucidating the monoUb proteome.
We plan to identify the intracellular repertoire of proteins that are modified by monoubiquitination following growth factor treatment (EGF), by a mass spectrometry approach. Depending on the rate of success of the initial trial, the procedure will be applied to a systematic dissection of the monoUb proteome under conditions of activation of different receptor tyrosine kinases (RTKs), or of activation of other signaling pathways (DNA damage, oxidative stress). A parallel effort using Saccaromyces cerevisiae as model system will be pursued.

Dissecting the role of monoubiquitination in endocytosis
Activated RTKs are able to induce monoubiquitination of various endocytic proteins, including eps15, eps15R, epsins, Hrs. We plan to investigate how the signal is delivered from activated RTKs to the ubiquitination machinery and what role monoubiquitination may play in endocytic proteins. We are using eps15 as a model system and subsequently we will extend our findings to other monoubiquitinated endocytic proteins.

Fig2: Ubiquitin in endocytosis [+zoom]

Elucidating the molecular mechanisms of coupled monoubiquitination
Many proteins, referred to as ubiquitin (Ub) receptors, harbor short motifs/domains that interact with Ub (Ub-binding domains, UBD). At least nine families of UBDs have been identified; these domains confer the Ub-binding activity on Ub receptors which are consequently able to bind to proteins that have been modified by a single Ub (monoubiquitination) or by Ub chains (polyubiquitination). Ub receptors are themselves frequently modified by monoubiquitination, by a process that requires the presence of a functional UBD, and is referred to as coupled monoubiquitination. Three classes of UBDs, the UIM (Ubiquitin Interacting Motif), the CUE, and the GAT, can sustain coupled monoubiquitination through molecular mechanisms that are still obscure. We are addressing this question by a paired approach of mutagenesis and in vitro enzymatic assays.