Our research is directed at furthering the understanding of protein kinase regulation with a particular focus towards the underlying structural mechanism that imparts specificity to signaling function. We seek to uncover the structural basis for novel catalytic switching, substrate recognition and down-stream phosphoregulatory mechanisms. In addition, we are interested in uncovering the structure and function of new proteins and activities that impact protein kinase function. These efforts have recently led our lab into the area of ubiquitin directed proteolysis. Our long-term goal is to make use of what we learn about protein kinases and phosphoregulatory systems to develop drugs to treat human disease.

Below is a sampling of active projects in our laboratory:

RAF signaling

A dimerization-dependent mechanism drives RAF catalytic activation.
Rajakulendran T, Sahmi M, Lefrançois M, Sicheri F, Therrien M.
Nature. 2009 Sep 24;461(7263):542-5. Epub 2009 Sep 2.

The RAF family kinases are amongst the most prolific human oncogenes. Understanding the mechanisms that underlie RAF family function would facilitate approaches to RAF inhibition that would afford an effective tool against many cancers. In collaboration with Dr. Marc Therrien at the IRIC (Institute for Research in Immunology and Cancer, University of Montreal), we undertook a multifaceted approach utilizing structural biology, biochemistry and cell-based studies to elucidate the mechanism by which RAF kinases are activated. We discovered that the activation of the catalytic (kinase) domain of RAF kinases is controlled principally by an allosteric interaction between two kinase domains in a specific side-to-side dimer configuration. The acquisition of the side-to-side dimer configuration is essential for aberrant RAF signaling in cancers, suggesting future RAF inhibition strategies could be aimed at preventing dimer formation. RAF inhibition strategies aimed at disrupting the dimer would represent a major advance over current generation of RAF inhibitors – one that it would eliminate the unwanted off-target effects of ATP-competitive inhibitors, as well as circumventing the paradoxical activation of RAF that results upon treatment with certain ATP-competitive inhibitors.

The UPR sensor IRE1

Structure of the dual enzyme Ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing.
Lee KP, Dey M, Neculai D, Cao C, Dever TE, Sicheri F.
Cell. 2008 Jan 11;132(1):89-100

Certain physiological conditions, such as high secretory load, lead to the build-up of harmful misfolded protein clusters in an intracellular compartment called the endoplasmic reticulum (ER); cells in this state are said to be experiencing ER stress. Cells have evolved an adaptive mechanism termed the Unfolded Protein Response (UPR) to detect and resolve ER stress. IRE1 functions as a key sensor and signal transducer of ER stress in all nucleated organisms.

In response to ER stress, IRE1 activates a signaling protein called XBP1 that upregulates UPR genes to combat ER stress. IRE1 was initially recognized as a receptor protein kinase, which are enzymes that attach phosphate groups onto proteins such as signaling proteins to augment their function. IRE1 does not put phosphates on XBP1. Instead, to activate XBP1, IRE1 excises an internal segment (an intron) from messenger RNA (mRNA) coding for XBP1. How IRE1, previously known only as a receptor kinase, eliminates the ER stress responsive intron from XBP1 mRNA has been a mystery since this phenomenon was described more than a decade ago.

To investigate the mechanism of IRE1 function, we determined the X-ray crystal structure of the cytosolic effector region of IRE1 protein from Baker's yeast. By studying this structure we gained new insight into the inner-workings of IRE1, which were subsequently confirmed using biochemical, biophysical and genetic approaches. We discovered that when IRE1 is activated by misfolded proteins, the effector regions of two IRE1 molecules attach together in a precise back-to-back arrangement. This arrangement composes a catalytic surface spanning the paired IRE1 molecules, which allows IRE1 to make incisions in XBP1 mRNA. We also discovered how the association of IRE1 effector regions is controlled by binding of a stimulatory molecule called ADP, which in turn is regulated by the ability of IRE1 to attach phosphates onto itself.

Our findings provide a deeper understanding of how IRE1 communicates ER stress signals across the ER membrane. Our discoveries also illuminate potential avenues to modulate IRE1 function pharmacologically; interest in this area is gaining momentum given the emerging connection between IRE1 and numerous human diseases including cancer and diabetes.

Mechanism of action of the culin nedylation factor Dcn1

Dcn1 functions as a scaffold-type E3 ligase for cullin neddylation.
Kurz T, Chou YC, Willems AR, Meyer-Schaller N, Hecht ML, Tyers M, Peter M, Sicheri F.
Mol Cell. 2008 Jan 18;29(1):23-35

Ubiquitination plays a central regulatory role in eukaryotes, controlling a broad range of biological processes, including cell cycle progression, protein trafficking, and signal transduction. Cullin-based E3 ubiquitin ligases are activated through covalent modification of the cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 is an essential regulator of cullin neddylation and thus ubiquitin ligase activity. We have solved the 1.9 Å X-ray crystal structure of Dcn1 and demonstrated that Dcn1 is required for both direct binding to cullins and for cullin neddylation in vitro and in vivo.

Role of KEOPS in telomere maintenance

Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine.
Mao DY, Neculai D, Downey M, Orlicky S, Haffani YZ, Ceccarelli DF, Ho JS, Szilard RK, Zhang W, Ho CS, Wan L, Fares C, Rumpel S, Kurinov I, Arrowsmith CH, Durocher D, Sicheri F.
Mol Cell. 2008 Oct 24;32(2):259-75

The KEOPS complex is comprised of four evolutionarily conserved proteins (ie. Bud32, Cgi121, Kae1, Pcc1) that help to maintain the length of telomeric ends in eukaryotes.  Mutation of KEOPS in yeast leads to striking telomere defects via an unknown mechanism.  We set out to solve the crystal structure of KEOPS to better understand its functional and biochemical roles in telomere length maintenance.  To this end, we solved the atomic structure of an Archaeal-derived KEOPS complexes involving Kae1, Bud32, Pcc1, and Cgi121 subunits.  Our study sheds light on the functional interdependency of the KEOPS complex and its four subunits and provides the essential framework upon which future genetic and biochemical studies will stand.

The role of multimierization in SCF ligase function:

Suprafacial orientation of the SCFCdc4 dimer accommodates multiple geometries for substrate ubiquitination.
Tang X, Orlicky S, Lin Z, Willems A, Neculai D, Ceccarelli D, Mercurio F, Shilton BH, Sicheri F, Tyers M. Cell. 2007 Jun 15;129(6):1165-76

SCF ubiquitin ligases recruit substrates for degradation via F-box protein adaptor subunits. WD40 repeat F-box proteins, such as Cdc4 and b-TrCP, contain a conserved dimerization motif called the D-domain. We have discovered that the D-domain of yeast Cdc4 and human b-TrCP form a super-helical homotypic dimer. Disruption of the D-domain compromises the activity of yeast SCFCdc4 towards the CDK inhibitor Sic1 and other substrates. SCF-Cdc4 dimerization has little effect on the affinity for Sic1 but markedly stimulates ubiquitin conjugation. A model of the dimeric holo-SCF-Cdc4 complex based on small angle X-ray scatter measurements reveals a suprafacial configuration, in which substrate binding sites and E2 catalytic sites lie in the same plane with a separation of 64 Å within and 102 Å between each SCF monomer. This spatial variability may accommodate diverse acceptor lysine geometries in both substrates and the elongating ubiquitin chain, and thereby increase catalytic efficiency.