The Kyriakis laboratory studies the mechanisms by which inflammatory and proliferative stimuli alter cell function. We are interested in how polypeptide mitogens, proinflammatory cytokines and microbial pathogens trigger activation of protein kinases and, in turn, how these protein kinases influence gene expression in chronic and acute inflammation and in the stimulation of cell proliferation. Our work is focused especially on the mitogen-activated protein kinases (MAPKs). We believe that elucidating these pathways, and their biological functions will enable the identification of novel drug targets and the development of improved treatments for diseases such as atherosclerosis, cancer, and the complications of diabetes.
Research Focus and Highlights
The mitogen-activated protein kinases (MAPKs) are a large evolutionarily conserved family of protein kinases present in all eukaryotic cells. All eukaryotic cells possess multiple MAPK pathways that enable integrated responses to divergent stimuli. MAPK pathways all consist of a central three tiered core wherein MAPKs are activated by MAPK kinases (MKKs) which, in turn, are activated by MKK kinases (MAP3Ks). Understanding the regulation of MAP3Ks is key to dissecting the mechanisms by which specific extracellular stimuli recruit distinct MAPK core pathways to affect cell function. Understanding MAP3K regulation will enable the development of specific treatment modalities that target specifically signaling pathways that are involved in disease pathology while leaving beneficial signaling intact.
In 1991, we discovered a subfamily of the MAPKs, the c-Jun N-terminal kinases, JNKs (also called stress-activated protein kinases, SAPKs). The JNKs participate in protein phosphorylation/signal transduction cascades that are homologous to, but largely distinct from the more familiar Ras-extracellular signal-regulated kinase (ERK) pathway. The JNKs and a related MAPK pathway, the p38 pathway, are activated in response to inflammatory cytokines (TNF, IL-1), pathogen-associated molecular patterns (PAMPs, conserved molecular moieties such as bacterial lipopolysaccharide [LPS] and peptidoglycans produced by microbial pathogens), danger-associated molecular patterns (DAMPs, conserved molecular species such as oxidized LDL [oxLDL] produced endogenously by cells in response to stress), environmental stresses, (heat shock, UV light g radiation), chemical stress (protein synthesis inhibition), and ischemic reperfusion injury. The JNKs and p38s can also be activated by polypeptide mitogens. Once activated, the JNKs can, among other things, phosphorylate and activate the trans activation function of c-Jun and activating transcription factor-2 (ATF2), components of the activator protein-1 (AP-1) heterodimeric transctription factor.
1) Dissection of a mechanism by which PAMPs/DAMPs recruit JNK and p38 to contribute to the inflammatory response. Germinal center kinase (GCK) and the regulation of JNK and p38 by PAMPs/DAMPs.
GCK is the founding member of a protein kinase group distantly related to S. cerevisiae Ste20. We have made a number of observations concerning GCK as a regulator of JNK and p38.
- Transient expression of GCK potently activates coexpressed JNK.
- GCK is activated selectively by PAMPs and DAMPs via a novel mechanism. Extracellular stimuli do not increase GCK's intrinsic kinase activity per se. Instead, GCK is constitutively ubiquitinated and degraded by the proteasome. Activating stimuli lead to sequestration of GCK from the proteasome. Accordingly, in response to PAMP signals, GCK accumulates in the cell to a level at which it can initiate signaling.
- GCK stabilization requires the signaling adapter protein TNF receptor-associated factor (TRAF)-6 to which GCK binds in a PAMP-stimulated manner.
- Activated/stabilized GCK can, in turn, bind and directly activate the JNK and p38-specific MAP3Ks mixed lineage kinases (MLKs)-2 and –3.
- MLKs-2 and –3 are required for coupling GCK to JNK and p38.
We have since generated gck-/- mice. Disruption of gck substantially impairs MLK2/3, JNK and p38 activation by a subset of clinically relevant PAMPs (LPS, peptidoglycan, for example) and DAMPs (oxLDL). Consistent with this, GCK is required for PAMP induction of macrophage cytokines. Most importantly, hematopoietic cell GCK is required for the development of lethal systemic inflammation upon in vivo administration of LPS.
Inasmuch as GCK is activated by oxLDL, we are currently studying the potential role of GCK in atherogenesis. In addition, given the emerging role of inflammation in the pathogenesis of obesity-induced insulin resistance and type-2 diabetes, we are studying the role of GCK in metabolic disease.
2) The role of mammalian sterile 20-like kinase-2 (Mst2) in regulation of the Ras-ERK pathway.
Mst2 is one of two conserved mammalian orthologues of the Drosophila Ste20-like kinase Hippo. The mammalian Hippo pathway, like its Drosophila cousin, is a tumor suppressor mechanism. In this capacity, Mst2 (and Mst1) phosphorylate and activate the tumor suppressor kinases large tumor suppressor (Lats)-1 and -2 (orthologues of Drosophila Warts). Lats1/2 phosphorylate and modulate the function of yes-associated protein (Yap, an orthologue of Drosophila Yorkie), an oncogenic transcriptional regulator.
Despite the known role of Mst2 as a tumor suppressor, we find that RNAi silencing of Mst2 impairs cell proliferation via suppression of mitogen activation of ERK. This suppression involves inhibition of the ERK-specific MAP3K Raf-1.
Our ongoing studies suggest that Mst2 functions to reduce the inhibitory phosphorylation of Raf-1 at Ser259, as well as the activating phosphorylation of the pro-oncogenic kinase Akt. This process is mediated by Mst2-dependent stabilization of the catalytic subunit of protein phosphatase-2A (PP2A-c), which functions to dephosphorylate Raf-1 Ser259 and to dephosphorylate and deactivate Akt. Mst2-dependent stabilization of PP2A-c requires Lats1/2 and involves attenuation of the ubiquitin-dependent proteolysis of the PP2A-c polypeptide.
3) The small basic helix-loop-helix (bHLH) protein p8, a transcriptional modulator with divergent effects on cell function.
In 2002, we identified p8 in a screen for genes induced in diabetic renal disease and required for renal mesangial cell hypertrophy. Since that time, we have shown that p8 functions as a transcriptional modulator that cooperates with transcription factors to affect gene expression.
In this capacity, p8 is required for endothelin-stimulated cardiomyocyte hypertrophy, and, in line with this, binds to the ANF promoter where it is required for ANF expression. Likewise, TNF induction of cardiac fibroblast matrix metalloprotease-9 (MMP9) requires p8. p8 associates with the MMP9 promoter in vicinity of two critical AP-1 sites. We are currently using p8-/- mice to explore the role of p8 in several cardiovascular pathologies.
Our recent cellular studies indicate that p8 functions to suppress autophagy by down regulating the expression of key pro-autophagic genes. Autophagy is a cellular process of self-digestion that, in times of nutrient paucity, enables the production of metabolic raw materials needed to maintain metabolic homeostasis.
Research Administrator: Dionne Bradford