Table of Contents
2. Fences keep neighborhoods friendly
3. How these pathways collide: AMPK is the hammer and GIV is the nail
4. Why stability matters
5. The anti-cancer mechanism of Metformin
6. The dark side: GIV mutations and tumor development
For a few lucky gold miners, one tiny nugget of gold glinting on the ground reveals an entire gold mine. The same can be true in science. The most unlikely of findings can propel fields to entirely unexpected directions. A recent example is highlighted by work from a group of cell biologists at UC-San Diego. In the on-line journal eLife, these researchers connect a well-characterized metabolic pathway, LKB1-AMPK, to a previously unknown effector protein, GIV and in doing so uncover very significant discoveries about cancer biology. First they reveal a long sought after anti-cancer mechanism for the widely used diabetes drug, Metformin and next they link tumor development and metastases to possible mutations in this novel pathway.
Fences keep neighborhoods friendly
We build fences to keep things out and our bodies have a similar approach. Specialized epithelial cells form tight and rigid barriers to protect areas such as the gut, lungs, brain and oral cavities. Their structure is imposed by rigid tight junction (TJ) complexes, which connect neighboring cells together. This organization also imposes restricted movement and restricted cell proliferation. As single cell layers, each surface is exposed to a different environment, creating an asymmetrical polarized cell. The apical end is outward facing and the basal surface attaches to the tissue below. Apical and basal surfaces perform critical and distinct cellular functions and when this polarity is lost, the barrier falls apart.
How these pathways collide: AMPK is the hammer and GIV is the nail
Our bodies run on energy derived from ATP. Degradation of ATP creates the intracellular byproduct AMP. The ratios of ATP to AMP relate to the energy state of individual cells in time. AMPK, AMP-activated protein kinase, is the critical sensor of this ratio and is phosphorylated by LKB1 during states of energetic stress when AMP concentration rises.
In polarized epithelial cells, maintenance of the structural TJ requires significant energy costs, making regulation of the LKB1-AMPK pathway critical for homeostasis. Either functional loss or expression reductions in AMPK are associated with a breakdown in epithelial linings by a weakening of the TJ complex. Although this relationship has been well described, the precise molecular mechanism for AMPK protection of TJ complexes was still mysterious.
Sitting at the intersection between intracellular AMPK and the membrane localized TJ, is the multimodular scaffolding protein GIV/Girdin. Originally discovered in 2005, GIV (G-alpha interacting vesicle associated protein) is a large and complex enigmatic protein. Other multifunctional proteins are known, but GIV takes multi-tasking to the extreme. The C-terminus has both GEF (guanine nucleotide exchange factor) and GDI (guanine nucleotide dissociation inhibitor) capabilities to regulate G-protein signaling. While the N-terminus has some TJ protein binding domains, no one had put AMPK and GIV together in a single pathway until now.
Using bioinformatics to determine putative phosphorylation sites within the N-terminus, these scientists, lead by senior author Dr. Pradipta Ghosh, MD, found one specific serine (S245) was directly phosphorylated by activated AMPK in response to energetic stress in cultured epithelial cells. Phosphorylation of S245 on GIV was sufficient to stabilize TJ complexes and maintain barrier functions, a key missing link in the known mechanism of AMPK-mediated protection.
Why stability matters
With a mechanism for TJ stability in hand, Ghosh et al, tackled a long standing mystifying observation of clinicians: long-term treatment with the AMPK activator Metformin (Glucophage) has beneficial anti-cancer side effects. Using Metformin as a direct activator for AMPK or Compound C, a known inhibitor of AMPK activation as a negative control, they were able to demonstrate that specific phosphorylation of GIV S245 increases stability of TJs even in glucose starvation conditions. Stability equates to homeostasis. Initiation of oncogenic progression is preceded by a loss of cellular polarity and Metformin apparently staves off this cellular change by activating the AMPK-GIV pathway.
However, while this tumor suppression finding is optimistic and encouraging, there is a dark side as well. While screening several cancer databases for GIV mutations, areas within and surrounding the AMPK-binding domain frequently popped up with noteworthy changes. One particular mutation, L249P, four residues from S245 and within the consensus-binding site for AMPK, abolishes the phosphorylation of GIV by allosteric hindrance. Cell expressing this mutant GIV will fail to stabilize TJs under energetic stress, will lose polarity and can initiate early oncogenic changes. Even treatment with Metformin will not prevent this progression, providing yet another example of the value of personalized medicine in the field of cancer treatment.
Aznar, N., Patel, A., Rohena, C.C., Ying, D., Joosen, J.P., Taupin, V., Kufareva, I., Farquahar, M.G., Ghosh, P. (2016) AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin. DOI: http://dx.doi.org/10.7554/eLife.20795 eLife 2016;5:e20795