PI(3)P and Exocytosis

The classic opinion regarding PI(3)P’s role in intracellular trafficking is that it is synthesized and functions primarily on early endosomes. Several recent publications have highlighted a potential second role for this lipid in exocytosis. Extending previous work by this group and others on the positive role of PI3K-C2a in neurosecretory pathways (1,2) and GLUT4 exoctyosis (3,4) this paper from Tania Maffucci's group interrogates the role of this lipid kinase in insulin secretion in INS1 cells. Combined these results suggest a general role for PI3K-C2a and its product, PI(3)P in exocytotic events.

This group has been investigating the role of the class II PI3K isoforms in exocytosis in a variety of systems. In this paper they investigate the role of PI3K-C2a in insulin secretion using INS1 rat insulinoma cells as a model. Stable knockdown cells did not show any defects in proliferation, calcium signaling, intracellular insulin levels or the expression levels and sub-cellular localization of exocytotic proteins. The PI3K-C2a knockdown cells did however show significant defects in insulin secretion, stimulated by either a secretagogue cocktail or potassium chloride.

Mechanistically, the authors show that there is no defect in insulin granules proximal to the plasma membrane at the resting state. They do detect a decrease in the amount of SNAP25 hydrolysis induced by the secretagogue cocktail. SNAP25 hydrolysis has been proposed to be an important step in the fusion of exocytic vesicles (5). This proteolytic event has not been established as a major mechanism in exocytosis, and it may only correlate with defects in PI3K-C2a signaling. However if reduced SNAP25 proteolysis is the mechanistic defect resulting from PI3K-C2a knockdown, then this suggests a role in protease regulation by PI(3)P or another PI(3)P-derived molecule as a key part of the general exocytotic machinery. It is also possible that there is another, as of yet unstudied role of PI3K-C2a and PI(3)P in exocytosis.



Dominguez, V., Raimondi, C., Somanath, S., Bugliani, M., Loder, M., Edling, C., Divecha, N., da Silva-Xavier, G., Marselli, L., Persaud, S., Turner, M., Rutter, G., Marchetti, P., Falasca, M., & Maffucci, T. (2010). Class II Phosphoinositide 3-Kinase Regulates Exocytosis of Insulin Granules in Pancreatic Cells Journal of Biological Chemistry, 286 (6), 4216-4225 DOI: 10.1074/jbc.M110.200295

References

(1) Meunier, Frederic, Shona Osborne, Gerald Hammond, Frank Cooke, Peter Parker, Jan Domin, and Giampietro Schiavo. “Phosphatidylinositol 3-Kinase C2{alpha} Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis.” Molecular Biology of the Cell 16, no. 10 (2005): 4841-4851. PubMed, DOI.

(2) Wen, Peter J, Shona L Osborne, Isabel C Morrow, Robert G Parton, Jan Domin, and Frederic A Meunier. “Ca2+-regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-kinase C2alpha on neurosecretory vesicles.” Molecular biology of the cell 19, no. 12 (December 2008): 5593-603. PMC, PubMed, DOI.

(3) Maffucci, Tania, Anna Brancaccio, Enza Piccolo, Robert C Stein, and Marco Falasca. “Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation.” The EMBO journal 22, no. 16 (August 15, 2003): 4178-89. PubMed, DOI.

(4) Falasca, Marco, William E Hughes, Veronica Dominguez, Gianluca Sala, Florentia Fostira, Michelle Q Fang, Rosanna Cazzolli, Peter R Shepherd, David E James, and Tania Maffucci. “The role of phosphoinositide 3-kinase C2alpha in insulin signaling.” The Journal of biological chemistry 282, no. 38 (September 21, 2007): 28226-36. PubMed, DOI.

Teaching, whats the point?

First a caveat, I dont teach right now, and havent formally taught in several years, even then it was a GTA sort of experience not a lecture hall. I'm also actively looking or jobs with a teaching component, so its not like I have a totally unbiased perspective here.

However, a recent trip forced me to think (in slightly more coherent terms) about the role of teaching in a scientists life. In my field at least, there seems to be, at times, a selection against competent teachers ever getting in front of undergrads. The people who are excellent communicators, listeners, original thinkers, have the best breadth of knowledge about their topic and are the hardest workers (ie the best scientists) are often directly or indirectly encouraged to do only science. These skills are of course the things that make the best educators.

Now I used to think primarily about a teaching load as what that experience could do for me. I could teach a course relevant to my field of interest and I could harvest the fact-questioning and creativity of the students to further my own research program. That sounds great for me, but maybe not so great for them. There is another perspective though, which I think might be disregarded quite a bit. The public has funded my training as a scientist (through both University funding and more directly through NSERC in Canada and NIH/ADA in the States) and its my responsibility to pay that investment back.

Its great to think that my research someday may cure some devastating disease, or will directly improve the human condition. At the end of a scientific career, even a very good one, how many people can point to a specific direct impact. Most researchers (especially basic scientists) can improve a field, and provide insights that then may go on to help people more directly but most of us are not inclined, or capable to directly impact the public.

How then can we have an impact and pay back the public investment in our careers. It seems to me that a teaching responsibility might be the most efficient way of repaying that investment. An inspiring teacher, who can convey both the benefits of the scientific enterprise and point out how it improves the human condition can have an incredible impact on an undergraduate's future opinions of science. If that student does not end up as a scientist (as most won't) that experience will carry with them to wherever they go and whatever they do. If that student has learned an appreciation for science (or a more specific field in science) then they will carry that to their colleagues and their friends and their families and will spread that appreciation. In the end that kind of viral spreading will help pay back that investment in me much more efficiently than anything I am likely to directly accomplish through research.

IRS Serine Phosphorylation and Insulin Sensitivity?

This is part of my last year in science series. Click here for the rationale.

Insulin signalling passes through a cascade of signaling proteins starting with the recruitment of the Insulin Receptor Substrate (IRS) to the Insulin Receptor.  Insulin resistance, or impaired insulin signaling is a hallmark of obesity and diabetes.  One of the ways in which was thought to happen was through phosphorylation of Serine 307 on IRS, a phosphorylation event which has been proposed to be inhibitory to insulin signaling.  As an example, it has been proposed that inflammation (via JNK or IKKbeta), overnutrition (via S6K) or several other factors can lead to the phosphorylation of this protein.  Since several of these factors correlate with reduced insulin signaling, and the ablation of these factors leads to both increased insulin signaling and reduced serine phosphorylation, the obvious hypothesis was that serine phosphorylation of IRS is causative of insulin signaling.

Correlation does not equal causation, so in order to test this hypothesis in vivo, Morris White's group at Harvard Medical School generated knockin mice, in which Serine 307 of IRS1 is mutated to an alanine (and is therefore unable to be phosphorylated).  The idea would be that these mice would be unable to phosphorylated IRS1 on Serine 307, and therefore would be resistant to the deleterious effects of this phosphorylation.  This serine to alanine knockin model is considered the gold standard for translating an observational protein phosphorylation site into an in vivo phenotype.  Unfortunately for the prevailing hypothesis, the opposite was true.

The paper, from Copps et al. published in January of 2010 show that on a normal diet S307A mice showed modest reductions in insulin sensitivity, and increased fasted insulin levels.  Both of these effects were amplified by high fat diet, and were associated with a reduction in weight gain, in contradiction to the previous hypothesis, that these effects would be ameliorated.  Mechanistically, when on a high fat diet (or coupled with liver specific IRS2 knockout) the S307A mouse had decreased tyrosine phosphorylation of IRS, but no effect of the downstream insulin targets Akt and S6K. Therefore it is unclear exactly how the insulin intolerance is propagated into effects on glucose homeostasis.

In the past year this article has been cited 10 times (according to Google Scholar), mostly in review articles, but the major upshot here, is that the models which showed effects on IRS Serine 307 phosphorylation and insulin resistance, and concluded that insulin resistance was mediated by increased phosphorylation may need re-interpretation.  IRS is phosphorylated on several other sites, so the general hypothesis that serine phosphorylation of IRS causes insulin resistance could still be true, but that again might need to wait until such a knockin model can be generated.  This work also points out the risks of correlating phenotypes with incompletely characterized phosphorylation sites.



Copps KD, Hancer NJ, Opare-Ado L, Qiu W, Walsh C, & White MF (2010). Irs1 serine 307 promotes insulin sensitivity in mice. Cell metabolism, 11 (1), 84-92 PMID: 20074531 DOI