Molecular basis of class II PI3-kinase function

Lipids are important components of biological membranes that surround the cell and organelles inside the cell. Special lipids such as Phosphatidylinositol (PI) are not only responsible to be a building block of the membrane but also act as signaling molecules and denominators of membrane identity to control cell growth, survival, differentiation, migration and intracellular membrane dynamics among other functions. Depending on the modification state, like phosphorylation, they are changing its signaling potential and can only be found at certain locations inside the cell or membranes. Among the seven different phosphorylated Phosphatidylinositol (PIP) species found in mammals phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] arguably is the least understood, synthesized by class II PI 3-kinases (PI3KC2). Recent landmark studies from the host laboratory revealed that these kinases are regulating uptake of molecules into the cell (endocytosis) and are further reflecting the cellular nutrient status. Dysregulation of PI3KC2a-c is associated with diabetes and cancer but the mechanism that regulate PI3KC2a-c activity remain poorly understood. In my proposed research I will fill this important knowledge gap by capitalizing on the outstanding opportunities provided by the host lab to dissect the molecular basis of class II PI3K function. Specifically, I will build on reagents and experimental procedures recently established by the host lab to produce high amounts of pure and active PI3KC2 suitable for biochemical characterization and X-ray crystallography approaches. The availability of high amounts of soluble active PI3KC2 opens the door for the further biochemical, structural biological and functional characterization of this important class of enzymes. With the financial support of the Schrödinger-Stipend I aim to determine the structure and mechanism(s) of activation of PI3KC2a and PI3KC2b by using a variety of different molecular biological approaches, all of which are well established in host lab. Furthermore, over the last 8 years I obtained ample experience and training in protein crystallography that will help me to produce high quality protein crystals suitable to solve the structure of PI3KC2a- and b using X-ray diffraction experiments. Such 3D-structures will shed light on our understanding of PI3KC2a/b regulation and function in endocytosis and nutrient signaling and thereby pave the way for the pharmacological manipulation of these enzymes to establish novel avenues for the treatment of myotubular myopathy and, possibly, diabetes and cancer.

Lipids are important components of biological membranes, crucial to separate the cell and its intracellular organelles from the exterior. Phosphatidylinositol (PI) and its phosphorylated derivatives (PIPs) are special lipids, which act as signaling molecules and denominators of membrane identity to control cell growth, survival, differentiation, migration and intracellular membrane dynamics among other functions. Each of the seven PIP species found in mammals have specialized functions within the cell, whereas phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] is the least understood. Recent landmark studies from the host laboratory have identified the class II PI 3-kinases PI3KC2a and b as major PI(3,4)P2-synthesizing enzymes at the plasma membrane and within the endolysosomal system to spatiotemporally regulate endocytosis and nutrient signaling. In spite of their important cell physiological functions and the fact that dysregulation of PI3KC2a-c is associated with diabetes and cancer, the mechanism that regulate PI3KC2a-c activity remain poorly understood. In my proposed research I aimed to fill this important knowledge gap by capitalizing on the outstanding opportunities provided by the host lab to dissect the molecular basis of class II PI3K function. In order to understand the activation- and regulatory-mechanism outside of the cell (in vitro), I performed biochemical characterization approaches using recombinantly expressed protein. To do so, I initially established a proper expression and purification protocol, suitable to obtain high amounts of soluble active PI3KC2. With the use of a variety of different Protein constructs, I was able to identify the domains and its related amino acids which are essential to bind specific PIPs. Furthermore, I could identify PIPs, which are able to increase the kinase activity but, in contrast, not producing any phosphorylated products. Finally, within the host lab I designed constructs, suitable for structural characterization such as crystallography and cryo-EM. I was able to obtain initial crystals and established a protocol to record first negative stain images of the PI3KC2b. Additionally, such constructs were implemented in the screening experiments to find specific PI3KC2-inhibitors. With the financial support of the Schrödinger-Stipend I paved the way to determine the structure and mechanism(s) of activation of PI3KC2a and PI3KC2b in the near future by using a variety of different molecular biological approaches. Furthermore, within the host lab I could further strengthen my experience and training in structural biology, protein- and lipid-biochemistry and cell biology. Taken together, the unique opportunity of a research-stay at the FMP-Berlin, with the financial support of the FWF Erwin Schrödinger Auslandstipendium, provided me the possibility to establish my own independent research group in Austria and to finally hold a professor position in the near future.