Two Proteins Containing Tandem DIII Domains, Calpain 10 and Dictyostelium Cpl, are Involved in Cytoskeletal Regulation

by Czerwinski, Eric Paul

Polymorphism in the CAPN10 gene has been associated with metabolic changes related to type 2 diabetes through genetic studies in several populations. Reduced expression of calpain 10 apparently leads to insulin resistance and diabetes. Our initial studies were performed on the myxomycete, Dictyostelium discoideum, a model organism with properties useful for both genetic manipulation and biochemical preparations. It expresses a calpain 10-like protein, Cpl, which has tandem DIII domains, giving it a similar domain organization to calpain 10. To discover the physiologic function of Cpl, a cplA gene disruption strain, RM1, was created. This strain exhibits increased cell-surface attachment and decreased proliferation when grown on a surface rather than in shaking culture. Disruption of Cpl also caused effects on cell motility, endocytosis, chemotaxis, and an increase in cortical actin. The results demonstrate an effect of Cpl ablation on cellular processes governed by cytoskeleton-membrane interactions. The possibility of an analogous function of mammalian calpain 10 led us to investigate cytoskeletal or membrane-associated binding partners of calpain 10. Our laboratory identified the lipid raft protein flotillin-1 as having a strong interaction with the DIII domains of calpain 10 in yeast 2-hybrid assays. Using bacterially expressed constructs we show that the DIII domains are required for this interaction. Additionally, flotillin-1 is a substrate of endogenous and recombinant calpain 10 in the presence of Ca2+ and PIP3. SNAP-23, a SNARE protein required for GLUT4 vesicle fusion was also a substrate of calpain 10 under these conditions.