PROJECT SUMMARY Adipose tissue is critical for whole body energy homeostasis. Both obesity and lipodystrophy (caused by loss of fat) are associated with diabetes and insulin resistance. Our recent work identified a new mutant mouse, termed teeny, that exhibited growth retardation, lipodystrophy, extreme insulin resistance, severe diabetes with fasting glucose as high as 600-700 mg/dL, and fatty liver. The teeny phenotype is caused by a null mutation in the Kelch repeat and BTB (POZ) Domain containing 2 (KBTBD2), which has no previously assigned functions. We found that KBTBD2 operates as an E3 ubiquitin ligase to regulate the insulin signaling pathway by targeting the degradation of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85?. This proposal will further explore the role of KBTBD2 in lipodystrophy, insulin resistance, and diabetes, and will seek to identify mechanistically related regulators of diabetes and obesity. In the mentored K99 phase, mice will be generated to manipulate Kbtbd2 expression in different tissues to study its tissue-specific functions, and to dissect the role of each tissue in the development of teeny phenotype (Aim 1). I found that KBTBD2 harbors a YXXM motif, which may be phosphorylated in order to recruit p85?. The phosphorylation of KBTBD2 could explain why p85? is selectively degraded in different cells and tissues. This, in turn, would suggest that KBTDB2 itself might be subject to post-translational regulation depending upon tissue, developmental stage, and/or metabolic status. The focus of Aim 2 will be to study the post-translational modification of KBTBD2. In the independent R00 phase, I will further explore the biological consequences of the observed p85? mono-ubiquitination (Aim 3). The transcriptional suppression of Kbtbd2 in diet-induced obesity, leading to the accumulation of p85?, prompts us to search for deubiquitinases, which might be targeted by inhibitors to reduce the risk of diabetes in obesity. In preliminary studies, several candidate p85? deubiquitinases have been identified. To identify new genes that are involved in obesity and diabetes, we have established several forward genetic screens in mice and found interesting hits. Among these, a second BTB protein (RHOBTB2) may act within the overall framework we have built. In Aim 4, I will study the mechanism of deubiquitinases and other newly identified regulators in obesity and diabetes. Successful completion of the aims outlined in this proposal will improve our understanding of KBTBD2 and may provide new targets for diabetes treatment. To accomplish the proposed research, I will include Dr. Philipp Scherer (UT Southwestern Medical Center) as my co-mentor during the mentored K99 phase to gain more training in adipocyte and diabetes research. My mentor, Dr. Bruce Beutler, has established a unique forward genetic initiative with the ability to identify causative mutations in real time. I will continue working on newly identified regulators of obesity and diabetes from the established forward genetic screens in the independent R00 phase. This training will allow me to expand my expertise and help my transition into a successful independent academic researcher.