The objective of this grant application is to test a novel hypothesis that AMPK inhibits angiogenesis by phosphorylation and inhibition of ALK1. Recent findings that treatment of type 2 diabetes with metformin, a well-known AMPK activator, reduces cancer incidence have prompted its use in clinical trials for cancer therapy. Thus far, more than 150 clinical trials (www.clinicaltrial.gov) are in progress with metformin for the treatment of different cancers. Although targeting AMPK for the therapy of metabolic syndrome and cancer is gaining more attention, its effects on angiogenesis, a crucial step for malignant cells to populate to a sizable tumor and a culprit for diabetic complications, are not clear. This grant proposal is aimed at advancing such understanding. Compelling evidence has demonstrated that activin receptor-like kinase 1 (ALK1) plays a key role in the development of vascular system. Additionally, recent studies have suggested that ALK1 is involved in pathogenic angiogenesis of several diseases (e.g. cancer and diabetic retinopathy), which provides an appealing rationale that ALK1 could be a valuable target in anti-angiogenesis therapy. Interestingly, the functional interaction between AMPK and ALK1 signaling has never been documented. In our preliminary experiments with human umbilical vein endothelial cells, we found that AMPK activation by metformin potently suppresses BMP2-induced phosphorylation of Smad1/5 and endothelial cell tube formation. Furthermore, sequence alignment points to a potential AMPK phosphorylation site (S269) in the L45 loop of the catalytic domain of ALK1, which is essential for Smad binding. Thus, we speculate that the phosphorylation of this site by AMPK may lead to disruption of ALK1 interaction with Smad1/5, and subsequent inhibition of angiogenesis. It is possible that dynamics of the phosphorylation and dephosphorylation of this site is maintained under physiological condition and disruption of the balance may lead to pathogenic angiogenesis. As a first step to test this hypothesis, we set out the following two specific aims: (1) To determine if AMPK inhibits ALK1- mediated angiogenesis and, (2) To identify AMPK phosphorylation site(s) on ALK1 and elucidate the molecular mechanism by which AMPK regulates ALK1. The proposed study will be the first to tether AMPK and ALK1 signaling pathways together to angiogenesis. Thus, completion of the study will add novel information to vascular biology. Moreover, since suppression of AMPK activity, upregulation of ALK1 signaling and dysregulated angiogenesis all have been found in such diseases as type 2 dabetes and cancer, the proposed study will be instrumental to understanding the pathogenesis of these diseases and to their treatment. Especially, given metformin has already been used clinically as a safe and inexpensive drug against type 2 diabetes, the outcome of the proposed study will help to expand its use to anti-angiogenesis therapies.