verbatim): This proposal is the competing renewal of the Applicant's R01 to study signaling by the type I receptor for transforming growth factor beta (TGFb). Members of the TGFb superfamily have been implicated as regulators of cell fate and positional information during cardiac embryogenesis, and as potential autocrine/paracrine factors in hypertrophied or failing hearts. For all members of this family, including TGFb itself and bone morphogenetic proteins (BMPs), signaling requires a heteromeric protein kinase complex, made of transmembrane proteins (type II and type I receptors) with characteristic serine/threonine kinase cytoplasmic domains. In the presence of ligand, the type II receptor phosphorylates the type I receptor, whereupon the type I receptor activates signaling by downstream, intracellular effector proteins. One established pathway for TGFb and BMP signaling involves the direct phosphorylation of Smad transcription factors by ligand-activated type I receptors. However, a co-existing or alternative mechanism has been described, via certain members of the mitogen-activated protein kinase (MAPK) family, including TGFb-activated kinase (TAK1), a MAP kinase kinase kinase (MAP3K) that leads to activation of the p38 and Jun N-terminal kinase (JNK) pathways. Preliminary studies by the Applicant have demonstrated that: TAK1 is activated by TGFb in cardiac muscle cells; TAK1 is necessary for TGFb signaling to a marker of hypertrophy (using a kinase-deficient mutation); TAK1 is activated in vivo during hypertrophy induced by load; and TAK1 is sufficient to trigger heart failure in transgenic mice (using a constitutive mutation, at levels of activity similar to those provoked by aortic banding). Two pivotal questions remain to be answered. First, does endogenous TAK1 have an essential function in cardiac myocytes, or in embryogenesis more generally? Second, how does endogenous TAK1 become activated by TGFb family members, and by mechanical load? The Specific Aims for this competing renewal are: (1) Using conventional (germline) and conditional (Cre-mediated) gene targeting, to test the hypothesis that endogenous TAK1 plays an essential role in TGFb signal transduction and the early or late aspects of cardiac organogenesis. (2) Using viral gene transfer in vitro, and gene targeting in vivo, to test two potential pathways for activation of TAK1 in the myocardium-via a TAK1-associated protein, TAB1, versus an upstream MAP3K kinase (MAP4K).
