Multi-PI: Titus J. Boggon and David A. Calderwood TITLE: Signaling mechanisms of the cerebral cavernous malformations protein complex ABSTRACT Loss of function mutations in the genes encoding KRIT1 (CCM1), CCM2 (OSM), or CCM3 (PDCD10) cause Cerebral Cavernous Malformations (CCM). The presentation of this disease includes dilated leaky blood vessels, especially in the neurovasculature, resulting in stroke, focal neurological defects, seizures and vascular abnormalities. Each of the multi-domain CCM proteins functions as a molecular scaffold and importantly, CCM2 recruits the MAP kinase kinase kinase, MEKK3, to the multi-protein CCM signaling complex. Recruitment of MEKK3 to the CCM complex is thought to result in suppression of MEKK3 activation of the MEK5-ERK5 pathway and a reduction in KLF2/4 levels. In CCM disease this suppression is lost. Despite extensive research on CCM proteins and their intersection with MEKK3 major gaps in understanding remain, including 1) the fundamental organization of the CCM complex and how this impacts its activity, and 2) the mechanisms by which the CCM complex controls the MEKK3 MAP kinase signaling cascade. In this highly-collaborative, multi-PI proposal the Boggon and Calderwood laboratories will address these outstanding important gaps in knowledge. We will do this in two Aims. In Aim 1, building on extensive preliminary data, we propose a revision of canonical models of the CCM complex and will assess CCM complex stoichiometry as a determinant of signal transduction. In Aim 2, we will discover how CCM proteins act as determinants of MEKK3 signaling outcomes. Our structure-directed functional studies will reveal the normal formation of and define the basis for targeted CCM complex modulation of MEKK3-MEK5-ERK5 signaling.