Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. Here, we use the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. We show that inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. We further show that Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/beta-catenin complex and that shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating beta-catenin localization during gland morphogenesis. Our results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis.

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Several classes of signaling proteins contain autoinhibitory domains that prevent unwarranted signaling and coordinate the induction of activity in response to external cues. CARD11, a scaffold protein critical for antigen receptor signaling to NF-B, undergoes autoregulation by a poorly understood inhibitory domain (ID), which keeps CARD11 inactive in the absence of receptor triggering through inhibitory intramolecular interactions. This autoinhibitory strategy makes CARD11 highly susceptible to gain-of-function mutations that are frequently observed in diffuse large B cell lymphoma (DLBCL) and that disrupt ID-mediated autoinhibition, leading to constitutive NF-B activity, which can promote lymphoma proliferation. Although DLBCL-associated CARD11 mutations in the caspase recruitment domain (CARD), LATCH domain, and coiled coil have been shown to disrupt intramolecular ID binding, surprisingly, no gain-of-function mutations in the ID itself have been reported and validated. In this study, we solve this paradox and report that the CARD11 ID contains an unusual array of four repressive elements that function cooperatively with redundancy to prevent spontaneous NF-B activation. Our quantitative analysis suggests that potent oncogenic CARD11 mutations must perturb autoinhibition by at least three repressive elements. Our results explain the lack of ID mutations in DLBCL and reveal an unusual autoinhibitory domain structure and strategy for preventing unwarranted scaffold signaling to NF-B.Many signaling pathways that control cell proliferation and differentiation have evolved inhibitory checkpoints that prevent uncontrolled signaling. Checkpoints can involve inhibitory proteins that provide negative feedback once signaling begins or that actively suppress signaling prior to receptor engagement. For several pathways, checkpoint function is achieved by an autoinhibitory domain, which governs the activity of the key pathway component in which it is found. Such autoinhibitory domains have been described for several classes of signaling proteins, including tyrosine (1, 2) and serine/threonine kinases (3-5), guanine nucleotide exchange factors (6), phospholipases (7,8), and E3 ubiquitin ligases (9 -11), among others (12, 13). Although an autoinhibitory domain can provide robust, concentration-independent regulation in cis, the presence of an autoinhibitory domain in a signaling protein can make it especially susceptible to mutations that disrupt autoinhibition and lead to constitutive signaling and potentially pathological cell behavior. A mechanistic understanding of autoinhibitory domain function is thus critical for understanding how the output of signaling pathways is determined and how the dysregulation of autoinhibition by mutation can promote oncogenic transformation.

The CARD11 signaling scaffold transmits signaling between antigen receptors on B and T lymphocytes and the transcription factor NF-B during the adaptive immune response. CARD11 activity is controlled by an inhibitory domain (ID), which participates in intramolecular interactions and prevents cofactor binding prior to receptor triggering. Oncogenic CARD11 mutations associated with the activated B cell-like subtype of diffuse large B cell lymphoma somehow perturb ID-mediated autoinhibition to confer CARD11 with the dysregulated spontaneous signaling to NF-B that is required for the proliferation and survival of the lymphoma. Here, we investigate how the four repressive elements (REs) we have discovered in the CARD11 ID function to inhibit CARD11 activity with cooperativity and redundancy. We find that each RE contributes to the maintenance of the closed inactive state of CARD11 that predominates in the absence of receptor engagement. Each RE also contributes to the prevention of Bcl10 binding in the basal unstimulated state. RE1, RE2, and RE3 participate in intramolecular interactions with other CARD11 domains and share domain targets for binding. Remarkably, diffuse large B cell lymphoma-associated gain-of-function mutations in the caspase recruitment domain, LATCH, or coiled coil can perturb intramolecular interactions mediated by multiple REs, suggesting how single amino acid oncogenic CARD11 mutations can perturb or bypass the action of redundant inhibitory REs to achieve the level of hyperactive CARD11 signaling required to support lymphoma growth.