Federico Coluccio Leskow

Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein

In humans, SHH haploinsufficiency results in holoprosencephaly (HPE), a defect in anterior midline formation. Despite the importance of maintaining SHH transcript levels above a critical threshold, we know little about the upstream regulators of SHH expression in the forebrain. Here we describe a rare nucleotide variant located 460 kb upstream of SHH in an individual with HPE that resulted in the loss of Shh brain enhancer-2 (SBE2) activity in the hypothalamus of transgenic mouse embryos. Using a DNA affinity-capture assay, we screened the SBE2 sequence for DNA-binding proteins and identified members of the Six3 and Six6 homeodomain family as candidate regulators of Shh transcription. Six3 showed reduced binding affinity for the mutant compared to the wild-type SBE2 sequence. Moreover, Six3 with HPE-causing alterations failed to bind and activate SBE2. These data suggest a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE.

Structural Mechanism for Lipid Activation of the Rac-Specific GAP, β2-Chimaerin

The lipid second messenger diacylglycerol acts by binding to the C1 domains of target proteins, which translocate to cell membranes and are allosterically activated. Here we report the crystal structure at 3.2 A resolution of one such protein, beta2-chimaerin, a GTPase-activating protein for the small GTPase Rac, in its inactive conformation. The structure shows that in the inactive state, the N terminus of beta2-chimaerin protrudes into the active site of the RacGAP domain, sterically blocking Rac binding. The diacylglycerol and phospholipid membrane binding site on the C1 domain is buried by contacts with the four different regions of beta2-chimaerin: the N terminus, SH2 domain, RacGAP domain, and the linker between the SH2 and C1 domains. Phospholipid binding to the C1 domain triggers the cooperative dissociation of these interactions, allowing the N terminus to move out of the active site and thereby activating the enzyme.

Phospholipase Cγ/diacylglycerol‐dependent activation of β2‐chimaerin restricts EGF‐induced Rac signaling

Although receptor‐mediated regulation of small G‐proteins and the cytoskeleton is intensively studied, the mechanisms for attenuation of these signals are poorly understood. In this study, we have identified the Rac‐GAP β2‐chimaerin as an effector of the epidermal growth factor receptor (EGFR) via coupling to phospholipase Cγ (PLCγ) and generation of the lipid second messenger diacylglycerol (DAG). EGF redistributes β2‐chimaerin to promote its association with the small GTPase Rac1 at the plasma membrane, as determined by FRET. This relocalization and association with Rac1 were impaired by disruption of the β2‐chimaerin C1 domain as well as by PLCγ1 RNAi, thus defining β2‐chimaerin as a novel DAG effector. On the other hand, GAP‐deficient β2‐chimaerin mutants show enhanced translocation and sustained Rac1 association in the FRET assays. Remarkably, RNAi depletion of β2‐chimaerin significantly extended the duration of Rac activation by EGF, suggesting that β2‐chimaerin serves as a mechanism that self‐limits Rac activity in response to EGFR activation. Our results represent the first direct evidence of divergence in DAG signaling downstream of a tyrosine‐kinase receptor via a PKC‐independent mechanism.

Differential endocytosis and signaling dynamics of insulin receptor variants IR-A and IR-B

Insulin signaling comprises a complex cascade of events, playing a key role in the regulation of glucose metabolism and cellular growth. Impaired response to insulin is the hallmark of diabetes, whereas upregulated insulin activity occurs in many cancers. Two splice variants of the insulin receptor (IR) exist in mammals: IR-A, lacking exon 11, and full-length IR-B. Although considerable biochemical data exist on insulin binding and downstream signaling, little is known about the dynamics of the IR itself. We created functional IR transgenes fused with visible fluorescent proteins for use in combination with biotinamido-caproyl insulin and streptavidin quantum dots. Using confocal and structured illumination microscopy, we visualized the endocytosis of both isoforms in living and fixed cells and demonstrated a higher rate of endocytosis of IR-A than IR-B. These differences correlated with higher and sustained activation of IR-A in response to insulin and with distinctive ERK1/2 activation profiles and gene transcription regulation. In addition, cells expressing IR-B showed higher AKT phosphorylation after insulin stimulation than cells expressing IR-A. Taken together, these results suggest that IR signaling is dependent on localization; internalized IRs regulate mitogenic activity, whereas metabolic balance signaling occurs at the cell membrane.

Identification of an Autoinhibitory Mechanism That Restricts C1 Domain-mediated Activation of the Rac-GAP α2-Chimaerin

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in α2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type α2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize α2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the α2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders α2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of α2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-α2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced α2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of α2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, α2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.

β2-chimaerin provides a diacylglycerol-dependent mechanism for regulation of adhesion and chemotaxis of T cells

The small GTPase Rac contributes to regulation of cytoskeletal rearrangement during chemokine-induced lymphocyte adhesion and migration in a multi-step process that is very precisely coordinated. Chimaerins are Rac1-specific GTPase-activating proteins of unknown biological function, which have a canonical diacylglycerol C1-binding domain. Here we demonstrate endogenous expression of β2-chimaerin in T lymphocytes and study the functional role of this protein in phorbol ester and chemokine (CXCL12)-regulated T-cell responses. We used green fluorescent protein-tagged β2-chimaerin and phorbol ester stimulation to investigate changes in protein localization in living lymphocytes. Our results demonstrate that active Rac cooperates with C1-dependent phorbol ester binding to induce sustained GFP-β2-chimaerin localization to the membrane. Subcellular distribution of GFP β2-chimaerin in living cells showed no major changes following CXCL12 stimulation. Nonetheless Rac1-GTP levels were severely inhibited in GFP-β2-chimaerin-expressing cells, which displayed reduced CXCL12-induced integrin-dependent adhesion and spreading. This effect was dependent on chimaerin GTPase-activating protein function and required diacylglycerol generation. Whereas β2-chimaerin overexpression decreased static adhesion, it enhanced CXCL12-dependent migration via receptor-dependent diacylglycerol production. These studies demonstrate that β2-chimaerin provides a novel, diacylglycerol-dependent mechanism for Rac regulation in T cells and suggest a functional role for this protein in Rac-mediated cytoskeletal remodeling.