We co-transfected TG2 WT or TG2 CD with Bcr and performed assays for levels of activated endogenous Rac. Bcr GTPase-activating activity. == Introduction == Transglutaminase 2 (TG2,2also called tissue transglutaminase) is a member of the transglutaminase family that selectively catalyzes the Ca2+-dependent formation of covalent bonds between -carboxamide groups of glutamine residues and -amino groups of lysine residues or primary amines. Unlike other family members, TG2 is expressed in many tissues and cell types, also functions as a G protein in transmembrane signaling, and acts as a cell surface adhesion mediator (14). TG2 has been the focus of numerous studies that show it plays an important role in a variety of biological functions including differentiation, apoptosis, signaling, adhesion, migration, wound healing, inflammation, and phagocytosis of apoptotic cells. Although TG2 appears to have many functional domains, studies have mainly concentrated on its cross-linking activity, VWF with little investigation into its non-enzymatic roles (3). Bcr was originally identified through its involvement in chronic myeloid leukemia (5). Subsequent studies established that it contains a domain with GTPase-activating protein (GAP) activity for the Rho family of small GTPases that includes Rho, Rac and Cdc42 (6). Although the purified GAP domain of Bcr and of the highly related Abr are active toward both Rac and Cdc42in vitro(7), they only act on Racin vivo(810). Rho family members are critical regulators of a variety of cellular functions including actin cytoskeleton rearrangement, growth, differentiation, and membrane trafficking (1114). They act as molecular switches that cycle WAY-262611 between an active, GTP-bound and an inactive, GDP-bound form. This cycle is tightly controlled by GAPs such as Bcr and Abr, by guanine nucleotide exchange factors (GEFs), and by guanine nucleotide dissociation inhibitors (GDIs). WAY-262611 Although many studies have focused on activation of Rho GTPases, the deactivation by GAPs plays an equally important critical role in their regulation (1517). For example, loss of the tumor suppressor DLC1, a RhoGAP, is associated with the development of hepatocellular carcinoma in man (18). How the GAP activity of such proteins is regulated is not completely understood. The Bcr protein contains multiple domains that could be involved in regulation of the GAP activity. We recently identified a direct interaction with RhoGDI as one regulatory mechanism (19). However, it is likely that Bcr is regulated though multiple, different interactions. In an alternative approach to investigate how Bcr is regulated, we sought to identify Bcr-interacting proteins in a yeast two-hybrid screen, using the entire Bcr protein as bait, and isolated TG2. We here report that TG2 functions as a regulator of the BcrGAP activity, and, through it, controls levels of activated Rac. Furthermore, GTP-bound TG2 has reduced affinity for Bcr and reduced ability to inhibit the Bcr GAP activity. == EXPERIMENTAL PROCEDURES == == == == == == Plasmids and Antibodies == The yeast two-hybrid screen has been previously described (20). Full-length human TG2 wild-type and C277S cDNAs in pcDNA3.1(+) were kindly provided by Gail Johnson (University of Rochester). Xpress-tagged wild type, CT (residues 1460), and NT (residues 1139) TG2 were subcloned into pcDNA3.1/HisC vectors through the polymerase chain reaction (PCR) using pcDN3.1(+)-TG2 wild type WAY-262611 as template. Xpress-tagged TG2 CT (residues 463687) was subcloned into pcDNA3.1/HisB. To generate Bcr GAP, a full-length humanBCRcDNA clone in pSK flanked by EcoRI sites (B1/SK) was digested with EcoRI HindIII, and the 2 2.8-kb fragment was subcloned into pSK digested with the same enzymes. The insert was removed by digestion with XbaI KpnI and subcloned into pCDE digested with WAY-262611 the same enzymes. The HindIII site inBCRis located in the GAP domain, and this construct lacks amino acid residues 10041271. BcrPK was constructed by isolating the N-terminal end ofBCRas a 0.4-kb SalI-StuI fragment from B1/SK. This fragment includes the first 39 amino acid residues of the oligomerization domain. The 3-end ofBCRwas purified as a NaeI SalI fragment from B1/SK (with the EcoRI insert in a different orientation). The NaeI site is located.