The Small GTP-Binding Protein Rho Potentiates AP-1 Transcription in T Cells

The Ras-related Rho family members are involved in thymic development, cell transformation, actin cytoskeletal rearrangement, and cell polarity (17, 26, 35, 36, 41, 47). The Rho family is comprised of several related proteins, including Rac1, Rac2, RhoA, RhoB, RhoC, Cdc42Hs, and TC10 (18, 19), which share structural similarity with Ras. These proteins contain intrinsic GTPase activity and bind GTP and GDP in a manner that is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs) (43, 46). Several GEFs for the Rho family, such as Ost (23), Tiam (29), and the faciogenital dysplasia gene product (FGD1 [39]), have been isolated and shown to promote binding of GTP to Rho. Bcr (11), p190 (8, 45), and Cdc42GAP (7) have been demonstrated to act as GAPs for the Rho family, promoting the conversion of GTP to GDP. The importance of Rho family members in cellular activation and growth has been underscored by several recent studies. In NIH 3T3 cells, coexpression of oncogenic Ras (61L) with activated Rho (63L) enhances morphological transformation and cell motility. Overexpression of dominant-negative (DN) mutants of Rac or Rho reduces oncogenic Ras transforming activity, indicating that activation of Rho is required for Ras transformation (26, 40). Roles for Rho in gene regulation and cell cycle progression have also been demonstrated. Microinjection of activated forms of Rho, Rac, and Cdc42Hs stimulates cell cycle progression and subsequent DNA synthesis. Serum-induced DNA synthesis and progression through the G1 phase can be blocked by microinjection of C3 exoenzyme (a specific inhibitor of Rho) or by expression of DN Rac or Cdc42Hs (38). In addition, thymuses lacking functional Rho isolated from transgenic mice that overexpress C3 exoenzyme are small and show markedly decreased cellularity (17). Other studies have demonstrated that Rho is required for survival of early pre-T cells and regulates cell cycle progression in late pre-T cells (20). The mechanism(s) by which Rho regulates such diverse cellular processes is not well understood. One possibility is that Rho mediates distinct cellular functions through control of transcriptional activation. Consistent with this hypothesis, reports have demonstrated that activated Rho regulates c-fos promoter activity by serum response factor (SRF) and that this activity can be blocked by the addition of C3 exoenzyme (1, 21). Fos interacts with c-Jun and subsequently controls the transcriptional activation of a number of other genes involved in many cell programs. Protein kinase C (PKC) consists of a family of structurally related serine/threonine kinases that play an important role in cell proliferation, differentiation, and transformation (32, 33). PKCs are divided into three major subgroups (conventional, novel, and atypical) defined by their structures and their abilities to be regulated by calcium and/or phorbol myristate acetate (PMA). Conventional PKCs contain a C-terminal catalytic domain and an N-terminal regulatory domain that is composed of a pseudosubstrate site, a C1 domain that binds diacylglycerol (DAG) or its analog PMA, and a C2 domain that binds calcium and phospholipid. The cellular roles of the different PKC isozymes remain unclear, but accumulated evidence has shown that individual PKC isoforms may play distinct roles in response to various activating stimuli. For example, overexpression of PKCɛ enhances the growth rate of NIH 3T3 cells, while overexpression of PKCδ decreases cell growth rate (31). The important role of PKCs in signal transduction is evident from their substrate specificity, subcellular localization, sensitivity to downregulation via agonist induction and regulation of cell growth and differentiation (33). Interaction of the T-cell antigen receptor (TCR) with antigen in the context of major histocompatibility complex proteins initiates a signal transduction cascade that leads to transcriptional activation, lymphokine production, cell proliferation, and induction of the cell’s effector function. In addition to the TCR, other costimulatory surface molecules, such as CD28, contribute to T-cell signaling (14). T-cell activation via anti-TCR and anti-CD28 antibody-mediated costimulation markedly enhances the production of several cytokines, including interleukin 2 (IL-2), IL-3, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha (51), and also results in cellular proliferation and functional activation. The binding of transcription factors to specific sites within the promoters of these cytokine genes is responsible for their upregulation. A 300-bp IL-2 promoter (IL-2P) region upstream of the transcription start site is sufficient to transcriptionally regulate IL-2 expression in response to TCR stimulation (42). Several transcription factors, such as NFAT, Oct-1, AP-1, and NF-κB, have been shown to bind to identified enhancer elements in the IL-2P. In lymphocytes, Fos and Jun form a competent transcription factor complex that activates the AP-1 enhancer elements within the promoters for several genes of diverse function, such as granzyme B, CD11c, CD40L, and CD69 genes, as well as the cytokine promoters. Thus, it is apparent that regulation of the binding activity of the AP-1 complex is integral to the control of many aspects of immune function, including cytokine production, cytolytic effector function, adhesion, and cell survival and proliferation. In this report, we demonstrate that overexpression of activated Rho synergizes with PMA to augment AP-1 transcriptional activity. Overexpression of CAAX-deleted activated Rho abolishes the enhancement. This effect is not mediated through the mitogen-activated protein kinase (MAPK) pathway, as it is not affected by overexpression of DN MEK or by the addition of a MEK inhibitor. The plasma membrane-associated Rho binds to PKCα but does not bind to PKCβ, -γ, or -ɛ. In addition, expression of the N-terminal regulatory domain of PKCα blocks activated Rho-plus-PMA-enhanced AP-1 transcriptional activation. These data suggest that the small GTP-binding protein, Rho, potentiates AP-1 enhancer element activity and may augment AP-1-containing gene transcription following T-cell activation.