p21ras initiates Rac-1 but not phosphatidyl inositol 3 kinase/PKB, mediated signaling pathways in T lymphocytes Elisabeth Genot 3 , Karin Reif 4 , Sarah Beach 3 , Ijsbrand Kramer 2 and Doreen Cantrell 1 1 Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK; 2 Department of Pharmacology, University College London, Gower Street, London WC1E 6BT p21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Eector pathways for this guanine nucleotide binding protein in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In ®broblasts, an important eector for the Ras oncogene is Phosphatidylinositol 3- kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3- kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3- kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sucient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3- kinase and Akt/PKB are not universal Ras eector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity. Keywords: p21ras; Akt/PKB; signalling pathways; T cells Introduction p21ras functions as a molecular switch in activating downstream eector pathways that control growth and dierentiation. This guanine nucleotide binding protein plays an important role in T lymphocyte activation by coupling the T cell antigen receptor (TCR) to the signaling pathways that regulate the activity of transcription factors important for the expression of cytokine genes (Cantrell, 1996). These include the ETS family protein Elk-1, the serum response factor (SRF), AP1 and Nuclear Factor of Activated T cells (NFAT) (Genot et al., 1996; Woodrow et al., 1993). p21ras signals are also important for thymocyte development and have been shown to regulate the positive selection of thymocytes (Swan et al., 1995). The Raf-1/MEK/ Erk2 pathway is one eector system for p21ras in T cells (Izquierdo et al., 1993, 1994). However, other Ras eector pathways must exist in lymphocytes. For example, activation of MEK is not required for TCR induced proliferative responses of mature thymocytes whereas p21ras signals are indispensable (Alberola-Ila, 1996; Alberola-Ila et al., 1995). Moreover, signals generated by activated p21ras but not MEK are sucient to induce the transition of Rag7/7 thymocytes from CD4/CD8 double negative to CD4/ CD8 double positive status (Crompton, 1996; Swat et al., 1996). In addition, activation of the MEK pathway can eciently couple the TCR to the Erk2 pathway but this is not sucient for p21ras activation of the transcription factors AP1 or NFAT (Genot et al., 1996). Finally, in mast cells activation of Erkl/2 MAP kinases is is not required for FceR1 regulation of NFAT at all, although activation of Ras is essential (Genot et al., 1996; Turner and Cantrell, 1997). These studies highlight that lymphocyte activation involves the coordinated action of multiple Ras eector pathways. A strong candidate for regulating Ras eector pathways in haematopoietic cells is the Rho family GTPase Rac-1. In particular, Rac-1 couples p21ras to the signaling pathways that induce the activity of the transcription factors AP1 and NFAT (Genot et al., 1996; Turner and Cantrell, 1997). The role of Rac-1 in Ras responses in lymphocytes has clear parallels with studies in ®broblasts where it is evident that signaling networks mediated by Rho-family GTPases such as Rac-1 and RhoA (Qiu et al., 1995a,b; Westwick et al., 1997) are important for Ras induction of cell transformation and DNA synthesis. In ®broblasts there has been some characterisation of the mechan- ism that couples p21ras to the GTPase Rac-1: notably, one key p21ras eector molecule is PtdIns 3-kinase which binds directly to active GTP bound p21ras resulting in activation of the lipid kinase (Rodriguez- Viciana et al., 1994, 1996). The PH domains of Rac/ Rho guanine nucleotide exchange proteins (GEFs) Correspondence: E Genot Current addresses: 3 Department of Immunology, Imperial College, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK 4 Department of Microbiology and Immunology, 513 Parnassus, Box 0414, Rm HSE301, University of California, San Francisco California 94143-0414, USA Received 9 September 1997; revised 5 May 1998; accepted 5 May 1998 Oncogene (1998) 17, 1731 ± 1738 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc