The T-Cell Receptor Regulates Akt (Protein Kinase B) via a Pathway Involving Rac1 and Phosphatidylinositide 3-Kinase

Abstract

The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.Engagement of the T-cell antigen receptor (TCR) by antigen in a major histocompatibility complex context or by antibodies that cross-link this receptor triggers a complex series of signaling events that lead to reorganization of the cytoskeleton as well as transcriptional activation of multiple genes and culminate in T-lymphocyte activation and proliferation (9). One of the earliest events triggered by TCR engagement is the activation of protein tyrosine kinases (PTKs). Activation of the Src tyrosine kinase Lck is necessary to phosphorylate the cytoplasmic tails of the CD3 complex on tyrosine residues within the immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the ITAMs provides docking sites for the Src homology domains (SH2) of the Syk family PTKs which, once recruited, become activated and cause subsequent tyrosine phosphorylation of multiple substrates. One such substrate is the integral membrane protein LAT (linker for activation of T cells), whose phosphorylation allows recruitment of a whole range of signaling molecules, including Grb2, PLC-␥, GADs, SLP-76, Cbl, Vav, and the regulatory subunit p85 of phosphatidylinositide (PI) 3-kinase, through either direct or indirect interactions (46). With respect to PI 3-kinase, the TCR is endowed with at least two other putative modes of activation: via a direct mechanism, by binding of the p85 regulatory subunit of PI 3-kinase to the tyrosine phosphorylated ITAM (11, 25), or in an indirect way, through activation of Ras (12), which in turn could in...

Rac1 and RhoG promote cell survival by the activation of PI3K and Akt, independently of their ability to stimulate JNK and NF-κB

“…In line with our present results, whereas early work indicated that small G proteins of the Rho family do not play a role in Akt regulation (Klippel et al, 1996), more recent studies have shown that Rac1 and 2 can indeed activate Akt in cells of hematopoietic origin, such as Jurkat and BaF3 cells (Genot et al, 2000;Nishida et al, 1999). In T cells, Rac activation of Akt participates in the formation of membrane spikes (Genot et al, 2000), and in BaF3 cells in the viability upon IL-3 removal (Nishida et al, 1999).…”

“…In T cells, Rac activation of Akt participates in the formation of membrane spikes (Genot et al, 2000), and in BaF3 cells in the viability upon IL-3 removal (Nishida et al, 1999). Furthermore, in Rac2-de®cient mast cells, the survival response to growth factors was diminished, and that correlated with a decreased activation of Akt and enhanced BAD/Bcl-X L expression .…”

T-Cell Receptor-Induced NF-κB Activation Is Negatively Regulated by E3 Ubiquitin Ligase Cbl-b

“…Furthermore, Vav-1 has been shown to be required for the membrane targeting of PKC-(50). It was also suggested that PI3-K may act downstream of Rac-1, a downstream substrate of Vav-1 (9). Therefore, the relationship between PI3-K, Vav-1, and PKC-is still not fully defined.…”

“…It was reported that PI3-K may regulate the exchange activity of Vav-1 in vitro through its product phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), which binds to the PH domain of Vav-1 and recruits Vav-1 to the plasma membrane (10,30). However, it has also been reported that PI3-K may act downstream of Rac-1, a downstream substrate of Vav-1 (9). Therefore, the relationship among Vav-1, PI3-K, and PKC-in primary T cells is not fully characterized.…”

Activation of CD4 T cells by Raf-independent effectors of Ras

“…This result is consistent with a previous study arguing that PI3K is an effector of Rac GTPase rather than Ras in T cells. Hence, an overexpression of Ras V12C40 may not necessarily mimic physiological conditions that control PI3K function in these cells (27).…”