Signaling through CD5 Activates a Pathway Involving Phosphatidylinositol 3-Kinase, Vav, and Rac1 in Human Mature T Lymphocytes

Abstract

CD5 acts as a coreceptor on T lymphocytes and plays an important role in T-cell signaling and T-cell-B-cell interactions. Costimulation of T lymphocytes with anti-CD5 antibodies results in an increase of the intracellular Ca2؉ levels, and subsequently in the activation of Ca 2؉ /calmodulin-dependent (CaM) kinase type IV. In the present study, we have characterized the initial signaling pathway induced by anti-CD5 costimulation. The activation of phosphatidylinositol (PI) 3-kinase through tyrosine phosphorylation of its p85 subunit is a proximal event in the CD5-signaling pathway and leads to the activation of the lipid kinase activity of the p110 subunit. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit the CD5-induced response as assessed in interleukin-2 (IL-2) secretion experiments. The expression of an inactivated Rac1 mutant (Rac1 ⅐ N17) in T lymphocytes transfected with an IL-2 promoter-driven reporter construct also abrogates the response to CD5 costimulation, while the expression of a constitutively active Rac1 mutant (Rac1-V12) completely replaces the CD5 costimulatory signal. The Rac1-specific guanine nucleotide exchange factor Vav is heavily phosphorylated on tyrosine residues upon CD5 costimulation, which is a prerequisite for its activation. The CD5 receptor, which is expressed on the surface of all T lymphocytes as well as on a subset of B lymphocytes, is a 67-kDa monomeric transmembrane glycoprotein that belongs to the scavenger receptor cysteine-rich family of extracellular domain-like structures (1,11,28). The counterreceptor of CD5 has been identified as CD72, a dimeric receptor which is commonly expressed on B lymphocytes (63). A second ligand of CD5, termed CD5L, is present on activated splenic B lymphocytes (4). In view of its involvement in the interactions between T and B lymphocytes and also between different subsets of B lymphocytes, it has been proposed that CD5 plays an important role in the regulation of the immune response (4,11,13,44,57).The CD5 receptor on T lymphocytes is associated with the T-cell receptor (TCR)-CD3-CD4 (or CD8) complex, which also comprises the protein tyrosine kinases p56 lck and p59 fyn , and it depends on this physical association for its functional activity (3,8,30,40,54). Once the TCR-CD3 complex is engaged by ligand, the cytoplasmic domain of CD5 becomes rapidly phosphorylated on tyrosine residues, as are the TCR chains (8,17,44). These tyrosines are present in a potential tyrosine kinase phosphorylation motif (Y-X 11 -Y-XX), which is similar to the immunoreceptor tyrosine activation motifs as found in the TCR and CD3 chains (3,44). Once the tyrosine residues in this motif became phosphorylated, they can serve as docking sites for SH2 (Src homology 2) domain-containing proteins (44, 53). The protein tyrosine kinase p56 lck , which is associated with CD4 or CD8 (50), seems to be responsible for the phosphorylation of the tyrosine residues in the cytoplasmic domain of the CD5 receptor upon TCR engagement. It has also been demonstrated that p56 lck ...

Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components

“…The following plasmids were used : pIRE LUC containing two copies of the IL-6 response element (IRE) of the ICAM-1 promoter in front of the herpes simplex virus thymidine kinase promoter and the luciferase gene [41] ; pSG5-STAT3 which expresses STAT3 from the SV40 promoter ; pSG5-STAT3β, which expresses a dominant-negative isoform of STAT3 lacking the 55 C-terminal amino-acid residues ; pSG5-STAT3 Ser(#( Ala, which expressed a mutant STAT3 in which the Ser(#( is replaced by Ala ; pCS2j-RacN17 and pCS2j-RacV12 expressing the dominant-negative and constitutive-active mutant variants of Rac ; pSEK-1\MKK-4(A-L) which expresses dominant-negative SEK-1\MKK-4 [42] ; pCDNA3-MEKK∆(K432M) which expresses a dominant-negative MEKK [43] ; pCDNA3-HA-JNK-1 which expresses HA-tagged JNK-1 (p46) ; pGEX-1-cJun(1-135) expressing glutathione S-transferase (GST)-c-Jun ; and the dominant-negative and constitutive-active mutants pEFmyc-Vav-C [44] and pMEX-myc-Vav-A(∆1-65) [39,45]. The expression vector encoding a dominant-negative mutant of Raf kinase, N∆Raf, was provided by Dr P. Coffer (Department of Pulmonary Diseases, University Hospital Utrecht, Utrecht, The Netherlands) [46].…”

Rac2 Is an Essential Regulator of Neutrophil Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activation in Response to Specific Signaling Pathways

“…Phosphorylation of JNK was also detectable after stimulation with either PMA or fMLP, which was consistently decreased in fMLP-activated rac2 Ϫ/Ϫ cells. Previous studies have suggested that Rac is downstream of phosphoinositide-3 kinase (PI3K) activation induced by chemoattractants and other signals (39,40). In rac2 Ϫ/Ϫ mast cells, PI3K activation by stem cell factor is normal, but phosphorylation of PI3-dependent kinase Akt (41) is decreased (29).…”

Rac and Phosphatidylinositol 3-Kinase Regulate the Protein Kinase B in FcεRI Signaling in RBL 2H3 Mast Cells

“…Although several studies show that Rac is activated by PI3-kinase, and many studies give evidence for a control of PI3-kinase by Rac, our findings are in line with the recent observation that in a murine T cell line inhibition of Rho GTPases by C. difficile toxin B attenuates PI3-kinase activity (60). By contrast, in CD5-stimulated T-cells, it was shown that Vav and Rac function downstream of PI3-kinase, i.e., inhibition by wortmannin could be overcome by overexpression of constitutively active Vav or Rac (61). In bone marrow-derived mast cells (BMMC), it was shown that PI3-kinase is involved in activation of the Rac/Jun kinase pathway (62).…”