Induction of FucT-VII by the Ras/MAP kinase cascade in Jurkat T cells

Abstract

Induction of the ␣1,3-fucosyltransferase FucT-VII in T lymphocytes is crucial for selectin ligand formation, but the signaling and transcriptional pathways that govern FucT-VII expression are unknown. Here, using a novel, highly phorbol myristate acetate (

H-Ras and Phosphoinositide 3-Kinase Cooperate to Induce α(1,3)-Fucosyltransferase VII Expression in Jurkat T Cells

“…The Rac1 G12V and Rac2 Q61L constitutively active mutants were gifts of Gary Bokoch, Scripps Research Institute, La Jolla, CA. The RafBxB cDNA has been described previously (17). All constructs were verified by bidirectional sequencing and cloned into the retroviral vectors as described earlier.…”

“…cDNA Constructs and Site-directed Mutagenesis-The constitutively active form of H-Ras (v-H-Ras) is the double point-mutant G12R/A59T, which has been described previously (17). The H-Ras activation mutant G12V, G12R, and the double point-mutant G12V/A59T were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).…”

“…Retroviral Infections-Production of recombinant retrovirus pseudotyped with the vesicular stomatitis virus glycoprotein G was performed as described previously (17). Briefly, cDNA encoding the different proteins was subcloned into murine stem cell virus (MSCV)-IRES-GFP (35) upstream of the internal ribosome entry site (IRES) and enhanced green fluorescent protein (GFP).…”

“…Ras can stimulate the Raf serine/threonine kinases, subsequently activating the ERK mitogen-activated protein kinases (18), which have been shown to be essential for FucT-VII induction (17). Apart from Raf, other known effectors include the phosphoinositide 3-kinase (PI3K), protein kinase C⑀, RIN1, AF6, MEKK1, and Nore1 (18,31).…”

“…We have recently shown that enforced expression of constitutively active Ras in Jurkat T cells leads to the expression of FucT-VII, implicating Ras as a FucT-VII regulator (17). Ras proteins are small guanine nucleotide-binding proteins that function as molecular switches in signal transduction cascades, regulating cell proliferation, survival, and differentiation (18,19).…”