Two YxxL segments of a single immunoreceptor tyrosine-based activation motif in the CD3 zeta molecule differentially activate calcium mobilization and mitogen-activated protein kinase family pathways

Immunoreceptor tyrosine-based activation motifs (ITAM), consisting of two Y xxL segments, transmit signals leading to IL-2 gene activation in T cells. We investigated here the functional difference in signal transduction betwe en these two YxxL segments in the CDBT, membrane-proximal ITAM. N-terminal YxxL mutants failed to induce ZAP-70 phosphorylation, elevation of intracel lular Ca2+ concentration ([Ca2+](i)) or extracellular signal-regulated kina se (ERK) activation even in the presence of CD28 co-stimulation, whereas a mutant of the leucine residue at the C-terminal YxxL segment retained the a bility to induce these events although this mutation abrogated the ability to induce IL-2 gene activation. In marked contrast to ERK activation, c-Jun N-terminal kinase (JNK) activation was observed in all mutants when costim ulated with CD28. The mutant of the leucine residue at the C-terminal YxxL segment had a defect in the transcriptional activation at the NF-AT cis-ele ment, which was restored to wild-type level by addition of a Ca2+ ionophore , suggesting that the intensity and/or duration of [Ca2+](i) elevation defi nes the threshold of T cell activation in this mutant. Our data collectivel y indicate that the activation pathways of ERK, JNK and Ca2+ mobilization a re differentially regulated through YxxL segments of an ITAM.