Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton.

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na(+)/H(+) exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG-EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

The epitope recognized by pan-HLA class I-reactive monoclonal antibody W6/32 and its relationship to unusual stability of the HLA-B27/beta2-microglobulin complex.

A broadly used pan-HLA class I-reactive monoclonal antibody W6/32 is believed to recognize a conformational epitope dependent on association between heavy chains and beta2-microglobulin (beta2m). However, in the present study we report that W6/32 does recognize at least some free HLA class I heavy chains under the partially denaturating conditions of nonreducing Western blotting, namely nearly all HLA-B allelic products. Furthermore, we confirm and largely extend our previous observation that complexes of beta2m with heavy chains of a few HLA class I allelic forms (most notably HLA-B27) exhibit unusual resistance to dissociation by SDS, which is reminiscent of MHC class II molecules. In addition, our data indicate the existence of covalent (disulfide-linked) heterodimers of certain HLA class I heavy chains (namely Cw1 and Cw4) and beta2m.

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation.

According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood alpha/beta T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood alpha/beta T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor-mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG-Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.

GPI-microdomains: a role in signalling via immunoreceptors.

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids are assembled on the leukocyte surface within membrane microdomains, which also accommodate a set of cytoplasmic signalling molecules (Src family kinases, G-proteins, linker proteins). Recent results suggest that these membrane specializations mediate not only signal transduction via GPI-proteins and glycolipids but also play important roles in initiation of signalling via immunoreceptors.

Signal transduction in leucocytes via GPI-anchored proteins: an experimental artefact or an aspect of immunoreceptor function?

Membrane proteins anchored in the membrane via a glycolipid glycosylphosphatidylinositol (GPI) as well as some glycolipids are able to transduce signals and induce diverse functional responses in cells upon their cross-linking via antibodies or natural ligands. In some cases this signaling capacity seems to be due to associations of these molecules with specific transmembrane proteins. GPI-anchored proteins are components of membrane microdomains enriched in glycosphingolipids and cholesterol and devoid of most transmembrane proteins. These membrane specializations are relatively resistant to solubilization in solutions of some mild detergents at low temperatures. These 'GPI-microdomains' contain also cytoplasmic signaling molecules such as Src-family protein tyrosine kinases and trimeric G-proteins. Thus, at least some signaling elicited upon cross-linking of GPI-anchored proteins and glycolipids may be due to perturbation of the signaling molecules associated with these microdomains. It is suggested that these specialized areas of the membrane rich in signaling molecules interact with immunoreceptors (TCR, BCR, Fc receptors) cross-linked upon their interactions with ligands and importantly contribute to initiation of proximal phases of their signaling pathways.

T cell receptor signalling results in rapid tyrosine phosphorylation of the linker protein LAT present in detergent-resistant membrane microdomains.

Stimulation of human T cell line Jurkat results in rapid tyrosine phosphorylation of a 35-38 kDa protein that is found in large and buoyant detergent-resistant membrane microdomains containing also glycosylphosphatidylinositol (GPI)-anchored proteins, glycolipids and Src-family protein tyrosine kinases ("GPI-microdomains"). The pp35-38 was found to be identical to LAT, a recently cloned key component of the T-cell receptor signalling pathway. Moreover, a modified form of protein tyrosine kinase Lck (pp60) was newly detected in the GPI-microdomains of the anti-CD3-stimulated Jurkat cells. These data support the idea that GPI-microdomains play important roles in immunoreceptor signalling.