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

Abstract

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 α/β 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 α/β 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.

Src family tyrosine kinases-driven colon cancer cell invasion is induced by Csk membrane delocalization

“…For example, the cytoskeletal-associated protein, paxillin, recruits Csk to the focal adhesion sites for the regulation of cell migration (Schaller and Parsons, 1995), whereas the signalling protein, Dok-1, induces Csk translocation to the plasma membrane for mitogenic regulation (Zhao et al, 2006). Additional Csk-binders have been identified, including the structural protein of caveolae, caveolin-1 (Lee et al, 2000), the junctional proteins VE-cadherin (Baumeister et al, 2005) and ZO-1 and the transmembrane protein phosphoprotein associated with glycosphingolipidenriched microdomain (PAG)/Csk-binding protein (Cbp) (Brdicka et al, 2000;Kawabuchi et al, 2000) localized in cholesterol-enriched membrane domains or rafts (named PAG in this study). Interestingly, SFK phosphorylation of these proteins triggers their binding to Csk through a SH2-pTyr-dependent mechanism, which creates a negative feedback regulatory loop.…”

Csk-binding protein (Cbp) negatively regulates epidermal growth factor-induced cell transformation by controlling Src activation

“…Epidermal growth factor-induced Cbp tyrosine phosphorylation depends on SFK activity There are a total of ten tyrosine residues in the amino acid sequence of human Cbp, and nine of them are located within potential consensus sequences for phosphorylation by SFK (YXXV/L/I) (Brdicka et al, 2000).…”

“…When tyrosine is phosphorylated, Cbp recruits Csk from the cytosol to the microdomains, where Csk suppresses SFK activity. This has been observed mainly in lymphocytes (Brdicka et al, 2000;Ohtake et al, 2002;Baumgartner et al, 2003;Davidson et al, 2003).…”

“…Csk (C-terminal Src kinase)-binding protein (Cbp), which is also called phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), is a ubiquitously expressed transmembrane adaptor protein (Kawabuchi et al, 2000;Brdicka et al, 2000). Cbp is exclusively localized to microdomains, where Src family kinase (SFK) is also found to be enriched (Simons and Ikonen, 1997;Brdicka et al, 2000).…”

“…Cbp is exclusively localized to microdomains, where Src family kinase (SFK) is also found to be enriched (Simons and Ikonen, 1997;Brdicka et al, 2000). When tyrosine is phosphorylated, Cbp recruits Csk from the cytosol to the microdomains, where Csk suppresses SFK activity.…”