Cutting edge: Fyn is essential for tyrosine phosphorylation of Csk-binding protein/phosphoprotein associated with glycolipid-enriched microdomains in lipid rafts in resting T cells

In resting T cells, Csk is constitutively localized in lipid rafts by virtue of interaction with a phosphorylated adaptor protein, Csk-binding protein (Cbp)/phosphoprotein associated with glycolipid-enriched microdomains, and sets an activation threshold in TCR signaling. In this study, we examined a kinase responsible for Cbp phosphorylation in T cell membrane rafts. By analyzing T cells from Fyn-/- mice, we clearly demonstrated that Fyn, but not Lck, has its kinase activity in membrane rafts, and plays a critical role in Cbp phosphorylation, Cbp-Csk interaction, and Csk kinase activity. Naive CD44(low)CD62 ligand(high) T cells were substantially reduced in Fyn-/- mice, presumably due to the inhibition of Cbp phosphorylation. Thus, Fyn mediates Cbp-Csk interaction and recruits Csk to rafts by phosphorylating Cbp. Csk recruited to rafts would then be activated and inhibit the kinase activity of Lck to keep resting T cells in a quiescent state. Our results elucidate a negative regulatory role for Fyn in proximal TCR signaling in lipid rafts.     

The lipid raft-anchored adaptor protein Cbp controls the oncogenic potential of c-Src

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.     

The ubiquitously expressed Csk adaptor protein Cbp is dispensable for embryogenesis and T-cell development and function

Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of "lipid rafts" is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.     

Release from tonic inhibition of T cell activation through transient displacement of C-terminal Src kinase (Csk) from lipid rafts

In resting peripheral T cells, Csk is constitutively present in lipid rafts through an interaction with the Csk SH2-binding protein, PAG, also known as Cbp. Upon triggering of the T cell antigen receptor (TCR), PAG/Cbp is rapidly dephosphorylated leading to dissociation of Csk from lipid rafts. However, tyrosine phosphorylation of PAG/Cbp resumes after 3--5 min, at which time Csk reassociates with the rafts. Cells overexpressing a mutant Csk that lacks the catalytic domain, but displaces endogenous Csk from lipid rafts, have elevated basal levels of TCR-zeta-chain phosphorylation and spontaneous activation of an NFAT-AP1 reporter from the proximal interleukin-2 promoter as well as stronger and more sustained responses to TCR triggering than controls. We suggest that a transient release from Csk-mediated inhibition by displacement of Csk from lipid rafts is important for normal T cell activation.     

Combined spatial and enzymatic regulation of Csk by cAMP and protein kinase a inhibits T cell receptor signaling

Raft-associated Csk controls signaling through the T cell receptor (TCR) and was mainly anchored to Cbp/PAG (phosphoprotein associated with glycosphingolipid-enriched membrane domains). Treatment of cells with the cAMP-elevating agent prostaglandin E(2) (PGE(2)) augmented the level of Cbp/PAG phosphorylation with a concomitant increase in amounts of Csk bound to Cbp/PAG. While TCR-triggering resulted in transient dissociation of Csk from Cbp/PAG/rafts allowing TCR-induced tyrosine phosphorylation to occur, pretreatment with PGE(2) reduced Csk dissociation upon TCR triggering. This correlated with lowered TCR-induced phosphorylation of CD3 zeta-chain and linker for activation of T cells. Moreover, competition of endogenous Csk from lipid rafts abolished PGE(2)-mediated inhibition of TCR-induced zeta-chain phosphorylation and activation of the nuclear factor of activated T cells (NFAT) activator protein 1 (AP-1). Finally, raft-associated Csk already activated via Cbp/PAG binding, gained additional increase in phosphotransferase activity upon protein kinase A-mediated phosphorylation of Csk. We propose that cAMP regulates Csk via both spatial and enzymatic mechanisms, thereby inhibiting signaling through the TCR.     

Removal of C-terminal SRC kinase from the immune synapse by a new binding protein

The Csk tyrosine kinase negatively regulates the Src family kinases Lck and Fyn in T cells. Engagement of the T-cell antigen receptor results in a removal of Csk from the lipid raft-associated transmembrane protein PAG/Cbp. Instead, Csk becomes associated with an approximately 72-kDa tyrosine-phosphorylated protein, which we identify here as G3BP, a phosphoprotein reported to bind the SH3 domain of Ras GTPase-activating protein. G3BP reduced the ability of Csk to phosphorylate Lck at Y505 by decreasing the amount of Csk in lipid rafts. As a consequence, G3BP augmented T-cell activation as measured by interleukin-2 gene activation. Conversely, elimination of endogenous G3BP by RNA interference increased Lck Y505 phosphorylation and reduced TCR signaling. In antigen-specific T cells, endogenous G3BP moved into a intracellular location adjacent to the immune synapse, but deeper inside the cell, upon antigen recognition. Csk colocalization with G3BP occurred in this "parasynaptic" location. We conclude that G3BP is a new player in T-cell-antigen receptor signaling and acts to reduce the amount of Csk in the immune synapse.     

Cutting Edge: Transmembrane phosphoprotein Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains as a negative feedback regulator of mast cell signaling through the FcepsilonRI

Tyrosine phosphorylation in the cytoplasmic domains of FcepsilonRI by the Src family kinase Lyn initiates a signaling cascade leading to mast cell activation. In this study, we show that a recently identified transmembrane protein, Csk-binding protein (Cbp), also known as phospoprotein associated with glycosphingolipid-enriched microdomains (PAG), negatively regulates FcepsilonRI signaling. In rat basophilic leukemia (RBL)-2H3 cells, the levels of tyrosine phosphorylation of Cbp/PAG and its association with Csk, a negative regulator for Lyn, significantly elevate immediately after aggregation of FcepsilonRI. An overexpression of Cbp/PAG in RBL-2H3 cells inhibits FcepsilonRI-mediated cell activation. This is accompanied with decreased levels of tyrosine phosphorylation of FcepsilonRI, association of FcepsilonRI with Lyn, and FcepsilonRI-associated tyrosine kinase activity. These findings combined with the fact that Cbp/PAG, Lyn, and aggregated FcepsilonRI are localized to lipid rafts, suggest that upon FcepsilonRI aggregation Cbp/PAG down-regulates the receptor-associated Lyn activity through relocating Csk to rafts, thereby efficiently mediating feedback inhibition of FcepsilonRI signaling.     

PAG-associated FynT regulates calcium signaling and promotes anergy in T lymphocytes

Phosphoprotein associated with glycolipid-enriched membranes (PAG), also named Csk-binding protein (Cbp), is a transmembrane adaptor associated with lipid rafts. It is phosphorylated on multiple tyrosines located in the cytoplasmic domain. One tyrosine, tyrosine 314 (Y314) in the mouse, interacts with Csk, a protein tyrosine kinase that negatively regulates Src kinases. This interaction enables PAG to inhibit T-cell antigen receptor (TCR)-mediated T-cell activation. PAG also associates with the Src-related kinase FynT. Genetic studies indicated that FynT was required for PAG tyrosine phosphorylation and binding of PAG to Csk in T cells. Herein, we investigated the function and regulation of PAG-associated FynT. Our data showed that PAG was constitutively associated with FynT in unstimulated T cells and that this association was rapidly lost in response to TCR stimulation. Dissociation of the PAG-FynT complex preceded PAG dephosphorylation and PAG-Csk dissociation after TCR engagement. Interestingly, in anergic T cells, the association of PAG with FynT, but not Csk, was increased. Analyses of PAG mutants provided evidence that PAG interacted with FynT by way of tyrosines other than Y314. Enforced expression of a PAG variant interacting with FynT, but not Csk, caused a selective enhancement of TCR-triggered calcium fluxes in normal T cells. Furthermore, it promoted T-cell anergy. Both effects were absent in mice lacking FynT, implying that the effects were mediated by PAG-associated FynT. Hence, besides enabling PAG tyrosine phosphorylation and the PAG-Csk interaction, PAG-associated FynT can stimulate calcium signals and favor T-cell anergy. These data improve our comprehension of the function of PAG in T cells. They also further implicate FynT in T-cell anergy.     

Modulation of proximal signaling in normal and transformed B cells by transmembrane adapter Cbp/PAG

The transmembrane protein Cbp/PAG (Csk binding protein/phospho-protein associated with glycosphingolipid-enriched microdomains) has a negative regulatory role in T cell activation as an adapter for C-terminal Src kinase, Csk. In T cells, membrane docking of Csk is promoted by binding to FynT-phosphorylated Cbp/PAG (pTyr317) to allow targeting of substrates residing in lipid rafts. Here, we investigate a potential parallel position for Cbp/PAG and the Src kinase Lyn in early B cell receptor signaling. Using normal and transformed B cells, we have compared signal profiles of BCR-triggered responses created by phospho-specific flow cytometry. In human normal B cells, our data show that reduced Cbp/PAG levels leads to enhanced and prolonged activation of proximal signaling mediators, while over-expression of the adapter in normal, EBV-transformed cells results in reduced calcium flux. Taken together, our findings support a negative regulatory function for Cbp/PAG in proximal BCR signaling in these cells.     

Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp,a lipid raft-associated transmembrane adaptor

PAG/Cbp (hereafter named PAG) is a transmembrane adaptor molecule found in lipid rafts. In resting human T cells, PAG is tyrosine phosphorylated and associated with Csk, an inhibitor of Src-related protein tyrosine kinases. These modifications are rapidly lost in response to T-cell receptor (TCR) stimulation. Overexpression of PAG was reported to inhibit TCR-mediated responses in Jurkat T cells. Herein, we have examined the physiological relevance and the mechanism of PAG-mediated inhibition in T cells. Our studies showed that PAG tyrosine phosphorylation and association with Csk are suppressed in response to activation of normal mouse T cells. By expressing wild-type and phosphorylation-defective (dominant-negative) PAG polypeptides in these cells, we found that the inhibitory effect of PAG is dependent on its capacity to be tyrosine phosphorylated and to associate with Csk. PAG-mediated inhibition was accompanied by a repression of proximal TCR signaling and was rescued by expression of a constitutively activated Src-related kinase, implying that it is due to an inactivation of Src kinases by PAG-associated Csk. We also attempted to identify the protein tyrosine phosphatases (PTPs) responsible for dephosphorylating PAG in T cells. Through cell fractionation studies and analyses of genetically modified mice, we established that PTPs such as PEP and SHP-1 are unlikely to be involved in the dephosphorylation of PAG in T cells. However, the transmembrane PTP CD45 seems to play an important role in this process. Taken together, these data provide firm evidence that PAG is a bona fide negative regulator of T-cell activation as a result of its capacity to recruit Csk. They also suggest that the inhibitory function of PAG in T cells is suppressed by CD45. Lastly, they support the idea that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation.     

Protein tyrosine phosphatase alpha regulates Fyn activity and Cbp/PAG phosphorylation in thymocyte lipid rafts

A role for the receptor protein tyrosine phosphatase alpha (PTPalpha) in immune cell function and regulation of Src family kinases was investigated using thymocytes from PTPalpha-deficient mice. PTPalpha-null thymocytes develop normally, but unstimulated PTPalpha-/- cells exhibit increased tyrosine phosphorylation of specific proteins, increased Fyn activity, and hyperphosphorylation of Cbp/PAG that promotes its association with C-terminal Src kinase. Elevated Fyn activity in the absence of PTPalpha is due to enhanced phosphorylation of Fyn tyrosines 528 and 417. Some PTPalpha is localized in lipid rafts of thymocytes, and raft-associated Fyn is specifically activated in PTPalpha-/- cells. PTPalpha is not a Cbp/PAG phosphatase, because it is not required for Cbp/PAG dephosphorylation in unstimulated or anti-CD3-stimulated thymocytes. Together, our results indicate that PTPalpha, likely located in lipid rafts, regulates the activity of raft Fyn. In the absence of PTPalpha this population of Fyn is activated and phosphorylates Cbp/PAG to enhance association with C-terminal Src kinase. Although TCR-mediated tyrosine phosphorylation was apparently unaffected by the absence of PTPalpha, the long-term proliferative response of PTPalpha-/- thymocytes was reduced. These findings indicate that PTPalpha is a component of the complex Src family tyrosine kinase regulatory network in thymocytes and is required to suppress Fyn activity in unstimulated cells in a manner that is not compensated for by the major T cell PTP and SFK regulator, CD45.     

Transmembrane phosphoprotein Cbp positively regulates the activity of the carboxyl-terminal Src kinase, Csk

Csk (carboxyl-terminal Src kinase) is a cytoplasmic tyrosine kinase that phosphorylates a critical tyrosine residue in each of the Src family kinases (SFKs) to inhibit their activities. Recently, we identified a transmembrane protein, Cbp (Csk-binding protein), that, when phosphorylated, can recruit Csk to the membrane where the SFKs are located. The Cbp-mediated relocation of Csk to the membrane may play a role in turning off the signaling events initiated by SFKs. To further characterize the Csk-Cbp interaction, we have generated a reconstituted system using soluble, highly purified proteins. Csk and phosphorylated Cbp were co-purified as a large protein complex consisting of at least four Csk.Cbp units. The addition of the phosphorylated, but not nonphosphorylated, Cbp to an in vitro assay stimulated Csk activity toward Src. Csk was also activated by a phosphopeptide containing the tyrosine in Cbp that binds to Csk (Tyr-314). Kinetic analysis revealed that Cbp or the phosphopeptide induced up to a 6-fold reduction in the K(m) for Src, indicating that the Csk.Cbp complex has a greater affinity for Src than free Csk. These findings suggest that Cbp is involved in the regulation of SFKs not only by relocating Csk to the membrane but also by directly activating Csk.     

Impairment in a negative regulatory system for TCR signaling in CD4+ T cells from old mice

To examine the involvement of lipid rafts in an age-associated decline in T cell function, we analyzed the effect of aging on the constituents of lipid rafts in resting mouse CD4(+) T cells. We found a pronounced, age-dependent reduction in PAG/Cbp, which is involved in the regulation of Src family kinases (SFKs) by recruiting Csk (a negative regulator of SFKs) to lipid rafts. This reduction is specific for T cells and is attributed, at least in part, to the reduction in its mRNA level. The reduction of PAG accompanies marked impairment in recruiting Csk to lipid rafts and a concomitant decrease in the inactive forms of SFKs. These findings indicate that old mouse CD4(+) T cells have a defect in a negative SFK regulatory system.     

Activation of c-Src and Fyn kinases by protein-tyrosine phosphatase RPTPalpha is substrate-specific and compatible with lipid raft localization

Src family tyrosine kinases (SFKs) function in multiple signaling pathways, raising the question of how appropriate regulation and substrate choice are achieved. SFK activity is modulated by several protein-tyrosine phosphatases, among which RPTPalpha and SHP2 are the best established. We studied how RPTPalpha affects substrate specificity and regulation of c-Src and Fyn in response to epidermal growth factor and platelet-derived growth factor. We find that RPTPalpha, in a growth factor-specific manner, directs the specificity of these kinases toward a specific subset of SFK substrates, particularly the focal adhesion protein Paxillin and the lipid raft scaffolding protein Cbp/PAG. A significant fraction of RPTPalpha is present in lipid rafts, where its targets Fyn and Cbp/PAG reside, and growth factor-mediated SFK activation within this compartment is strictly dependent on RPTPalpha. Forced concentration of RPTPalpha into lipid rafts is compatible with activation of Fyn. Finally, RPTPalpha-induced phosphorylation of Paxillin and Cbp/PAG induces recruitment of the SFK inhibitory kinase Csk, indicative of negative feedback loops limiting SFK activation by RPTPalpha. Our findings indicate that individual SFK-controlling PTPs play important and specific roles in dictating SFK substrate specificity and regulatory mechanism.     

The transmembrane adaptor Cbp/PAG1 controls the malignant potential of human non-small cell lung cancers that have c-src upregulation

The tyrosine kinase c-Src is upregulated in various human cancers, although the precise regulatory mechanism underlying this upregulation is unclear. We previously reported that a transmembrane adaptor Csk-binding protein (Cbp; PAG1) plays an important role in controlling the cell transformation that is induced by the activation of c-Src. To elucidate the in vivo role of Cbp, we examined the function of Cbp in lung cancer cell lines and tissues. In this study, we found that Cbp was markedly downregulated in human non-small cell lung cancer (NSCLC) cells. The ectopic expression of Cbp suppressed the anchorage-independent growth of the NSCLC cell lines (A549 and Lu99) that had upregulated c-Src, whereas the Cbp expression had little effect on other NSCLC cell lines (PC9 and Lu65) that express normal levels of c-Src. The expression of Cbp suppressed the kinase activity of c-Src in A549 cells by recruiting c-Src and its negative regulator, C-terminal Src kinase (Csk), to lipid rafts. The treatment with Src inhibitors, such as PP2, dasatinib, and saracatinib, also suppressed the growth of A549 cells. Furthermore, Cbp expression attenuated the ability of A549 cells to form tumors in nude mice, invade in vitro, and metastasize in vivo. In addition, we found a significant inverse correlation between the level of Cbp expression and the extent of lymph node metastasis in human lung cancers. These results indicate that Cbp is required for the Csk-mediated inactivation of c-Src and may control the promotion of malignancy in NSCLC tumors that are characterized by c-Src upregulation.     

Phosphoprotein associated with glycosphingolipid-enriched microdomains differentially modulates SRC kinase activity in brain maturation

Src family kinases (SFK) control multiple processes during brain development and function. We show here that the phosphoprotein associated with glycosphigolipid-enriched microdomains (PAG)/Csk binding protein (Cbp) modulates SFK activity in the brain. The timing and localization of PAG expression overlap with Fyn and Src, both of which we find associated to PAG. We demonstrate in newborn (P1) mice that PAG negatively regulates Src family kinases (SFK). P1 Pag1 ^-/- mouse brains show decreased recruitment of Csk into lipid rafts, reduced phosphorylation of the inhibitory tyrosines within SFKs, and an increase in SFK activity of >/ = 50%. While in brain of P1 mice, PAG and Csk are highly and ubiquitously expressed, little Csk is found in adult brain suggesting altered modes of SFK regulation. In adult brain Pag1-deficiency has no effect upon Csk-distribution or inhibitory tyrosine phosphorylation, but kinase activity is now reduced (−20–30%), pointing to the development of a compensatory mechanism that may involve PSD93. The distribution of the Csk-homologous kinase CHK is not altered. Importantly, since the activities of Fyn and Src are decreased in adult Pag1 ^-/- mice, thus presenting the reversed phenotype of P1, this provides the first in vivo evidence for a Csk-independent positive regulatory function for PAG in the brain.     

Csk-binding protein mediates sequential enzymatic down-regulation and degradation of Lyn in erythropoietin-stimulated cells

We have shown previously that the Src family kinase Lyn is involved in differentiation signals emanating from activated erythropoietin (Epo) receptors. The importance of Lyn to red cell maturation has been highlighted by Lyn-/- mice developing anemia. Here we show that Lyn interacts with C-terminal Src kinase-binding protein (Cbp), an adaptor protein that recruits negative regulators C-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk). Lyn phosphorylated Cbp on several tyrosine residues, including Tyr314, which recruited Csk/Ctk to suppress Lyn kinase activity. Intriguingly, phosphorylated Tyr314 also bound suppressor of cytokine signaling 1 (SOCS1), another well characterized negative regulator of cell signaling, resulting in elevated ubiquitination, and degradation of Lyn. In Epo-responsive primary cells and cell lines, Lyn rapidly phosphorylated Cbp, suppressing Lyn kinase activity via Csk/Ctk within minutes of Epo stimulation; hours later, SOCS1 bound to Cbp and was involved in the ubiquitination and turnover of Lyn protein. Thus, a single phosphotyrosine residue on Cbp coordinates a two-phase process involving distinct negative regulatory pathways to inactivate, then degrade, Lyn.     

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti‐ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C‐terminal src kinase)‐binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent‐resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti‐ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over‐expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314‐phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314‐phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP‐43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.     

Mechanism of Csk-mediated down-regulation of Src family tyrosine kinases in epidermal growth factor signaling

The Src family tyrosine kinases (SFKs) play pivotal roles as molecular switches that link a variety of extracellular cues to intracellular signaling pathway. The function of SFK is regulated by phosphorylation at the C-terminal regulatory site mediated by Csk. Recently a novel SFK target Cbp (or PAG) was identified as a membrane-anchored scaffold protein for Csk. To establish the mechanism of Csk/Cbp-mediated regulation of SFK in vivo, we observed dynamic changes in the interaction of Csk with Cbp by utilizing fusion proteins with modified green fluorescent proteins: cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). Upon SFK activation induced by epidermal growth factor stimulation, fluorescent resonance energy transfer (FRET) response was detected transiently at membrane ruffles in COS1 cells co-expressing CFP-Csk and Cbp-YFP and in cells expressing a single-molecule FRET indicator consisting of CskSH2 and Cbp. Suppression of SFK by PP2 or use of a mutant Cbp that lacks the Csk binding site abolished the FRET response, although a dominant-negative form of Csk enhanced and sustained the FRET response, demonstrating that the FRET response is dependent upon the SFK activity. These observations show that Csk/Cbp-mediated down-regulation of SFK takes place at membrane ruffles in an early stage of epidermal growth factor signaling and suggest that the Csk/Cbp-based FRET indicators are useful for monitoring the status of SFK in living cells.