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.     

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.     

Coordinated traffic of Grb2 and Ras during epidermal growth factor receptor endocytosis visualized in living cells

Activation of the epidermal growth factor receptor (EGFR) triggers multiple signaling pathways and rapid endocytosis of the epidermal growth factor (EGF)-receptor complexes. To directly visualize the compartmentalization of molecules involved in the major signaling cascade, activation of Ras GTPase, we constructed fusions of Grb2, Shc, H-Ras, and K-Ras with enhanced cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), and used live-cell fluorescence imaging microscopy combined with the fluorescence resonance energy transfer (FRET) technique. Stimulation of cells by EGF resulted in the accumulation of large pools of Grb2-CFP and YFP-Shc in endosomes, where these two adaptor proteins formed a complex with EGFR. H-Ras and K-Ras fusion proteins were found at the plasma membrane, particularly in ruffles and lamellipodia, and also in endosomes independently of GTP/GDP loading and EGF stimulation. The relative amount of endosomal H-Ras was higher than that of K-Ras, whereas K-Ras predominated at the plasma membrane. On application of EGF, Grb2, and Ras converge in the same endosomes through the fusion of endosomes containing either Grb2 or Ras or through the joint internalization of two proteins from the plasma membrane. To examine the localization of the GTP-bound form of Ras, we used a FRET assay that exploits the specific interaction of GTP-bound CFP-Ras with the YFP-fused Ras binding domain of c-Raf. FRET microscopy revealed that GTP-bound Ras is located at the plasma membrane, mainly in ruffles and at the cell edges, as well as in endosomes containing EGFR. These data point to the potential for endosomes to serve as sites of generation for persistent signaling through Ras.     

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.     

Src family kinases: regulation of their activities, levels and identification of new pathways

While the Src family of protein tyrosine kinases (SFK), and the main ancillary molecules involved in their regulation, have been studied for many years, the details of their interplay are not fully understood and thus remain under active investigation. Additionally, new players that coordinate their regulation and direct their signalling cascades are also being uncovered, shedding new light on the complexity of these signalling networks. Through the utilization of novel interaction assays, several new interconnecting mediators that are helping to show the elegance of Src family kinase regulation have been discovered. This review outlines SFK regulation, the discovery of the Csk binding protein (Phosphoprotein Associated with Glycosphingolipid-enriched microdomains, Cbp/PAG), and its role in regulating SFK kinase activity status, as well as protein levels. Further, details of the methods used to identify this dual mode of regulation can be applied to delineate the full gamut of SH2/SH3-directed SFK pathways and, indeed, those of any tyrosine kinase. Using Lyn as a model SFK, we and others have shown that Cbp recruits negative regulators of COOH-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk) after Lyn is activated and bound to Cbp. Lyn phosphorylates Cbp on multiple tyrosine residues, including two that can bind Lyn's SH2 domain with high affinity. Lyn also phosphorylates Y314, which recruits Csk/Ctk to phosphorylate Lyn at its Y508 negative site, allowing an inactive conformation to form. However, the pY508 site has a low affinity for Lyn's SH2 domain, while the Cbp sites have high affinity. Thus, until these Cbp sites are dephosphorylated, Lyn can remain active. Intriguingly, phosphorylated Y314 also binds the suppressor of cytokine signalling 1 (SOCS1), resulting in elevated ubiquitination and degradation of Lyn. Thus, a single phosphotyrosine residue within Cbp co-ordinates a two-phase process involving distinct negative regulatory pathways that allow inactivation, followed by degradation, of SFKs.     

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.     

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.     

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.     

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn.     

Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility.     

Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy

The interaction of activated epidermal growth factor receptor (EGFR) with the Src homology 2 (SH2) domain of the growth-factor-receptor binding protein Grb2 initiates signaling through Ras and mitogen-activated protein kinase (MAP kinase) [1,2]. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyze the spatiotemporal regulation of EGFR-Grb2 interactions in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) and Grb2 fused to yellow fluorescent protein (YFP). Efficient energy transfer between CFP and YFP should only occur if CFP and YFP are less than 50A apart, which requires direct interaction of the EGFR and Grb2 fused to these fluorescent moieties [3]. Stimulation by EGF resulted in the recruitment of Grb2-YFP to cellular compartments that contained EGFR-CFP and a large increase in FRET signal amplitude. In particular, FRET measurements indicated that activated EGFR-CFP interacted with Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signaling via EGFRs can occur in the endosomal compartment. The work also highlights the potential of FRET microscopy in the study of subcellular compartmentalization of protein-protein interactions in living cells.     

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.     

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.     

Regulation of the Src Family Kinases by Csk

The non-receptor tyrosine kinase Csk serves as an indispensable negative regulator of the Src family tyrosine kinases (SFKs) by specifically phosphorylating the negative regulatory site of SFKs, thereby suppressing their oncogenic potential. Csk is primarily regulated through its SH2 domain, which is required for membrane translocation of Csk via binding to scaffold proteins such as Cbp/PAG1. The binding of scaffolds to the SH2 domain can also upregulate Csk kinase activity. These regulatory features have been elucidated by analyses of Csk structure at the atomic levels. Although Csk itself may not be mutated in human cancers, perturbation of the regulatory system consisting of Csk, Cbp/PAG1, or other scaffolds, and certain tyrosine phosphatases may explain the upregulation of SFKs frequently observed in human cancers. This review focuses on the molecular bases for the function, structure, and regulation of Csk as a unique regulatory tyrosine kinase for SFKs.     

Transforming Potential of Src Family Kinases Is Limited by the Cholesterol-Enriched Membrane Microdomain

The upregulation of Src family kinases (SFKs) has been implicated in cancer progression, but the molecular mechanisms regulating their transforming potentials remain unclear. Here we show that the transforming ability of all SFK members is suppressed by being distributed to the cholesterol-enriched membrane microdomain. All SFKs could induce cell transformation when overexpressed in C-terminal Src kinase (Csk)-deficient fibroblasts. However, their transforming abilities varied depending on their affinity for the microdomain. c-Src and Blk, with a weak affinity for the microdomain due to a single myristate modification at the N terminus, could efficiently induce cell transformation, whereas SFKs with both myristate and palmitate modifications were preferentially distributed to the microdomain and required higher doses of protein expression to induce transformation. In contrast, disruption of the microdomain by depleting cholesterol could induce a robust transformation in Csk-deficient fibroblasts in which only a limited amount of activated SFKs was expressed. Conversely, the addition of cholesterol or recruitment of activated SFKs to the microdomain via a transmembrane adaptor, Cbp/PAG1, efficiently suppressed SFK-induced cell transformation. These findings suggest that the membrane microdomain spatially limits the transforming potential of SFKs by sequestering them away from the transforming pathways.Src family kinases (SFKs) are membrane-associated, non-receptor protein tyrosine kinases involved in a variety of intracellular signaling pathways (5). SFKs are comprised of eight members in mammals: c-Src, Fyn, c-Yes, Lyn, Lck, Hck, c-Fgr and Blk. Among these, c-Src, Fyn, and c-Yes are ubiquitously expressed, whereas the others are relatively concentrated in hematopoietic cell lineages. The intracellular distribution of each SFK also varies depending on their unique N-terminal sequences and acyl modifications (5, 27). These distinctive features of SFKs suggest that each SFK member plays a unique role in particular tissues or cells. In contrast, it is also known that SFKs have redundant and pleiotropic functions in regulating critical cellular events, such as cell division, motility, adhesion, angiogenesis, and survival (26). In a variety of human cancers, protein levels and/or specific activities of c-Src and c-Yes are frequently upregulated (13, 35). Upregulation of Lyn, Lck, Hck, c-Fgr, or Blk is also observed in some leukemias and lymphomas (10, 16, 26). These observations imply a role for SFKs in cell transformation, tumorigenesis, and metastasis (31). However, because SFK genes are rarely mutated in human cancers (31), the mechanisms underlying their upregulation in these cancers remain unclear. Furthermore, the distinctive expression patterns and functional redundancy among SFK members have hampered concurrent analyses of their intrinsic transforming abilities and contribution to cancer progression.In normal cells, the kinase activity of SFKs is negatively regulated by the phosphorylation of its C-terminal regulatory Tyr residue by C-terminal Src kinase (Csk) (21, 22). The cytoplasmic Csk requires Csk-binding scaffold proteins to gain efficient access to membrane-bound SFKs. Previously, we identified a transmembrane adaptor protein, Cbp (also known as PAG1), as a specific Csk-binding protein. Cbp/PAG1 is exclusively localized to a membrane microdomain enriched by cholesterol and sphingolipids and plays a scaffolding role for Cbp/PAG1 in Csk-mediated negative regulation of SFKs (3, 15). We also reported that expression of Cbp/PAG1 is noticeably downregulated by c-Src transformation and in some human cancer cells and that reexpression of Cbp/PAG1 can suppress c-Src-induced transformation and tumorigenesis (23). In addition, we showed that Cbp/PAG1 suppressed c-Src function independently of Csk by directly sequestering activated c-Src in the membrane microdomain. These findings suggest a potential role for Cbp/PAG1 as a suppressor for c-Src-mediated cancer progression. However, whether Cbp/PAG1 would serve as a suppressor for other SFK members and whether other microdomain adaptors, such as LIME (4, 11), would also contribute to the suppression of SFK-mediated transformation have yet to be examined.The membrane microdomain has been regarded as a signaling platform that harbors various signaling molecules and positively transduces cell signaling evoked by activated receptors (29). This model has been best exemplified in immunoreceptor-mediated signaling (8). Moreover, it was reported that SFKs could function positively when bound to Cbp/PAG1 in the microdomain (30, 32). Such positive roles of the microdomain in cell signaling are apparently inconsistent with its suppressive role in Src-mediated transformation. However, this discrepancy rather raises the possibility that the membrane microdomain would function to segregate or protect the normal signaling pathway from the transforming pathways. To prove this hypothesis, more extensive analysis of the role of the membrane microdomain in controlling cell transformation remains to be performed (28).To elucidate the role of the membrane microdomain in regulating the functions of SFKs, we first compared the transforming abilities of all SFK members using Csk-deficient cells, a reconstitution system in which wild-type SFKs can induce cell transformation (24), and we evaluated the relevance of the membrane distribution of SFKs to their transforming activities. We then investigated the role of the microdomain by disrupting or enhancing its function using methyl-β-cyclodextrin (MβCD) and a microdomain-specific adaptor, Cbp/PAG1, respectively. Our results show that the membrane microdomain and Cbp/PAG1 spatially limit the oncogenic potential of SFKs by sequestering them away from the transforming pathways.     

The Csk-binding protein PAG regulates PDGF-induced Src mitogenic signaling via GM1

Spatial regulation is an important feature of signal specificity elicited by cytoplasmic tyrosine kinases of the Src family (SRC family protein tyrosine kinases [SFK]). Cholesterol-enriched membrane domains, such as caveolae, regulate association of SFK with the platelet-derived growth factor receptor (PDGFR), which is needed for kinase activation and mitogenic signaling. PAG, a ubiquitously expressed member of the transmembrane adaptor protein family, is known to negatively regulate SFK signaling though binding to Csk. We report that PAG modulates PDGFR levels in caveolae and SFK mitogenic signaling through a Csk-independent mechanism. Regulation of SFK mitogenic activity by PAG requires the first N-terminal 97 aa (PAG-N), which include the extracellular and transmembrane domains, palmitoylation sites, and a short cytoplasmic sequence. We also show that PAG-N increases ganglioside GM1 levels at the cell surface and, thus, displaces PDGFR from caveolae, a process that requires the ganglioside-specific sialidase Neu-3. In conclusion, PAG regulates PDGFR membrane partitioning and SFK mitogenic signaling by modulating GM1 levels within caveolae independently from Csk.     

Csk-binding protein can regulate Lyn signals controlling cell morphology

The Src family kinase Lyn is involved in differentiation signals emanating from activated erythropoietin (Epo) receptors, it interacts with COOH-terminal Src kinase-binding protein (Cbp), an adaptor protein that recruits negative regulators COOH-terminal Src kinase (Csk) and suppressor of cytokine signaling-1 (SOCS1). Lyn phosphorylates Cbp on several tyrosine residues, including Tyr314, which recruits Csk/SOCS1, as well as Tyr381 and Tyr409 that bind Lyns own SH2 domain. We show that Cbp alters not only the ability of erythroid cells to differentiate but also their colony morphology. Consequently, we detailed the ability of Cbp to interact with and influence Lyns ability to initiate changes in cellular architecture, which affect cell–cell and cell–substratum interactions. Over-expression of active Lyn promotes filopodia formation while inactive Lyn promotes lamellipodia formation. Conversely, Cbp over-expression, which inhibits Lyn activity, promotes lamellipodia formation, while Cbp mutants preventing its interaction/signaling consequently allow Lyn to promote filopodia formation. Thus, the Lyn–Cbp pathway and subsequent regulation of Lyn signaling and cell morphology involves a dynamic and complex series of interactions.     

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.     

The effects of membrane compartmentalization of csk on TCR signaling

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described “heavy rafts” or non-raft membrane. Our data show that only Csk targeted into “classical” raft but not to “heavy raft” or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.     

Dok-R mediates attenuation of epidermal growth factor-dependent mitogen-activated protein kinase and Akt activation through processive recruitment of c-Src and Csk

Dok-R has previously been shown to associate with the epidermal growth factor receptor (EGFR) and become tyrosine phosphorylated in response to EGF stimulation. The recruitment of Dok-R to the EGFR, which is mediated through its phosphotyrosine binding (PTB) domain, results in attenuation of mitogen-activated protein kinase (MAPK) activation. Dok-R's ability to attenuate EGF-driven MAPK activation is independent of its ability to recruit rasGAP, a known attenuator of MAPK activity, suggesting an alternate Dok-R-mediated pathway. Herein, we have determined the structural determinants within Dok-R that are required for its ability to attenuate EGF signaling and to associate with c-Src and with the Src family kinase (SFK)-inhibitory kinase, Csk. We demonstrate that Dok-R associates constitutively with c-Src through an SH3-dependent interaction and that this association is essential to Dok-R's ability to attenuate c-Src activity and diminish MAPK and Akt/PKB activity. We further illustrate that EGF-dependent phosphorylation of Dok-R requires SFK activity and, more specifically, that SFK-dependent phosphorylation of tyrosine 402 on Dok-R facilitates the inducible recruitment of Csk. We propose that recruitment of Csk to Dok-R serves to bring Csk to c-Src and down-regulate its activity, resulting in a concomitant attenuation of MAPK and Akt/PKB activity. Furthermore, we demonstrate that Dok-R can abrogate c-Src's ability to protect the breast cancer cell line SKBR3 from anoikis and that an association with c-Src and Csk is required for this activity. Collectively these results demonstrate that Dok-R acts as an EGFR-recruited scaffolding molecule that processively assembles c-Src and Csk to attenuate signaling from the EGFR.