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.     

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.     

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.     

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.     

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.     

Interactions between the Fyn SH3-domain and adaptor protein Cbp/PAG derived ligands, effects on kinase activity and affinity

Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched domains is a transmembrane adaptor protein primarily involved in negative regulation of T-cell activation by recruitment of C-terminal Src kinase (Csk), a protein tyrosine kinase which represses Src kinase activity through C-terminal phosphorylation. Recruitment of Csk occurs via SH2-domain binding to PAG pTyr317, thus, the interaction is highly dependent on phosphorylation performed by the Src family kinase Fyn, which docks onto PAG using a dual-domain binding mode involving both SH3- and SH2-domains of Fyn. In this study, we investigated Fyn SH3-domain binding to 14-mer peptide ligands derived from Cbp/PAG-enriched microdomains sequence using biochemical, biophysical and computational techniques. Interaction kinetics and dissociation constants for the various ligands were determined by SPR. The local structural impact of ligand association has been evaluated using CD, and molecular modelling has been employed to investigate details of the interactions. We show that data from these investigations correlate with functional effects of ligand binding, assessed experimentally by kinase assays using full-length PAG proteins as substrates. The presented data demonstrate a potential method for modulation of Src family kinase tyrosine phosphorylation through minor changes of the substrate SH3-interacting motif.     

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.     

Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.     

Domain interactions in protein tyrosine kinase Csk.

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in downregulation of Src family members. It is composed of three principal domains: an SH3 (Src homology 3) domain, an SH2 (Src homology 2) domain, and a catalytic domain. The impact of the noncatalytic domains on kinase catalysis was investigated. The Csk catalytic domain was expressed in Escherichia coli as a recombinant glutathione S-transferase-fusion protein and demonstrated to have 100-fold reduced catalytic efficiency. Production of the catalytic domain by proteolysis of full-length Csk afforded a similar rate reduction. This suggested that the reduction in catalytic efficiency of the recombinant catalytic domain was intrinsic to the sequence and not an artifact related to faulty expression. This rate reduction was similar for peptide and protein substrates and was due almost entirely to a reduced k(cat) rather than to effects on substrate K(m)s. Viscosity experiments on the catalytic fragment kinase reaction demonstrated that the chemical (phosphoryl transfer) step had a reduced rate. While the Csk SH2 domain had no intermolecular effect on the kinase activity of the Csk catalytic domain, the SH3 domain and SH3-SH2 fragment led to a partial rescue (4-5-fold) of the lost kinase activity. This rescue was not achieved with two other SH3 domains (lymphoid cell kinase, Abelson kinase). The extrapolated K(d) of interaction for the Csk catalytic domain with the Csk SH3 domain was 2.2 microM and that of the Csk catalytic domain with the Csk SH3-SH2 fragment was 8.8 microM. Taken together, these findings suggest that there is likely an intramolecular interaction between the catalytic and SH3 domains in full-length Csk that is important for efficient catalysis. By employing a Csk SH3 specific type II polyproline helix peptide and carrying out site-directed mutagenesis, it was established that the SH3 surface that interacts with the catalytic domain was distinct from the surface that binds type II polyproline helix peptides. This finding suggests a novel mode of protein-protein interaction for an SH3 domain. The implications for Csk substrate selectivity, regulation, and function are discussed.     

Functional diversity of Csk, Chk, and Src SH2 domains due to a single residue variation

The C-terminal Src kinase (Csk) family of protein tyrosine kinases contains two members: Csk and Csk homologous kinase (Chk). Both phosphorylate and inactivate Src family kinases. Recent reports suggest that the Src homology (SH) 2 domains of Csk and Chk may bind to different phosphoproteins, which provides a basis for different cellular functions for Csk and Chk. To verify and characterize such a functional divergence, we compared the binding properties of the Csk, Chk, and Src SH2 domains and investigated the structural basis for the functional divergence. First, the study demonstrated striking functional differences between the Csk and Chk SH2 domains and revealed functional similarities between the Chk and Src SH2 domains. Second, structural analysis and mutagenic studies revealed that the functional differences among the three SH2 domains were largely controlled by one residue, Glu127 in Csk, Ile167 in Chk, and Lys200 in Src. Mutating these residues in the Csk or Chk SH2 domain to the Src counterpart resulted in dramatic gain of function similar to Src SH2 domain, whereas mutating Lys200 in Src SH2 domain to Glu (the Csk counterpart) resulted in loss of Src SH2 function. Third, a single point mutation of E127K rendered Csk responsive to activation by a Src SH2 domain ligand. Finally, the optimal phosphopeptide sequence for the Chk SH2 domain was determined. These results provide a compelling explanation for the functional differences between two homologous protein tyrosine kinases and reveal a new structure-function relationship for the SH2 domains.     

Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets

SFKs (Src family kinases) contribute importantly to platelet function in haemostasis. SFK activity is controlled by Csk (C-terminal Src kinase), which phosphorylates a C-terminal tyrosine residue on SFKs, resulting in inhibition of SFK activity. Csk is recruited to sites of SFK activity by tyrosine-phosphorylated Csk-binding proteins. Paxillin, a multidomain adaptor protein, has been shown to act as a Csk-binding protein and to inhibit Src activity during growth factor signalling. Human platelets express Hic-5, a member of the paxillin family; however, its ability to act as a Csk-binding protein has not been characterized. We sought to identify and characterize the ability of paxillin family members to act as Csk-binding proteins during platelet activation. We found that murine and human platelets differ in the complement of paxillin family members expressed. Human platelets express Hic-5, whereas murine platelets express paxillin and leupaxin in addition to Hic-5. In aggregating human platelets, Hic-5 was tyrosine phosphorylated and recruited Csk via its SH2 domains. In aggregating murine platelets, however, Csk bound preferentially to paxillin, even though both paxillin and Hic-5 were abundantly present and became tyrosine phosphorylated. The SFK Lyn, but not Src or Fyn, was associated with paxillin family members in resting and aggregated human and murine platelets. Lyn, however, was phosphorylated on its C-terminal inhibitory tyrosine residue only following platelet aggregation, which was coincident with recruitment of Csk to paxillin and/or Hic-5 in a manner dependent on prior alpha(IIb)beta3 engagement. These observations support the notion that Hic-5 and paxillin function as negative feedback regulators of SFKs in aggregated platelets and that, when both are present, paxillin is preferentially used.     

Theoretical Insights Reveal Novel Motions in Csk’s SH3 Domain That Control Kinase Activation

The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD) simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS) and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk’s activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk.     

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.     

Association of inhibitory tyrosine protein kinase p50csk with protein tyrosine phosphatase PEP in T cells and other hemopoietic cells.

p50csk is a tyrosine protein kinase (TPK) that represses the activity of Src family TPKs. We previously showed that Csk is a potent negative regulator of antigen receptor signaling in T lymphocytes and that its Src homology (SH) 3 and SH2 domains are required to inhibit these signals. To test the idea that the Csk SH3 and SH2 domains mediate interactions with other cellular proteins, we attempted to identify Csk-associated polypeptides using the yeast two-hybrid system. The results of our experiments demonstrated that Csk physically associates with PEP, a protein tyrosine phosphatase (PTP) expressed in hemopoietic cells. Further analyses revealed that this interaction was mediated by the Csk SH3 domain and by a proline-rich region (PPPLPERTP) in the non-catalytic C-terminal portion of PEP. The association between Csk and PEP was documented in transiently transfected Cos-1 cells and in a variety of cells of hemopoietic lineages, including T cells. Additional analyses demonstrated that the association between Csk and PEP is highly specific. Together, these data indicated that PEP may be an effector and/or a regulator of p50csk in T cells and other hemopoietic cells. Moreover, they allowed the identification of PEP as the first known ligand for the Csk SH3 domain.     

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.     

Proteomic identification of ZO-1/2 as a novel scaffold for Src/Csk regulatory circuit

To elucidate the regulatory mechanism of cell transformation induced by c-Src tyrosine kinase, we performed a proteomic analysis of tyrosine phosphorylated proteins that interact with c-Src and/or its negative regulator Csk. The c-Src interacting proteins were affinity-purified from Src transformed cells using the Src SH2 domain as a ligand. LC-MS/MS analysis of the purified proteins identified general Src substrates, such as focal adhesion kinase and paxillin, and ZO-1/2 as a transformation-dependent Src target. The Csk binding proteins were analyzed by a tandem affinity purification method. In addition to the previously identified Csk binding proteins, including Cbp/PAG, paxillin, and caveolin-1, we found that ZO-1/2 could also serve as a major Csk binding protein. ZO-2 was phosphorylated concurrently with Src transformation and specifically bound to Csk in a Csk SH2 dependent manner. These results suggest novel roles for ZO proteins as Src/Csk scaffolds potentially involved in the regulation of Src transformation.     

Distal Loop Flexibility of a Regulatory Domain Modulates Dynamics and Activity of C-Terminal Src Kinase (Csk)

The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Csk and SFKs share a modular design with the kinase domain downstream of the N-terminal SH2 and SH3 domains that regulate catalytic function and membrane localization. While the function of interfacial segments in these multidomain kinases are well-investigated, little is known about how surface sites and long-range, allosteric coupling control protein dynamics and catalytic function. The SH2 domain of Csk is an essential component for the down-regulation of all SFKs. A unique feature of the SH2 domain of Csk is the tight turn in place of the canonical CD loop in a surface site far removed from kinase domain interactions. In this study, we used a combination of experimental and computational methods to probe the importance of this difference by constructing a Csk variant with a longer SH2 CD loop to mimic the flexibility found in homologous kinase SH2 domains. Our results indicate that while the fold and function of the isolated domain and the full-length kinase are not affected by loop elongation, native protein dynamics that are essential for efficient catalysis are perturbed. We also identify key motifs and routes through which the distal SH2 site might influence catalysis at the active site. This study underscores the sensitivity of intramolecular signaling and catalysis to native protein dynamics that arise from modest changes in allosteric regions while providing a potential strategy to alter intrinsic activity and signaling modulation.     

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.     

The C-terminus of CIS defines its interaction pattern

Proteins of the SOCS (suppressors of cytokine signalling) family are characterized by a conserved modular structure with pre-SH2 (Src homology 2), SH2 and SOCS-box domains. Several members, including CIS (cytokine-inducible SH2 protein), SOCS1 and SOCS3, are induced rapidly upon cytokine receptor activation and function in a negative-feedback loop, attenuating signalling at the receptor level. We used a recently developed mammalian two-hybrid system [MAPPIT (mammalian protein-protein interaction trap)] to analyse SOCS protein-interaction patterns in intact cells, allowing direct comparison with biological function. We find that, besides the SH2 domain, the C-terminal part of the CIS SOCS-box is required for functional interaction with the cytokine receptor motifs examined, but not with the N-terminal death domain of the TLR (Toll-like receptor) adaptor MyD88. Mutagenesis revealed that one single tyrosine residue at position 253 is a critical binding determinant. In contrast, substrate binding by the highly related SOCS2 protein, and also by SOCS1 and SOCS3, does not require their SOCS-box.     

Functional analysis of Csk in signal transduction through the B-cell antigen receptor.

In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.