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.     

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.     

Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets.

Chk/Hyl is a recently isolated non-receptor tyrosine kinase with greatest homology to a ubiquitous negative regulator of Src family kinases, Csk. To understand the significance of co-expression of Chk and Csk in platelets, we examined the subcellular localization of each protein. Chk, but not Csk, was completely translocated from the Triton X-100-soluble to the Triton X-100-insoluble cytoskeletal fraction within 10 s of thrombin stimulation. Chk and Lyn, but not Csk and c-Src, co-fractionated in the higher density lysate fractions of resting platelets, with Chk being found to localize close to CD36 (membrane glycoprotein IV)-anchored Lyn. The kinase activity of co-fractionated Lyn was suppressed 3-fold. In vitro phosphorylation assays showed that Chk suppressed Lyn activity by phosphorylating its C-terminal negative regulatory tyrosine. Upon stimulation of platelets with thrombin, the rapid and complete translocation of Chk away from Lyn caused concomitant activation of Lyn. This activation was accompanied by dephosphorylation of Lyn at its C-terminal negative regulatory tyrosine in cooperation with a protein tyrosine phosphatase. These results suggest that Chk, but not Csk, may function as a translocation-controlled negative regulator of CD36-anchored Lyn in thrombin-induced platelet activation.     

HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction

Nef is an HIV-1 virulence factor that promotes viral pathogenicity by altering host cell signaling pathways. Nef binds several members of the Src kinase family, and these interactions have been implicated in the pathogenesis of HIV/AIDS. However, the direct effect of Nef interaction on Src family kinase (SFK) regulation and activity has not been systematically addressed. We explored this issue using Saccharomyces cerevisiae, a well defined model system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk suppressed SFK activity and reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and c-Src but did not detectably affect Fgr, Fyn, Lck, or Yes. Mutagenesis of the Nef PXXP motif essential for SH3 domain binding greatly reduced the effect of Nef on Hck, Lyn, and c-Src, suggesting that Nef activates these Src family members through allosteric displacement of intramolecular SH3-linker interactions. These data show that Nef selectively activates Hck, Lyn, and c-Src among SFKs, identifying these kinases as proximal effectors of Nef signaling and potential targets for anti-HIV drug discovery.     

A novel non-catalytic mechanism employed by the C-terminal Src-homologous kinase to inhibit Src-family kinase activity

Although C-terminal Src kinase (CSK)-homologous kinase (CHK) is generally believed to inactivate Src-family tyrosine kinases (SFKs) by phosphorylating their consensus C-terminal regulatory tyrosine (Tyr(T)), exactly how CHK inactivates SFKs is not fully understood. Herein, we report that in addition to phosphorylating Tyr(T), CHK can inhibit SFKs by a novel non-catalytic mechanism. First, CHK directly binds to the SFK members Hck, Lyn, and Src to form stable protein complexes. The complex formation is mediated by a non-catalytic Tyr(T)-independent mechanism because it occurs even in the absence of ATP or when Tyr(T) of Hck is replaced by phenylalanine. Second, the non-catalytic CHK-SFK interaction alone is sufficient to inactivate SFKs by inhibiting the catalytic activity of autophosphorylated SFKs. Third, CHK and Src co-localize to specific plasma membrane microdomains of rat brain cells, suggesting that CHK is in close proximity to Src such that it can effectively inactivate Src in vivo. Fourth, native CHK.Src complex exists in rat brain, and recombinant CHK.Hck complex exists in transfected HEK293T cells, implying that CHK forms stable complexes with SFKs in vivo. Taken together, our findings suggest that CHK inactivates SFKs (i) by phosphorylating their Tyr(T) and (ii) by this novel Tyr(T)-independent mechanism involving direct binding of CHK to SFKs. It has been documented that autophosphorylated SFKs can still be active, in some cases even when their Tyr(T) is phosphorylated. Thus, the ability of the Tyr(T)-independent mechanism to suppress the activity of both non-phosphorylated and autophosphorylated SFKs represents a fail-safe measure employed by CHK to down-regulate SFK signaling under all circumstances.     

Activation of Src family tyrosine kinases by ferric ions

The Src-family tyrosine kinases (SFKs) are oncogenic enzymes that contribute to the initiation and progression of many types of cancer. In normal cells, SFKs are kept in an inactive state mainly by phosphorylation of a consensus regulatory tyrosine near the C-terminus (Tyr⁵³⁰ in the SFK c-Src). As recent data indicate that tyrosine modification enhances binding of metal ions, the hypothesis that SFKs might be regulated by metal ions was investigated. The c-Src C-terminal peptide bound two Fe^3 + ions with affinities at pH 4.0 of 33 and 252 μM, and phosphorylation increased the affinities at least 10-fold to 1.4 and 23 μM, as measured by absorbance spectroscopy. The corresponding phosphorylated peptide from the SFK Lyn bound two Fe^3 + ions with much higher affinities (1.2 pM and 160 nM) than the Src C-terminal peptide. Furthermore, when Lyn or Hck kinases, which had been stabilised in the inactive state by phosphorylation of the C-terminal regulatory tyrosine, were incubated with Fe^3 + ions, a significant enhancement of kinase activity was observed. In contrast Lyn or Hck kinases in the unphosphorylated active state were significantly inhibited by Fe^3 + ions. These results suggest that Fe^3 + ions can regulate SFK activity by binding to the phosphorylated C-terminal regulatory tyrosine.     

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.     

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.     

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.     

Oxidative stress activates both Src-kinases and their negative regulator Csk and induces phosphorylation of two targeting proteins for Csk: caveolin-1 and paxillin

Csk negatively regulates Src family kinases (SFKs). In lymphocytes, Csk is constitutively active, and is transiently inactivated in response to extracellular stimuli, allowing activation of SFKs. In contrast, both SFKs and Csk were inactive in unstimulated mouse embryonic fibroblasts, and both were activated in response to oxidative stress. Csk modulated the oxidative stress-induced, but not the basal SFK activity in these cells. These data indicate that Csk may be more important for the return of Src-kinases to the basal state than for the maintenance of basal activity in some cell types. Csk must be targeted to its SFK substrates through an SH2-domain-mediated interaction with a phosphoprotein. Our data indicate that caveolin-1 is one of these targeting proteins. SFKs bind to caveolin-1 and phosphorylate it in response to oxidative stress and insulin. Csk binds specifically to the phosphorylated caveolin-1 and attenuates its stress-induced phosphorylation. Importantly, phosphocaveolin was one of two major phosphoproteins associated with Csk after incubation with peroxide or insulin. Paxillin was the other. Activation/rapid attenuation of SFKs by Csk is required for actin remodeling. Caveolin-1 is phosphorylated at the ends of actin fibers at points of contact between the actin cytoskeleton and the plasma membrane, where it could in part mediate this attenuation.     

Proteomic,functional and motif‐based analysis of C‐terminal Src kinase‐interacting proteins

C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.     

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.     

Signalling through Src family kinase isoforms is not redundant in models of thrombo‐inflammatory vascular disease

The Src family kinases (SFK) are a group of signalling molecules with important regulatory functions in inflammation and haemostasis. Leucocytes and platelets express multiple isoforms of the SFKs. Previous studies used broad‐spectrum pharmacological inhibitors, or murine models deficient in multiple SFK isoforms, to demonstrate the functional consequences of deficiencies in SFK signalling. Here, we hypothesized that individual SFK operate in a non‐redundant fashion in the thrombo‐inflammatory recruitment of monocyte during atherosclerosis. Using in vitro adhesion assays and single SFK knockout mice crossed with the ApoE^?/? model of atherosclerosis, we find that SFK signalling regulates platelet‐dependent recruitment of monocytes. However, loss of a single SFK, Fgr or Lyn, reduced platelet‐mediated monocyte recruitment in vitro. This translated into a significant reduction in the burden of atherosclerotic disease in Fgr^?/?/ApoE^?/? or Lyn^?/?/ApoE^?/? animals. SFK signalling is not redundant in thrombo‐inflammatory vascular disease and individual SFK may represent targets for therapeutic intervention.     

The regulation of N-methyl-D-aspartate receptors by Src kinase

Src family kinases (SFKs) play critical roles in the regulation of many cellular functions by growth factors, G-protein-coupled receptors and ligand-gated ion channels. Recent data have shown that SFKs serve as a convergent point of multiple signaling pathways regulating N-methyl-d-aspartate (NMDA) receptors in the central nervous system. Multiple SFK molecules, such as Src and Fyn, closely associate with their substrate, NMDA receptors, via indirect and direct binding mechanisms. The NMDA receptor is associated with an SFK signaling complex consisting of SFKs; the SFK-activating phosphatase, protein tyrosine phosphatase α; and the SFK-inactivating kinase, C-terminal Src kinase. Early studies have demonstrated that intramolecular interactions with the SH2 or SH3 domain lock SFKs in a closed conformation. Disruption of the interdomain interactions can induce the activation of SFKs with multiple signaling pathways involved in regulation of this process. The enzyme activity of SFKs appears 'graded', exhibiting different levels coinciding with activation states. It has also been proposed that the SH2 and SH3 domains may stimulate catalytic activity of protein tyrosine kinases, such as Abl. Recently, it has been found that the enzyme activity of neuronal Src protein is associated with its stability, and that the SH2 and SH3 domain interactions may act not only to constrain the activation of neuronal Src, but also to regulate the enzyme activity of active neuronal Src. Collectively, these findings demonstrate novel mechanisms underlying the regulation of SFKs.     

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.     

Mitochondrial processing peptidase: multiple-site recognition of precursor proteins

The Csk Homologous Kinase (CHK) has been shown to have an enzymatic activity similar to the tyrosine kinase Csk in that it down-regulates Src family kinase activity by causing phosphorylation of the Src C-terminal tyrosine residue. In megakaryocytic Mo7e cells, CHK associates with a specific phosphotyrosine juxtamembrane sequence of the SCF/KL-activated c-Kit receptor. Here, we show that in Mo7e cells, the major Src family kinase activity is p53/56(Lyn). Studies using immobilized c-Kit phosphopeptides show that Lyn is able to specifically associate with the tyrosine-phosphorylated juxtamembrane 568Y*VY*IDPT sequence of c-Kit which has previously been shown to associate with CHK. In cells over-expressing CHK by means of a recombinant vaccinia virus, we observed an elimination of the SCF/KL-stimulated Lyn kinase peak of activity observed at 2-5 minutes in cells infected with the helper T7-expressing vaccinia virus by itself. Examination of total tyrosine phosphorylation by Western blotting showed that over-expression of CHK resulted in a reduction in the levels of tyrosine phosphorylations in the range of 50-60 kDa, but had no apparent effect on c-Kit autophosphorylation. Taken together, these findings show that CHK is able to down-regulate SCF/KL-stimulated Lyn activity in megakaryocytes.     

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.     

Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development

Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling. Here, we report that the Src family kinases (SFKs) Fyn and Lyn regulate each of these distinct integrin-driven behaviors. Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation). However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation. The two SFKs respond to integrin ligation by different mechanisms: Lyn, by increased autophosphorylation of a catalytic tyrosine; and Fyn, by reduced Csk phosphorylation of the inhibitory COOH-terminal tyrosine. These findings illustrate how different SFKs can act as effectors for specific cell responses during development within a single cell lineage, and, furthermore, provide a molecular mechanism to explain similar region-specific hypomyelination in laminin- and Fyn-deficient mice.     

Inhibition of Lck: evidence for a novel natural Src family kinase inhibitor

Src family kinase (SFK) is a family of protein tyrosine kinases that play important roles in the development of various cancers. Here, we showed that a naturally occurring inhibitory factor of SFK can be extracted from the rat brain. This inhibitor strongly suppressed the activity of SFKs including Lck and Fyn. It did not inhibit other protein tyrosine kinases including Wee1 or serine/threonine kinases Mst2, Cdk5/p25, Cdk5/p35, and Cdk2/cyclin A. The inhibitor was not an ATPase, a phosphatase that dephosphorylates substrates of the SFK reaction, or a protease that degrades SFKs. Activity of mutant Lck with C-terminal tyrosine substituted with phenylalanine was also suppressed by the inhibitor to a similar extent of wild-type Lck, indicating that the inhibitor was not CSK. Gel filtration chromatography indicated that the molecular size of the prevalent form of this inhibitor was approximately 44 kDa.     

C-terminal Src kinase-homologous kinase (CHK), a unique inhibitor inactivating multiple active conformations of Src family tyrosine kinases

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.