The role of c-Src in platelet-derived growth factor alpha receptor internalization

We used two approaches to investigate the role of Src family kinases (SFKs) in ligand-stimulated internalization of the platelet-derived growth factor alpha receptor (alphaPDGFR). In cells that normally express SFKs, the internalization rate of the F72/74 mutant alphaPDGFR (which is unable to recruit or activate SFKs) was slower than that of the wild-type (WT) alphaPDGFR. When expressed in cells lacking SFKs (SYF cells), internalization of the WT and mutant receptors was indistinguishable, which indicated that there was not an inherent defect in the mutant receptor's ability to undergo ligand-driven internalization. The internalization difference between the WT and mutant receptors was seen again when c-Src was expressed in the SYF cells. Surprisingly, c-Src slowed the internalization of the mutant receptor but had little effect on WT receptor. We propose the following working hypothesis to explain these findings. In resting cells SFKs suppress internalization of the alphaPDGFR by phosphorylating a hypothetical protein X. This suppression is relieved when cells are exposed to PDGF because SFKs are recruited to the activated WT alphaPDGFR and thereby no longer actively phosphorylate protein X. The internalization of the mutant receptor is slow because it is unable to recruit SFKs and thereby fails to relieve the suppression of internalization. Our findings suggest a role for SFKs in regulating the permissiveness for internalization of the alphaPDGFR.     

A juxtamembrane tyrosine in the colony stimulating factor-1 receptor regulates ligand-induced Src association, receptor kinase function, and down-regulation

Recent literature implicates a regulatory function of the juxtamembrane domain (JMD) in receptor tyrosine kinases. Mutations in the JMD of c-Kit and Flt3 are associated with gastrointestinal stromal tumors and acute myeloid leukemias, respectively. Additionally, autophosphorylated Tyr559 in the JMD of the colony stimulating factor-1 (CSF-1) receptor (CSF-1R) binds to Src family kinases (SFKs). To investigate SFK function in CSF-1 signaling we established stable 32D myeloid cell lines expressing CSF-1Rs with mutated SFK binding sites (Tyr559-TFI). Whereas binding to I562S was not significantly perturbed, Y559F and Y559D exhibited markedly decreased CSF-1-dependent SFK association. All JMD mutants retained intrinsic kinase activity, but Y559F, and less so Y559D, showed dramatically reduced CSF-1-induced autophosphorylation. CSF-1-mediated wild-type (WT)-CSF-1R phosphorylation was not markedly affected by SFK inhibition, indicating that lack of SFK binding is not responsible for diminished Y559F phosphorylation. Unexpectedly, cells expressing Y559F were hyperproliferative in response to CSF-1. Hyperproliferation correlated with prolonged activation of Akt, ERK, and Stat5 in the Y559F mutant. Consistent with a defect in receptor negative regulation, c-Cbl tyrosine phosphorylation and CSF-1R/c-Cbl co-association were almost undetectable in the Y559F mutant. Furthermore, Y559F underwent reduced multiubiquitination and delayed receptor internalization and degradation. In conclusion, we propose that Tyr559 is a switch residue that functions in kinase regulation, signal transduction and, indirectly, receptor down-regulation. These findings may have implications for the oncogenic conversion of c-Kit and Flt3 with JMD mutations.     

A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.     

Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases

Src family kinases (SFKs) are abundant in chromaffin cells that reside in the adrenal medulla and respond to cholinergic stimulation by secreting catecholamines. Our previous work indicated that SFKs regulate acetylcholine- or nicotine-induced secretion, but the site of modulatory action was unclear. Using whole cell recordings, we found that inhibition of SFK tyrosine kinase activity by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine) treatment or expression of a kinase-defective c-Src reduced the peak amplitude of nicotine-induced currents in chromaffin cells or in human embryonic kidney cells ectopically expressing functional neuronal alpha3beta4alpha5 acetylcholine receptors (AChRs). Conversely, the phosphotyrosine phosphatase inhibitor, sodium vanadate, or expression of mutationally activated c-Src resulted in enhanced current amplitudes. These results suggest that SFKs and putative phosphotyrosine phosphatases regulate the activity of AChRs by opposing actions. This proposed model was supported further by the findings that SFKs physically associate with the receptor and that the AChR is tyrosine-phosphorylated.     

A mechanism for SRC kinase-dependent signaling by noncatalytic receptors

A fundamental issue in cell biology is how signals are transmitted across membranes. A variety of transmembrane receptors, including multichain immune recognition receptors, lack catalytic activity and require Src family kinases (SFKs) for signal transduction. However, many receptors only bind and activate SFKs after ligand-induced receptor dimerization. This presents a conundrum: How do SFKs sense the dimerization of receptors to which they are not already bound? Most proposals for resolving this enigma invoke additional players, such as lipid rafts or receptor conformational changes. Here we used simple thermodynamics to show that SFK activation is a natural outcome of clustering of receptors with SFK phosphorylation sites, provided that there is phosphorylation-dependent receptor-SFK association and an SFK bound to one receptor can phosphorylate the second receptor or its associated SFK in a dimer. A simple system of receptor, SFK, and an unregulated protein tyrosine phosphatase (PTP) can account for ligand-induced changes in phosphorylation observed in cells. We suggest that a core signaling system comprising a receptor with SFK phosphorylation sites, an SFK, and an unregulated PTP provides a robust mechanism for transmembrane signal transduction. Other events that regulate signaling in specific cases may have evolved for fine-tuning of this basic mechanism.     

Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.     

Phosphorylation of tyrosine 720 in the platelet-derived growth factor alpha receptor is required for binding of Grb2 and SHP-2 but not for activation of Ras or cell proliferation.

Following binding of platelet-derived growth factor (PDGF), the PDGF alpha receptor (alphaPDGFR) becomes tyrosine phosphorylated and associates with a number of signal transduction molecules, including phospholipase Cgamma-1 (PLCgamma-1), phosphatidylinositol 3-kinase (PI3K), the phosphotyrosine phosphatase SHP-2, Grb2, and Src. Here, we present data identifying a novel phosphorylation site in the kinase insert domain of the alphaPDGFR at tyrosine (Y) 720. We replaced this residue with phenylalanine and expressed the mutated receptor (F720) in Patch fibroblasts that do not express the alphaPDGFR. Characterization of the F720 mutant indicated that binding of two proteins, SHP-2 and Grb2, was severely impaired, whereas PLCgamma-1 and PI3K associated to wild-type levels. In addition, mutating Y720 to phenylalanine dramatically reduced PDGF-dependent tyrosine phosphorylation of SHP-2. Since Y720 was required for recruitment of two proteins, we investigated the mechanism by which these two proteins associated with the alphaPDGFR. SHP-2 bound the alphaPDGFR directly, whereas Grb2 associated indirectly, most probably via SHP-2, as Grb2 and SHP-2 coimmunoprecipitated when SHP-2 was tyrosine phosphorylated. We also compared the ability of the wild-type and F720 alphaPDGFRs to mediate a number of downstream events. Preventing the alphaPDGFR from recruiting SHP-2 and Grb2 did not compromise PDGF-AA-induced activation of Ras, initiation of DNA synthesis, or growth of cells in soft agar. We conclude that phosphorylation of the alphaPDGFR at Y720 is required for association of SHP-2 and Grb2 and tyrosine phosphorylation of SHP-2; however, these events are not required for the alphaPDGFR to activate Ras or initiate a proliferative response. In addition, these findings reveal that while SHP-2 binds to both of the receptors, it binds in different locations: to the carboxy terminus of the betaPDGFR but to the kinase insert of the alphaPDGFR.     

Integrin-induced tyrosine phosphorylation of protein-tyrosine phosphatase-alpha is required for cytoskeletal reorganization and cell migration

Protein-tyrosine phosphatase-alpha (PTPalpha) activates Src family kinases (SFKs) to promote the integrin-stimulated early autophosphorylation of focal adhesion kinase (FAK). We report here that integrin stimulation induces tyrosine phosphorylation of PTPalpha. PTPalpha was dephosphorylated upon fibroblast detachment from the substratum and rephosphorylated when cells were plated on the integrin ligand fibronectin. alpha PTP phosphorylation occurred at Tyr789 and required SFKs (Src or Fyn/Yes), FAK, and an intact cytoskeleton. It also required active PTPalpha or constitutively active Src. These observations indicate that PTPalpha activates SFKs and that the subsequently activated SFK.FAK tyrosine kinase complex in turn phosphorylates PTPalpha. Reintroduction of wild-type PTPalpha or unphosphorylatable PTPalpha(Y789F) (but not inactive PTPalpha) into PTPalpha-null fibroblasts restored defective integrin-induced SFK activation, FAK phosphorylation, and paxillin phosphorylation. PTPalpha(Y789F) and inactive PTPalpha could not rescue delayed actin stress fiber assembly and focal adhesion formation or defective cell migration. This study distinguishes two roles of PTPalpha in integrin signaling: an early role as an activator of SFKs and FAK with no requirement for PTPalpha phosphorylation and a later downstream role in cytoskeleton-associated events for which PTPalpha phosphorylation at Tyr789 is essential.     

Cellular settings mediating Src Substrate switching between focal adhesion kinase tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.     

Src family kinases regulate renal epithelial dedifferentiation through activation of EGFR/PI3K signaling

Dedifferentiation, a process by which differentiated cells become mesenchymal‐like proliferating cells, is the first step in renal epithelium repair and occurs in vivo after acute kidney injury and in vitro in primary culture. However, the underlying mechanism remains poorly understood. In this report, we studied the signaling events that mediate dedifferentiation of proximal renal tubular cells (RPTC) in primary culture. RPTC dedifferentiation characterized by increased expression of vimentin concurrent with decreased expression of cytokeratin‐18 was observed at 24 h after the initial plating of freshly isolated proximal tubules and persisted for 72 h. At 96 h, RPTC started to redifferentiate as revealed by reciprocal expression of cytokeratin‐18 and vimentin and completed at 120 h. Phosphorylation levels of Src, epidermal growth factor receptor (EGFR), AKT (a target of phosphoinositide‐3‐kinase (PI3K)), and ERK1/2 were increased in the early time course of culture (<72 h). Inhibition of Src family kinases (SFKs) with PP1 blocked EGFR, AKT, and ERK1/2 phosphorylation, as well as RPTC dedifferentiation. Inhibition of EGFR with AG1478 also blocked AKT and ERK1/2 phosphorylation and RPTC dedifferentiation. Although inactivation of the PI3K/AKT pathway with LY294002 inhibited RPTC dedifferentiation, blocking the ERK1/2 pathway with U0126 did not show such an effect. Moreover, inhibition of SFKs, EGFR, PI3K/AKT, but not ERK1/2 pathways abrogated RPTC outgrowth and SFK inhibition decreased RPTC proliferation and migration. These findings demonstrate a critical role of SFKs in mediating RPTC dedifferentiation through activation of the EGFR/PI3K signaling pathway. J. Cell. Physiol. 227: 2138–2144, 2012. © 2011 Wiley Periodicals, Inc.     

Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [(3)H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, and odc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.     

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.     

Mutual Regulation of Src Family Kinases and the Neurotrophin Receptor TrkB

The neurotrophin receptor tyrosine kinase TrkB is critical to diverse biological processes. We investigated the interplay of Src family kinases (SFKs) and TrkB to better understand mechanisms of TrkB signaling in physiological and pathological conditions. We compared and contrasted the role of SFKs in TrkB signaling following activation of TrkB by two mechanisms, its transactivation by zinc, and its activation by its prototypic neurotrophin ligand, brain-derived neurotrophic factor (BDNF). Using biochemical, pharmacological, and chemical genetic studies of cultured rodent neurons, we found that zinc promotes preferential phosphorylation of Tyr-705/Tyr-706 of TrkB by a SFK-dependent but TrkB kinase-independent mechanism, a signaling event critical for transactivation of TrkB by zinc. By contrast, SFK activity is not essential for BDNF-mediated activation of TrkB, yet SFK activity is increased as a consequence of TrkB activation by BDNF. Moreover, BDNF-induced phosphorylation of Tyr-705/Tyr-706 of TrkB was inhibited by SFK inhibitors, implicating a regulatory role of SFKs in TrkB activation by BDNF. In sum, SFKs are activated by TrkB and, in turn, SFKs can promote TrkB activation. We propose models depicting the mutual regulation of SFKs and TrkB following activation of TrkB by zinc and BDNF.     

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.     

The Interaction of Protein-tyrosine Phosphatase α (PTPα) and RACK1 Protein Enables Insulin-like Growth Factor 1 (IGF-1)-stimulated Abl-dependent and -independent Tyrosine Phosphorylation of PTPα

Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration.     

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.     

Regulation of Src family kinases involved in T cell receptor signaling by protein-tyrosine phosphatase CD148

CD148 is a receptor-like protein-tyrosine phosphatase known to inhibit transduction of mitogenic signals in non-hematopoietic cells. Similarly, in the hematopoietic lineage, CD148 inhibited signal transduction downstream of T cell receptor. However, it also augmented immunoreceptor signaling in B cells and macrophages via dephosphorylating C-terminal tyrosine of Src family kinases (SFK). Accordingly, endogenous CD148 compensated for the loss of the main SFK activator CD45 in murine B cells and macrophages but not in T cells. Hypothetical explanations for the difference between T cells and other leukocyte lineages include the inability of CD148 to dephosphorylate a specific set of SFKs involved in T cell activation or the lack of CD148 expression during critical stages of T cell development. Here we describe striking differences in CD148 expression between human and murine thymocyte subsets, the only unifying feature being the absence of CD148 during the positive selection when the major developmental block occurs under CD45 deficiency. Moreover, we demonstrate that similar to CD45, CD148 has both activating and inhibitory effects on the SFKs involved in TCR signaling. However, in the absence of CD45, activating effects prevail, resulting in functional complementation of CD45 deficiency in human T cell lines. Importantly, this is independent of the tyrosines in the CD148 C-terminal tail, contradicting the recently proposed phosphotyrosine displacement model as a mechanism of SFK activation by CD148. Collectively, our data suggest that differential effects of CD148 in T cells and other leukocyte subsets cannot be explained by the CD148 inability to activate T cell SFKs but rather by its dual inhibitory/activatory function and specific expression pattern.     

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.     

Ubiquitination of the common cytokine receptor gammac and regulation of expression by an ubiquitination/deubiquitination machinery

The common cytokine receptor gamma(c) is shared by the interleukin-2, -4, -7, -9, -15, and -21 receptors, and is essential for lymphocyte proliferation and survival. The regulation of gamma(c) receptor expression level is therefore critical for the ability of cells to respond to these cytokines. We previously reported that gamma(c) is efficiently constitutively internalized and addressed towards a degradation endocytic compartment. We show that gamma(c) is ubiquitinated and also associated to ubiquitinated proteins. We report that the ubiquitin-ligase c-Cbl induces gamma(c) down-regulation. In addition, the ubiquitin-hydrolase, DUB-2, counteracts the effect of c-Cbl on gamma(c) expression. We show that an increase in DUB-2 expression correlates with an increased gamma(c) half-life, resulting in the up-regulation of the receptor. Altogether, we show that gamma(c) is the target of an ubiquitination mechanism and its expression level can be regulated through the activities of a couple of ubiquitin-ligase/ubiquitin-hydrolase enzymes, namely c-Cbl/DUB-2.     

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

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