Ubiquitination and degradation of Syk and ZAP-70 protein tyrosine kinases in human NK cells upon CD16 engagement

Syk and ZAP-70 nonreceptor protein tyrosine kinases (PTKs) are essential elements in several cascades coupling immune receptors to intracellular responses. The critical role of these kinases in promoting the propagation of intracellular signaling requires a tight regulation of their activity, thus the existence of a negative feedback loop regulating their expression can be hypothesized. Herein, we have investigated whether ubiquitin-dependent proteolysis could be a mechanism responsible for controlling the fate of Syk and ZAP-70 after their immunoreceptor-induced activation. We found that both Syk and ZAP-70 become ubiquitinated in response to aggregation of the low affinity Fc receptor for IgG (CD16) on human natural killer cells. We confirmed the identity of the major in vivo ubiquitinated kinase species by performing an in vitro ubiquitination assay. In addition, we found that after CD16 stimulation, ubiquitinated forms of Syk and ZAP-70 associate with the receptor complex. After CD16 engagement, we also observed a decrease in the stability of Syk and ZAP-70 PTKs that is counteracted by pretreatment with either proteasome or lysosomal inhibitors. Moreover, in the presence of the proteasome inhibitor, epoxomicin, we observed an accumulation of ubiquitinated forms of both kinases. Our findings provide evidence of ligand-induced ubiquitination of nonreceptor PTKs belonging to the Syk family and propose the ubiquitin-dependent proteasome-mediated degradation pathway as a mechanism for attenuating the propagation of intracellular signaling initiated by immune receptor engagement.     

Receptor and Gβγ isoform-specific interactions with G protein-coupled receptor kinases

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free βγ subunits of heterotrimeric G proteins (Gβγ). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of Gβγ with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a β-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known Gβγ sequestrant. Furthermore, in cells overexpressing both GRK2 and Gβ₁γ₂, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/Gβγ complex. That Gβγ specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/Gβγ complex is formed after agonist occupancy of the lysophosphatidic acid and β-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a Gβγ complex after stimulation of all three GPCRs. This Gβγ binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds Gβ₁ and Gβ₂ but not Gβ₃, while the GRK3 fusion protein binds all three Gβ isoforms. This study provides a direct demonstration of a role for Gβγ in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.     

Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4)

We have identified a member of the VEGF family by computer-based homology searching and have designated it VEGF-D. VEGF-D is most closely related to VEGF-C by virtue of the presence of N- and C-terminal extensions that are not found in other VEGF family members. In adult human tissues, VEGF-D mRNA is most abundant in heart, lung, skeletal muscle, colon, and small intestine. Analyses of VEGF-D receptor specificity revealed that VEGF-D is a ligand for both VEGF receptors (VEGFRs) VEGFR-2 (Flk1) and VEGFR-3 (Flt4) and can activate these receptors. However, VEGF-D does not bind to VEGFR-1. Expression of a truncated derivative of VEGF-D demonstrated that the receptor-binding capacities reside in the portion of the molecule that is most closely related in primary structure to other VEGF family members and that corresponds to the mature form of VEGF-C. In addition, VEGF-D is a mitogen for endothelial cells. The structural and functional similarities between VEGF-D and VEGF-C define a subfamily of the VEGFs.     

The inhibitory effect of anti-CD33 monoclonal antibodies on AML cell growth correlates with Syk and/or ZAP-70 expression

OBJECTIVES: Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that can function as a downregulator of cell growth, mediating growth arrest and apoptosis. The protein kinase Syk is an essential element in several cascades coupling certain antigen receptors to cell responses. Recently we reported that CD33 recruits Syk for its signaling in AML cell lines. In this study, we further investigated the mechanism(s) of Syk engagement in CD33 signaling in primary AML samples. METHODS: We investigated 25 primary AML samples for their proliferative response (3H-thymidine incorporation) and biochemical changes (Western blot analysis) to anti-CD33 mAb treatment. RESULTS: Proliferation studies demonstrated that 14 (56%) of AML samples were responsive (R) while 11 (44%) were nonresponsive (n-R) to inhibitory antibody activity. Seven of 25 AML samples (28%) expressed undetectable levels of Syk. However, cells from two of these patients expressed the ZAP-70 protein kinase. In Syk/ZAP-70(+) samples, CD33 ligation inhibited proliferation in 70% of cases, while none of the Syk/ZAP-70(-) samples was responsive. There were significant biochemical differences between responder and nonresponder AML populations. In responder samples, CD33 ligation induced phosphorylation of CD33 andSyk and formation of the CD33/Syk complex. In nonresponder samples, CD33 was not phosphorylated, and Syk was in complex with the SHP-1 protein phosphatase constitutively. CONCLUSIONS: Syk is an important component in the regulation of proliferation in AML cells. The differential response of AML cells to CD33 ligation is associated with the level of the Syk expression.     

BAP-135, a target for Bruton’s tyrosine kinase in response to B cell receptor engagement

Bruton’s tyrosine kinase (Btk) is essential for B cell activation, but downstream targets of Btk have not been defined. We now describe a protein, BAP-135, that is associated with Btk in B cells and is a substrate for phosphorylation by Btk. BAP-135, which exhibits no detectable homology to known proteins, contains six occurrences of a hitherto undescribed amino acid repeat and two motifs, similar to the Src autophosphorylation site, that represent potential targets for tyrosine phosphorylation. The pleckstrin homology domain of Btk comprises the principal site of BAP-135 binding. Btk-dependent phosphorylation of BAP-135 is abolished by mutations that impair activation of Btk by Src-related kinases. Btk and BAP-135 exist in a complex before B cell antigen receptor (BCR) engagement; in response to BCR crosslinking, BAP-135 is transiently phosphorylated on tyrosine. Taken together, these observations suggest that BAP-135 may reside downstream of Btk in a signaling pathway originating at the BCR.     

G-protein-coupled receptors and tyrosine kinases: crossroads in cell signaling and regulation.

G-protein-coupled receptors and protein tyrosine kinases represent two prominent pathways for cellular signaling. As our knowledge of cell signaling pathways mediated by the superfamily of G-protein-coupled receptors and the smaller family of receptor tyrosine kinases expands, so does our appreciation of how these two major signaling platforms share information and modulate each other, otherwise termed "cross-talk". Cross-talk between G-protein-coupled receptors and tyrosine kinases can occur at several levels, including the receptor-to-receptor level, and at crucial downstream points (e.g. phosphatidylinositol-3-kinase, Akt/protein kinase B and the mitogen-activated protein kinase cascade). Regulation of G-protein-coupled receptors by non-receptor tyrosine kinases, such as Src family members, also operates in signaling. A broader understanding of how G-protein-coupled receptors and tyrosine kinases cross-talk reveals new insights into signaling modalities in both health and disease.     

Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells

Rotavirus contains two outer capsid viral proteins, the spike protein VP4 and major capsid component VP7, both of which are implicated in cell entry. We show that VP4 and VP7 contain tripeptide sequences previously shown to act as recognition sites for integrins in extracellular matrix proteins. VP4 contains the α2β1 integrin ligand site DGE. In VP7, the αxβ2 integrin ligand site GPR and the α4β1 integrin ligand site LDV are embedded in a novel disintegrin-like domain that also shows sequence similarity to fibronectin and the tie receptor tyrosine kinase. Microorganism sequence homology to these ligand motifs and to disintegrins has not been reported previously. In our experiments, peptides including these rotaviral tripeptides and mAbs directed to these integrins specifically blocked rotavirus infection of cells shown to express α2β1 and β2 integrins. Rotavirus VP4-mediated cell entry may involve the α2β1 integrin, whereas VP7 appears to interact with αxβ2 and α4β1 integrins.     

ZAP-70 enhances B-cell-receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells.

Expression of ZAP-70 is an important negative prognostic factor in chronic lymphocytic leukemia (CLL). This protein tyrosine kinase is a key mediator of T-cell receptor (TCR) signaling and is structurally homologous to Syk, which plays an analogous role in B-cell receptor (BCR) signaling. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is not completely understood. We have now compared antigen receptor-induced activation of ZAP-70 in B cells and T cells by analyzing phosphorylation of critical regulatory tyrosine residues. We show that BCR-mediated activation of ZAP-70 is very inefficient in CLL and lymphoma B cells and is negligible when compared to activation of Syk. Despite the inefficient catalytic activation, the ability of ZAP-70 to recruit downstream signaling molecules in response to antigen receptor stimulation appeared relatively preserved. Moreover, ectopic expression of ZAP-70 enhanced and prolonged activation of several key mediators of BCR signaling, such as the Syk, ERK, and Akt kinases, and decreased the rate of ligand-mediated BCR internalization. We conclude that the role of ZAP-70 in BCR signaling is quite distinct from its role in TCR signaling and is likely mediated by inhibition of events that terminate the signaling response.     

Src homology 2 (SH2)-containing 5'-inositol phosphatase localizes to podosomes, and the SH2 domain is implicated in the attenuation of bone resorption in osteoclasts

c-Src plays an important role in bone resorption by osteoclasts. Here, we show using wild-type and ship(-/-) osteoclasts that Src homology 2 (SH2)-containing 5'-inositol phosphatase (SHIP) appeared to negatively regulate bone resorption activated by c-Src. SHIP was found to localize to podosomes under the influence of c-Src, and the presence of either the amino-terminal region comprising the SH2 domain or the carboxyl-terminal region was sufficient for its localization. Although SHIP lacking a functional SH2 domain was still found in podosomes, it could not rescue the hyper-bone resorbing activity and hypersensitivity to receptor activator of nuclear factor-kappaB ligand in ship(-/-) osteoclasts, suggesting that the localization of SHIP to podosomes per se was not sufficient and the SH2 domain was indispensable for its function. Cas and c-Cbl, known to function in podosomes of osteoclasts, were identified as novel proteins binding to the SHIP SH2 domain by mass spectrometric analysis, and this interaction appeared to be dependent on the Src kinase activity. These results demonstrate that c-Src enhances the translocation of SHIP to podosomes and regulates its function there through the SH2 domain, leading to an attenuation of bone resorption.     

Activation of protein kinase C by tyrosine phosphorylation in response to H2O2

Protein kinase C (PKC) isoforms, α, βI, and γ of cPKC subgroup, δ and of nPKC subgroup, and ζ of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H₂O₂. These isoforms isolated from the H₂O₂-treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC α and δ, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC δ showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H₂O₂. These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.     

Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response

The delivery of signals from the activated T cell antigen receptor (TCR) inside the cell relies on the protein tyrosine kinase ZAP-70 (ζ-associated protein of 70 kDa). A recent crystal structure of inactive full-length ZAP-70 suggests that a central interface formed by the docking of the two SH2 domains of ZAP-70 onto the kinase domain is crucial for suppressing catalytic activity. Here we validate the significance of this autoinhibitory interface for the regulation of ZAP-70 catalytic activity and the T cell response. For this purpose, we perform in vitro catalytic activity assays and binding experiments using ZAP-70 proteins purified from insect cells to examine activation of ZAP-70. Furthermore, we use cell lines stably expressing wild-type or mutant ZAP-70 to monitor proximal events in T cell signaling, including TCR-induced phosphorylation of ZAP-70 substrates, activation of the MAP kinase pathway, and intracellular Ca²⁺ levels. Taken together, our results directly correlate the stability of the autoinhibitory interface with the activation of these key events in the T cell response.     

Impact of transgenic overexpression of SH2-containing inositol 5'-phosphatase 2 on glucose metabolism and insulin signaling in mice

SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a 5'-lipid phosphatase hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI(3,4,5)P(3) to PI(3,4)P(2) in the regulation of insulin signaling, and is shown to be increased in peripheral tissues of diabetic C57BL/KSJ-db/db mice. To clarify the impact of SHIP2 in the pathogenesis of insulin resistance with type 2 diabetes, we generated transgenic mice overexpressing SHIP2. The body weight of transgenic mice increased by 5.0% (P < 0.05) compared with control wild-type littermates on a normal chow diet, but not on a high-fat diet. Glucose tolerance and insulin sensitivity were mildly but significantly impaired in the transgenic mice only when maintained on the normal chow diet, as shown by 1.2-fold increase in glucose area under the curve over control levels at 9 months old. Insulin-induced phosphorylation of Akt was decreased in the SHIP2-overexpressing fat, skeletal muscle, and liver. In addition, the expression of hepatic mRNAs for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase was increased, that for sterol regulatory element-binding protein 1 was unchanged, and that for glucokinase was decreased. Consistently, hepatic glycogen content was reduced in the 9-month-old transgenic mice. Structure and insulin content were histologically normal in the pancreatic islets of transgenic mice. These results indicate that increased abundance of SHIP2 in vivo contributes, at least in part, to the impairment of glucose metabolism and insulin sensitivity on a normal chow diet, possibly by attenuating peripheral insulin signaling and by altering hepatic gene expression for glucose homeostasis.     

The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein.

Stimulation of the T-cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins, including phospholipase C (PLC) gamma 1 and the TCR zeta chain. We describe here a 70-kDa tyrosine phosphoprotein (ZAP-70) that associates with zeta within 15 sec following TCR stimulation. The phosphorylation of ZAP-70 and its association with zeta is independent of the other TCR chains since stimulation of a functional CD8/zeta chimeric receptor in a TCR-negative T cell leads to coprecipitation of ZAP-70 with the chimeric protein. In a Jurkat cell expressing the TCR and the CD8/zeta chimeric protein, tyrosine phosphorylation and association of ZAP-70 occurs exclusively with the stimulated receptor complex. In addition, a tyrosine kinase that does not appear to be fyn associates with the cytoplasmic domain of zeta and phosphorylates zeta and ZAP-70 in vitro.     

The B-cell receptor and ZAP-70 in chronic lymphocytic leukemia

In addition to the important observations relating immunoglobulin (Ig) mutation status to clinical behavior, studies on the Ig expressed in chronic lymphocytic leukemia (CLL) have revealed compelling evidence that antigen selection contributes to the pathogenesis of this disease. CLL cells that use unmutated Ig can generally be distinguished from CLL cells that use Ig with somatic mutations by expression of the 70-kD zeta-associated protein (ZAP-70). ZAP-70 apparently enhances the capacity of CLL cells to respond to antigen, and therefore might play a causal role in the relatively aggressive clinical behavior noted for patients who have CLL cells that use unmutated Ig. Clinical surveys have found that expression of ZAP-70 by CLL cells is apparently a stronger predictor of early disease progression than is the use by CLL cells of unmutated Ig. As such, strategies that respectively monitor or target Ig-receptor signaling in CLL might be very useful in the risk assessment or treatment of this disease.     

Cancer-associated mutations in the p85α N-terminal SH2 domain activate a spectrum of receptor tyrosine kinases

The phosphoinositide 3-kinase regulatory subunit p85α is a key regulator of kinase signaling and is frequently mutated in cancers. In the present study, we showed that in addition to weakening the inhibitory interaction between p85α and p110α, a group of driver mutations in the p85α N-terminal SH2 domain activated EGFR, HER2, HER3, c-Met, and IGF-1R in a p110α-independent manner. Cancer cells expressing these mutations exhibited the activation of p110α and the AKT pathway. Interestingly, the activation of EGFR, HER2, and c-Met was attributed to the ability of driver mutations to inhibit HER3 ubiquitination and degradation. The resulting increase in HER3 protein levels promoted its heterodimerization with EGFR, HER2, and c-Met, as well as the allosteric activation of these dimerized partners; however, HER3 silencing abolished this transactivation. Accordingly, inhibitors of either AKT or the HER family reduced the oncogenicity of driver mutations. The combination of these inhibitors resulted in marked synergy. Taken together, our findings provide mechanistic insights and suggest therapeutic strategies targeting a class of recurrent p85α mutations.

PIK3R1, which encodes the p85α regulatory subunit of phosphoinositide 3-kinases (PI3Ks), is frequently mutated in cancers. PIK3R1 mutations are associated with poor survival of cancer patients [the Genomic Data Commons (GDC) Data Portal] (1). Cancer-associated mutations have been detected in all five protein domains of p85α, namely, the Src homology 3 (SH3) domain, GTPase-activating protein (GAP) domain, N-terminal SH2 (nSH2) domain, inter-SH2 (iSH2) domain, and C-terminal SH2 (cSH2) domain (2). Hotspot PIK3R1 mutations cluster in the iSH2 and SH2 domains in agreement with the primary roles of these domains in stabilizing and inhibiting p110 in the p85α–p110 heterodimer (3).The first reported and characterized clusters of cancer patient–derived mutations were located in two regions of the iSH2 domain (i.e., the E439–K459 and D560–W583 regions) (4–6). Driver mutations in these regions can disrupt the inhibitory interaction between the iSH2 and p110 C2 domains (7), thereby alleviating the inhibition of p110 kinase activity by p85α (8). The other driver mutations target the inhibitory interactions between the nSH2 domain and p110 helical domain (9). The nSH2 domain driver mutations (G376R and K379E) have been suggested to play oncogenic roles by weakening this inhibitory interface (4). Importantly, all these p85α mutants retain the ability to physically bind to p110 and stabilize it. The cSH2 domain interacts with the p110 kinase domain and contributes to p110 inhibition (10). A cSH2 domain driver mutation, K674R, elevates AKT phosphorylation (11). However, the effect of this mutant on p110 remains to be elucidated. Apart from binding to p110, the nSH2 and cSH2 domains bind to phosphotyrosine (pY)-containing consensus sequences (pYXXM) in pY-phosphorylated receptor tyrosine kinases (RTK) or adaptor proteins (3). Engineered p85α mutations in the nSH2 (R358A, S361D) and cSH2 (R649A, S652D) domains impair binding to pY peptides by either removing charge pairing (R358A and R649A) or introducing charge repulsion (S361D, S652D) to the pY phosphate group (12, 13). Binding to the pY motifs upon RTK stimulation is incompatible with the p110-inhibiting interactions of the p85α SH2 domains. Therefore, binding of p85α to pY motifs allows the controlled activation of p110.In this study, we revealed an oncogenic mechanism evoked by a group of driver mutations in the nSH2 domain. These p85α mutations promote the stabilization of the HER3 protein, thereby activating multiple RTKs. The activation of both RTKs and PI3K/AKT by these nSH2 domain driver mutations should be considered to achieve therapeutic efficacy.     

Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2

CC chemokine receptor 2 (CCR2) is a prominent receptor for the monocyte chemoattractant protein (MCP) group of CC chemokines. Mice generated by gene targeting to lack CCR2 exhibit normal leukocyte rolling but have a pronounced defect in MCP-1-induced leukocyte firm adhesion to microvascular endothelium and reduced leukocyte extravasation. Constitutive macrophage trafficking into the peritoneal cavity was not significantly different between CCR2-deficient and wild-type mice. However, after intraperitoneal thioglycollate injection, the number of peritoneal macrophages in CCR2-deficient mice did not rise above basal levels, whereas in wild-type mice the number of macrophages at 36 h was ≈3.5 times the basal level. The CCR2-deficient mice showed enhanced early accumulation and delayed clearance of neutrophils and eosinophils. However, by 5 days neutrophils and eosinophils in both CCR2-deficient and wild-type mice had returned to near basal levels, indicating that resolution of this inflammatory response can occur in the absence of macrophage influx and CCR2-mediated activation of the resident peritoneal macrophages. After intravenous injection with yeast β-glucan, wild-type mice formed numerous large, well-defined granulomas throughout the liver parenchyma, whereas CCR2-deficient mice had much fewer and smaller granulomas. These results demonstrate that CCR2 is a major regulator of induced macrophage trafficking in vivo.