JNK2 modulates the CD1d-dependent and -independent activation of iNKT cells.

Invariant natural killer T (iNKT) cells play critical roles in autoimmune, anti-tumor, and anti-microbial immune responses, and are activated by glycolipids presented by the MHC class I-like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)-dependent iNKT cell activation is not well-known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d-mediated Ag presentation in APCs, but also contributes to CD1d-independent iNKT cell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKT cells in a CD1d-independent, but IL-12-dependent manner, we found the virus-induced loss of iNKT cells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wild-type (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKT cells were found to express less surface IL-12 receptors. As with a VV infection, an IL-12 injection also resulted in a smaller decrease in JNK2 KO iNKT cells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d-mediated Ag presentation and contributes to IL-12-induced iNKT cell activation and loss during viral infections.

Neurofibromin 1 Impairs Natural Killer T-Cell-Dependent Antitumor Immunity against a T-Cell Lymphoma.

Neurofibromin 1 (NF1) is a tumor suppressor gene encoding a Ras GTPase that negatively regulates Ras signaling pathways. Mutations in NF1 are linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. In terms of antitumor immunity, CD1d-dependent natural killer T (NKT) cells play an important role in the innate antitumor immune response. Generally, Type-I NKT cells protect (and Type-II NKT cells impair) host antitumor immunity. We have previously shown that CD1d-mediated antigen presentation to NKT cells is regulated by cell signaling pathways. To study whether a haploinsufficiency in NF1 would affect CD1d-dependent activation of NKT cells, we analyzed the NKT-cell population as well as the functional expression of CD1d in Nf1+/- mice. Nf1+/- mice were found to have similar levels of NKT cells as wildtype (WT) littermates. Interestingly, however, reduced CD1d expression was observed in Nf1+/- mice compared with their WT littermates. When inoculated with a T-cell lymphoma in vivo, Nf1+/- mice survived longer than their WT littermates. Furthermore, blocking CD1d in vivo significantly enhanced antitumor activity in WT, but not in Nf1+/- mice. In contrast, a deficiency in Type-I NKT cells increased antitumor activity in Nf1+/- mice, but not in WT littermates. Therefore, these data suggest that normal NF1 expression impairs CD1d-mediated NKT-cell activation and antitumor activity against a T-cell lymphoma.

A VP22-Null HSV-1 Is Impaired in Inhibiting CD1d-Mediated Antigen Presentation.

CD1d-restricted T (natural killer T [NKT]) cells are important for controlling a herpes simplex virus (HSV) infection. One of the mechanisms of immune evasion by HSV is to downregulate CD1d-mediated activation of NKT cells. VP22 is an HSV-1-encoded protein responsible for reorganizing the host cell's cytoskeletal network and viral spreading. We have previously shown that modification of the cytoskeleton can alter CD1d-mediated antigen presentation. In this study, we found that an HSV-1 lacking VP22 (ΔUL49) was impaired in its ability to inhibit CD1d-mediated antigen presentation compared with the wild-type (WT) virus; this was reversed by a repair virus (UL49R) in CD1d-expressing cells. We further demonstrated that CD1d recycling was inhibited by infection with WT and UL49R, but not the ΔUL49 virus. Ectopic expression of VP22 in CD1d-expressing cells complemented the VP22-deficient virus in inhibiting antigen presentation. Moreover, inhibiting viral protein synthesis rescued VP22-dependent inhibition of CD1d antigen presentation. In conclusion, our findings suggest that VP22 is required (but not sufficient) for the inhibition of CD1d-mediated antigen presentation by an HSV-1 infection.

STAT3 promotes CD1d-mediated lipid antigen presentation by regulating a critical gene in glycosphingolipid biosynthesis.

Cytokines that regulate the immune response signal through the Janus kinase / signal transducer and activation of transcription (JAK/STAT) pathway, but whether this pathway can regulate CD1d-mediated lipid antigen presentation to natural killer T (NKT) cells is unknown. Here, we found that STAT3 promotes antigen presentation by CD1d. Antigen-presenting cells (APCs) in which STAT3 expression was inhibited exhibited markedly reduced endogenous lipid antigen presentation to NKT cells without an impact on exogenous lipid antigen presentation by CD1d. Consistent with this observation, in APCs where STAT3 was knocked down, dramatically decreased levels of UDP glucose ceramide glucosyltransferase (UGCG), an enzyme involved in the first step of glycosphingolipid biosynthesis, were observed. Impaired lipid antigen presentation was reversed by ectopic expression of UGCG in STAT3-silenced CD1d(+) APCs. Hence, by controlling a fundamental step in CD1d-mediated lipid antigen presentation, STAT3 signalling promotes innate immune responses driven by CD1d.

Forming a complex with MHC class I molecules interferes with mouse CD1d functional expression.

CD1d molecules are structurally similar to MHC class I, but present lipid antigens as opposed to peptides. Here, we show that MHC class I molecules physically associate with (and regulate the functional expression of) mouse CD1d on the surface of cells. Low pH (3.0) acid stripping of MHC class I molecules resulted in increased surface expression of murine CD1d on antigen presenting cells as well as augmented CD1d-mediated antigen presentation to NKT cells. Consistent with the above results, TAP1-/- mice were found to have a higher percentage of type I NKT cells as compared to wild type mice. Moreover, bone marrow-derived dendritic cells from TAP1-/- mice showed increased antigen presentation by CD1d compared to wild type mice. Together, these results suggest that MHC class I molecules can regulate NKT cell function, in part, by masking CD1d.

Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant.

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and -active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored. Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavage by gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

Regulation of the actin cytoskeleton by Rho kinase controls antigen presentation by CD1d.

CD1d molecules are MHC class I-like molecules that present lipid Ags to NKT cells. Although we have previously shown that several different cell signaling molecules can play a role in the control of Ag presentation by CD1d, a defined mechanism by which a cell signaling pathway regulates CD1d function has been unclear. In the current study, we have found that the Rho kinases, Rho-associated, coiled-coil containing protein kinase (ROCK)1 and ROCK2, negatively regulate both human and mouse CD1d-mediated Ag presentation. Inhibition of ROCK pharmacologically, through specific ROCK1 and ROCK2 short hairpin RNA, or by using dendritic cells generated from ROCK1-deficient mice all resulted in enhanced CD1d-mediated Ag presentation compared with controls. ROCK regulates the actin cytoskeleton by phosphorylating LIM kinase, which, in turn, phosphorylates cofilin, prohibiting actin fiber depolymerization. Treatment of APCs with the actin filament depolymerizing agent, cytochalasin D, as well as knockdown of LIM kinase by short hairpin RNA, resulted in enhanced Ag presentation to NKT cells by CD1d, consistent with our ROCK inhibition data. Therefore, our overall results reveal a model whereby CD1d-mediated Ag presentation is negatively regulated by ROCK via its effects on the actin cytoskeleton.

EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling.

Epidermal growth factor (EGF) family peptides are ligands for the EGF receptor (EGFR). Here, we elucidate functional differences among EGFR ligands and mechanisms underlying these distinctions. In 32D/EGFR myeloid and MCF10A breast cells, soluble amphiregulin (AR), transforming growth factor alpha (TGFα), neuregulin 2 beta, and epigen stimulate greater EGFR coupling to cell proliferation and DNA synthesis than do EGF, betacellulin, heparin-binding EGF-like growth factor, and epiregulin. EGF competitively antagonizes AR, indicating that its functional differences reflect dissimilar intrinsic activity at EGFR. EGF stimulates much greater phosphorylation of EGFR Tyr1045 than does AR. Moreover, the EGFR Y1045F mutation and z-cbl dominant-negative mutant of the c-cbl ubiquitin ligase potentiate the effect of EGF but not of AR. Both EGF and AR stimulate phosphorylation of EGFR Tyr992. However, the EGFR Y992F mutation and phospholipase C gamma inhibitor U73122 reduce the effect of AR much more than that of EGF. Expression of TGFα in 32D/EGFR cells causes greater EGFR coupling to cell proliferation than does expression of EGF. Moreover, expression of EGF in 32D/EGFR cells causes these cells to be largely refractory to stimulation with soluble EGF. Thus, EGFR ligands are functionally distinct in models of paracrine and autocrine signaling and EGFR coupling to biological responses may be specified by competition among functionally distinct EGFR ligands.

The Q43L mutant of neuregulin 2ß is a pan-ErbB receptor antagonist.

The ErbB4 receptor tyrosine kinase possesses both tumour suppressor and oncogenic activities. Thus pharmacological agents are needed to help elucidate ErbB4 functions. However, limitations of existing ErbB4 agonists and antagonists have led us to seek novel ErbB4 antagonists. The Q43L mutant of the ErbB4 agonist NRG2ß (neuregulin 2ß) stimulates ErbB4 tyrosine phosphorylation, yet fails to stimulate ErbB4 coupling to cell proliferation. Thus in the present paper we hypothesize that NRG2ß/Q43L may be an ErbB4 antagonist. NRG2ß/Q43L competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation. NRG2ß/Q43L stimulates less ErbB4 tyrosine phosphorylation than does NRG2ß. In addition, NRG2ß stimulation of cell proliferation requires PI3K (phosphoinositide 3-kinase) activity and NRG2ß stimulates greater Akt phosphorylation than does NRG2ß/Q43L. Moreover, EGFR [EGF (epidermal growth factor) receptor] kinase activity (but not that of ErbB4) is critical for coupling ErbB4 to proliferation. Experiments utilizing ErbB4 splicing isoforms and mutants suggest that NRG2ß and NRG2ß/Q43L may differentially stimulate ErbB4 coupling to the transcriptional co-regulator YAP (Yes-associated protein). Finally, NRG2ß/Q43L competitively antagonizes agonist stimulation of EGFR and ErbB2/ErbB3, indicating that NRG2ß/Q43L is a pan-ErbB antagonist. Thus we postulate that NRG2ß/Q43L and other antagonistic ligands stimulate ErbB tyrosine phosphorylation on a set of residues distinct from that stimulated by agonists, thus suggesting a novel mechanism of ErbB receptor regulation. Moreover, NRG2ß/Q43L and related ligand-based antagonists establish a paradigm for the discovery of anti-ErbB therapeutics.

Anthrax lethal toxin impairs CD1d-mediated antigen presentation by targeting the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway.

Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis and an important means by which this bacterium evades the host's immune system. In this study, we demonstrate that CD1d-expressing cells treated with LT have reduced CD1d-mediated antigen presentation. We earlier showed an important role for the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulation of CD1d-mediated antigen presentation, and we report here that LT impairs antigen presentation by CD1d in an ERK1/2-dependent manner. Similarly, LT and the ERK1/2 pathway-specific inhibitor U0126 caused a decrease in major histocompatibility complex (MHC) class II-mediated antigen presentation. Confocal microscopy analyses revealed altered intracellular distribution of CD1d and LAMP-1 in LT-treated cells, similar to the case for ERK1/2-inhibited cells. These results suggest that Bacillus anthracis has the ability to evade the host's innate immune system by reducing CD1d-mediated antigen presentation through targeting the ERK1/2 pathway.

Statins impair CD1d-mediated antigen presentation through the inhibition of prenylation.

Statins are widely used as cholesterol-lowering agents that also decrease inflammation and target enzymes essential for prenylation, an important process in the activation and intracellular transport of proteins vital for a wide variety of cellular functions. Here, we report that statins impair a critical component of the innate immune response, CD1d-mediated Ag presentation. The addition of specific intermediates in the isoprenylation pathway reversed this effect, whereas specific targeting of enzymes responsible for prenylation mimicked the inhibitory effects of statins on Ag presentation by CD1d as well as MHC class II molecules. This study demonstrates the importance of isoprenylation in the regulation of Ag presentation and suggests a mechanism by which statins reduce inflammatory responses.

Mutational activation of ErbB family receptor tyrosine kinases: insights into mechanisms of signal transduction and tumorigenesis.

Signaling by the Epidermal Growth Factor Receptor (EGFR) and related ErbB family receptor tyrosine kinases can be deregulated in human malignancies as the result of mutations in the genes that encode these receptors. The recent identification of EGFR mutations that correlate with sensitivity and resistance to EGFR tyrosine kinase inhibitors in lung and colon tumors has renewed interest in such activating mutations. Here we review current models for ligand stimulation of receptor dimerization and for activation of receptor signaling by receptor dimerization. In the context of these models, we discuss ErbB receptor mutations that affect ligand binding and those that cause constitutive receptor phosphorylation and signaling as a result of constitutive receptor dimerization. We discuss mutations in the cytoplasmic regions that affect enzymatic activity, substrate specificity and coupling to effectors and downstream signaling pathways. Finally, we discuss how emergent mechanisms of ErbB receptor mutational activation could impact the search for clinically relevant ErbB receptor mutations.

The antibody sc-33040-R fails to specifically recognize phosphorylation of ErbB4 on tyrosine1056.

Phosphorylation state specific antibodies are important reagents for characterizing protein phosphorylation and signaling. However, these antibodies require proper validation to determine that they do not cross-react with the unphosphorylated peptide or with other phosphoproteins. We have previously shown that phosphorylation of tyrosine1056 of ErbB4 is critical for it to inhibit colony formation on plastic by human tumor cell lines. Thus, an antibody directed against this site would be useful for studying ErbB4 signaling and coupling to biological responses. Here, we demonstrate that a commercially available antibody raised against a phosphopeptide corresponding to the carboxyl-terminal domain of the ErbB4 receptor tyrosine kinase fails to exhibit appropriate specificity. Thus, this antibody does not appear to be suitable for studying ErbB4 phosphorylation or signaling.

Phosphorylation of ErbB4 on Tyr1056 is critical for inhibition of colony formation by prostate tumor cell lines.

We have previously demonstrated that the constitutively active Q646C mutant of the ErbB4 receptor tyrosine kinase inhibits colony formation by human prostate tumor cell lines. Here we use ErbB4 mutants to identify ErbB4 functions critical for inhibiting colony formation. A derivative of ErbB4 Q646 that lacks kinase activity fails to inhibit colony formation by prostate tumor cells. Likewise, an ErbB4 Q646C mutant in the context of the CT-b splicing isoform fails to inhibit colony formation. Mutation of tyrosine 1056 to phenylalanine abrogates inhibition of colony formation whereas an ErbB4 mutant that lacks all of the putative sites of tyrosine phosphorylation except for tyrosine 1056 still inhibits colony formation. Given that tyrosine 1056 is missing in the CT-b isoform, these results suggest that phosphorylation of tyrosine 1056 is critical for function. Indeed, an ErbB4 mutant that lacks kinase activity but has a glutamate phosphomimic residue substituted for tyrosine 1056 inhibits colony formation. Finally, 1-dimensional phosphopeptide mapping indicates that ErbB4 Q646C is phosphorylated on tyrosine 1056. These data suggest that phosphorylation of ErbB4 tyrosine 1056 is critical for coupling ErbB4 to prostate tumor suppression.

The epidermal growth factor receptor (EGFR)-S442F mutant displays increased affinity for neuregulin-2beta and agonist-independent coupling with downstream signalling events.

The EGFR (epidermal growth factor receptor; ErbB1) is frequently the subject of genetic changes in human tumours which contribute to the malignant phenotype by altering EGFR signalling. Examples of such genetic changes include overexpression, extracellular domain deletions and point mutations, and small deletions in the tyrosine kinase domain. We hypothesized that a point mutation in one of the EGFR ligand-binding domains would increase the affinity of EGFR for NRG2beta (neuregulin-2beta), which is not a potent stimulus of signalling by EGFR-Wt (wild-type EGFR). This mutation would permit NRG2beta stimulation of EGFR signalling in settings in which NRG2beta does not normally do so. To test this hypothesis, we have generated and evaluated various EGFR alleles containing mutations at Val441 and Ser442. NRG2beta is a much more potent stimulus of the EGFR-S442F mutant than of EGFR-Wt. Furthermore, the affinity of NRG2beta for the EGFR-S442F mutant is greater than the affinity of NRG2beta for EGFR-Wt. Finally, the EGFR-S442F mutant constitutively suppresses apoptosis via phosphoinositide 3-kinase and Akt signalling but is not highly tyrosine phosphorylated in the absence of ligand. These results suggest that mutations in the EGFR ligand-binding domain in tumours may permit potent stimulation of EGFR signalling by ligands that are not normally potent EGFR agonists, thereby providing for a novel mechanism by which EGFR signalling may be deregulated. These results also suggest that novel EGFR mutations and signalling activities may be responsible for deregulated EGFR signalling in tumour cells.

Phe45 of NRG2beta is critical for the affinity of NRG2beta for ErbB4 and for potent stimulation of ErbB4 signaling by NRG2beta*.

The Neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of growth factors. EGF family members regulate the signaling of ErbB family receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/HER2/Neu, ErbB3/HER3 and ErbB4/HER4. We have previously demonstrated that the EGF family hormone NRG2beta is a potent ErbB4 agonist, whereas NRG2alpha is a weak ErbB4 agonist. We have also previously demonstrated that Phe45 of NRG2beta regulates the potency of NRG2beta. Here, we address the hypotheses that Phe45 regulates the potency of NRG2beta by regulating the affinity of NRG2beta for ErbB4. We demonstrate that Phe45 of NRG2beta indeed regulates the affinity of NRG2beta for ErbB4. Furthermore, a hydrophobic or uncharged amino acid side chain at residue 45 contributes to NRG2beta binding to ErbB4. These data indicate that Phe45 of NRG2beta may regulate the affinity of NRG2beta for ErbB4 by interacting with hydrophobic amino acids in ErbB4.

Five carboxyl-terminal residues of neuregulin2 are critical for stimulation of signaling by the ErbB4 receptor tyrosine kinase.

The neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of peptide growth factors. These hormones are agonists for the ErbB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4. We recently observed that the EGF family hormone NRG2beta is a potent agonist for ErbB4. In contrast, NRG2alpha, a splicing isoform of the same gene that encodes NRG2beta, is a poor ErbB4 agonist. We hypothesized that carboxyl-terminal residues of NRG2beta are critical for stimulation of ErbB4 tyrosine phosphorylation and coupling to downstream signaling events. Here, we demonstrate that the substitution of a lysine residue for Phe45 in NRG2beta results in reduced ligand potency. We also demonstrate that substitution of a phenylalanine for Lys45 in NRG2alpha results in increased ligand potency. Finally, analyses of the gain-of-function NRG2alpha Chg5 mutant demonstrate that Gln43, Met47, Asn49, and Phe50 regulate ligand efficacy. Thus, these data indicate that carboxyl-terminal residues of NRG2beta are critical for activation of ErbB4 signaling. Moreover, these NRG2alpha and NRG2beta mutants reveal new insights into models for ligand-induced ErbB family receptor tyrosine phosphorylation and coupling to downstream signaling events.

A constitutively active ErbB4 mutant inhibits drug-resistant colony formation by the DU-145 and PC-3 human prostate tumor cell lines.

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. Several groups have hypothesized that signal transduction by the ErbB4 receptor tyrosine kinase is coupled to differentiation, growth arrest, and tumor suppression in mammary and prostate epithelial cells. In this report we demonstrate that a constitutively active ErbB4 mutant inhibits the formation of drug-resistant colonies by the DU-145 and PC-3 human prostate tumor cell lines. This is consistent with our hypothesis that ErbB4 signaling is growth inhibitory and may be coupled to tumor suppression in prostate cells.