Deletion of the carboxyl terminus of Tie2 enhances kinase activity,signaling, and function. Evidence for an autoinhibitory mechanism

Tie2 is an endothelial receptor tyrosine kinase that is required for both embryonic vascular development and tumor angiogenesis. There is considerable interest in understanding the mechanisms of Tie2 activation for therapeutic purposes. The recent solution of the Tie2 crystal structure suggests that Tie2 activity is autoinhibited by its carboxyl terminus. Here we investigated the role of the C tail in Tie2 activation, signaling, and function both in vitro and in vivo by deleting the C terminus of Tie2 (Delta CT). Compared to wild type Tie2, in vitro autophosphorylation and kinase activity were significantly enhanced by the Delta CT mutation. In NIH 3T3 cells expressing chimeric Tie2 receptors, both basal and ligand-induced tyrosine phosphorylation were markedly enhanced compared to wild type in several independent clones of Tie2-Delta CT. Moreover, the Delta CT mutation enhanced basal and ligand-dependent activation of Akt and extracellular signal-regulated kinase. Enhanced Akt activation correlated with significant inhibition of staurosporine-induced apoptosis. These findings demonstrate that the Tie2 C tail performs a novel negative regulatory role in Tie2 signaling and function, and they provide important insights into the mechanisms by which the Tie2 kinase is activated.     

Membrane-proximal region of the carboxyl terminus of the gonadotropin-releasing hormone receptor (GnRHR) confers differential signal transduction between mammalian and nonmammalian GnRHRs

Recently, we demonstrated that the mammalian type-I GnRH receptor (GnRHR) has a high preference for the phospholipase C/protein kinase C (PLC/PKC)-linked signaling pathway, whereas non-mammalian bullfrog (bf) GnRHRs couple to both adenylate cyclase/protein kinase A (AC/PKA)- and PLC/PKC-linked signaling pathways. In the pre-sent study, using AC/PKA-specific reporter (cAMP-responsive element-luciferase) and PLC/PKC-specific reporter (serum-responsive element-luciferase) systems, we attempted to identify the motif responsible for this difference. A deletion of the intracellular carboxyl-terminal tail (C tail) of bfGnRHR-1 remarkably decreased its ability to induce the AC/PKA-linked signaling pathway. Further dissection of the C tail indicated that an HFRK motif in the membrane-proximal sequence of bfGnRHR-1 C tail is a minimal requirement for the AC/PKA-linked signaling pathway as the addition of this motif to rat GnRHR or deletion of it from bfGnRHR-1 significantly affected the ability to induce the AC/PKA-linked signaling pathway. Deletion or addition of the HFRK motif, however, did not critically influence the PLC/PKC-linked signaling pathway. These results indicate that the HFRK motif in the membrane-proximal region confers the differential signal transduction pathways between mammalian and nonmammalian GnRHRs.     

Endoglin-Mediated Suppression of Prostate Cancer Invasion Is Regulated by Activin and Bone Morphogenetic Protein Type II Receptors

Mortality from prostate cancer (PCa) is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor β (TGFβ) superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFβ receptor, activin receptor-like kinase 2 (ALK2), and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFβ receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA) and bone morphogenetic protein receptor type II (BMPRII). Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII’s Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.     

Pseudomonas aeruginosa stimulates phosphorylation of the airway epithelial membrane glycoprotein Muc1 and activates MAP kinase

We reported previously that Muc1 on the surface of epithelial cells was a receptor for Pseudomonas aeruginosa (Lillehoj EP, Kim BT, and Kim KC. Am J Physiol Lung Cell Mol Physiol 282: L751-L756, 2002). Other studies showed that the Muc1 cytoplasmic tail (CT) contains multiple phosphorylation sites, some of which are phosphorylated constitutively and associated with signaling proteins. However, the relationship between extracellular P. aeruginosa binding and intracellular signaling is unknown. To investigate the signaling mechanism of Muc1, this study examined phosphorylation of its CT and activation of the extracellular signal-regulated kinase (ERK) in response to stimulation by P. aeruginosa or purified flagellin. Our results showed 1) the Muc1 CT was phosphorylated constitutively on serine and tyrosine, 2) serine phosphorylation was stimulated by bacterial cells or flagellin, and 3) binding of P. aeruginosa or flagellin to Muc1 induced phosphorylation of ERK. These results are the first to demonstrate Muc1 CT phosphorylation and ERK activation in response to a clinically important airway pathogen.     

The unique C-terminal tail of the mitogen-activated protein kinase ERK5 regulates its activation and nuclear shuttling

ERK5 is unique among mitogen-activated protein kinases (MAPKs) in that it contains a large C-terminal tail. We addressed the question of how this tail could affect the signaling capacity of ERK5. Gradual deletion of the C-terminal domains resulted in a drastic increase of ERK5 kinase activity, which was dependent on the up-stream MAPK cascade, thus indicating a possible auto-inhibitory function of the tail. It is interesting that ERK5 was able to autophosphorylate its own tail. Moreover, ERK5, which was found to be expressed in virtually all kinds of cell lines, localized to nuclear as well as cytoplasmic compartments. The localization of ERK5 was determined by its C-terminal domains, which were also required for appropriate nucleocytoplasmic shuttling. Taken together, these results indicate that ERK5 signaling is directed by the presence of its unique C-terminal tail, which might be the key to understanding the key role of ERK5 in MAPK signaling.     

The transmembrane domain of podoplanin is required for its association with lipid rafts and the induction of epithelial-mesenchymal transition

Podoplanin is a transmembrane glycoprotein that is upregulated in cancer and was reported to induce an epithelial-mesenchymal transition (EMT) in MDCK cells. The promotion of EMT was dependent on podoplanin binding to ERM (ezrin, radixin, moesin) proteins through its cytoplasmic (CT) domain, which led to RhoA-associated kinase (ROCK)-dependent ERM phosphorylation. Using detergent-resistant membrane (DRM) assays, as well as transmembrane (TM) interactions and ganglioside GM1 binding, we present evidence supporting the localization of podoplanin in raft platforms important for cell signalling. Podoplanin mutant constructs harbouring a heterologous TM region or lacking the CT tail were unable to associate with DRMs, stimulate ERM phosphorylation and promote EMT or cell migration. Similar effects were observed upon disruption of a GXXXG motif within the TM domain, which is involved in podoplanin self-assembly. In contrast, deletion of the extracellular (EC) domain did not affect podoplanin DRM association. Together, these data suggest that both the CT and TM domains are required for podoplanin localization in raft platforms, and that this association appears to be necessary for podoplanin-mediated EMT and cell migration.     

汉滩病毒G1蛋白胞质区ITAM样基序功能的研究

汉滩病毒(HTNV)的G1蛋白胞质区尾段包含保守的免疫受体酪氨酸活化基序(ITAM)样基序,该基序与许多重要的免疫受体胞质区ITAM基序同源性较高。为了研究HTNV的G1 ITAM样基序的免疫信号转导功能,首先人工合成了一段保守的酪氨酸残基磷酸化的G1 ITAM样基序多肽,应用体外蛋白激酶共沉淀实验,分别从Jur-kat细胞和Raji细胞裂解物中初筛到5~9种与该基序相互作用的磷酸化蛋白或激酶;然后通过突变体分析、体外磷酸化实验和体外激酶共沉淀-免疫印迹分析,进一步确证了G1 ITAM样基序在体外可以与Src家族蛋白酪氨酸激酶(PTK)Lyn、Fyn及其下游Syk家族激酶Syk、ZAP-70相互作用,而这种相互作用依赖于该基序中两个高度保守的酪氨酸残基的存在。上述研究表明,HTNV G1蛋白胞质区包含一个高度保守的功能性ITAM样基序,该基序在体外可以与TCR和BCR信号转导中关键的PTK相互作用,为进一步探讨HTNV G1蛋白ITAM样基序在肾综合征出血热(HFRS)免疫信号传递中的作用奠定了基础。     

A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.     

Lipid regulators of Pkh2 in Candida albicans,the protein kinase ortholog of mammalian PDK1

Pkh is the yeast ortholog of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Pkh phosphorylates the activation loop of Ypks, Tpks, Sch9 and also phosphorylates the eisosome components Lsp1 and Pil1, which play fundamental roles upstream of diverse signaling pathways, including the cell wall integrity and sphingosine/long-chain base (LCB) signaling pathways. In S. cerevisiae, two isoforms, ScPkh1 and ScPkh2, are required for cell viability, while only one ortholog exists in C. albicans, CaPkh2. In spite of the extensive information gathered on the role of Pkh in the LCB signaling, the yeast Pkh kinases are not known to bind lipids and previous studies did not identify PH domains in Pkh sequences. We now describe that the C-terminal region of CaPkh2 is required for its intrinsic kinase activity. In addition, we found that the C-terminal region of CaPkh2 enables its interaction with structural and signaling lipids. Our results further show that phosphatidylserine, phosphatidic acid, phosphatidylinositol (3,4 and 4,5)-biphosphates, and phosphatidylinositol (3,4,5)-trisphosphate inhibit Pkh activity, whereas sulfatide binds with high affinity but does not affect the intrinsic activity of CaPkh2. Interestingly, we identified that its human ortholog PDK1 also binds to sulfatide. We propose a mechanism by which lipids and dihydrosphingosine regulate CaPkh2 kinase activity by modulating the interaction of the C-terminal region with the kinase domain, while sulfatide-like lipids support localization CaPkh2 mediated by a C-terminal PH domain, without affecting kinase intrinsic activity.     

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2.     

Epitope Tags beside the N-Terminal Cytoplasmic Tail of Human BST-2 Alter Its Intracellular Trafficking and HIV-1 Restriction

BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif “KRXK” in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2.     

Mutational analysis of the cytoplasmic tail of jaagsiekte sheep retrovirus envelope protein

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a transmissible lung cancer in sheep, ovine pulmonary adenocarcinoma. JSRV is unique in that the envelope protein functions as an oncogene, since it can morphologically transform fibroblast and epithelial cells in culture and can induce lung tumors in mice. Previous studies indicated that the transmembrane (TM) protein is essential for transformation, and particular attention has focused on a YXXM motif in the cytoplasmic tail. In this study, we carried out systematic mutagenesis of the cytoplasmic tail of JSRV Env. Alanine scanning mutagenesis revealed four classes of mutants: mutants in which transformation was abrogated, those in which transformation was not affected, those with reduced transformation, and those with increased transformation (supertransformers). In general, the alanine mutations did not affect Env protein production or its localization to the plasma membrane. Three functional domains of the cytoplasmic tail were identified: an amphipathic helix at the N-terminal (juxtamembrane) side, a nonessential C-terminal region, and an internal region (including the YXXM motif) where mutations resulted in abrogation, decreases, or increases in transformation. Alanine mutations in the amphipathic helix in both the hydrophobic and hydrophilic faces generally abolished transformation. The mutation R591A showed partial transformation that was consistent with loss of signaling through the Akt-mTOR pathway and signaling predominantly through the Ras-Raf-MEK1/2-extracellular signal-regulated kinase 1/2 pathway. The supertransforming mutants generally showed increased signaling through Akt and reduced activation of p38 MAPK that is inhibitory for transformation. These mutants provide further insight into the role of the TM cytoplasmic tail in JSRV transformation.     

OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae

Somatic embryogenesis receptor kinase (SERK) proteins play pivotal roles in regulation of plant development and immunity. The rice genome contains two SERK genes, OsSerk1 and OsSerk2. We previously demonstrated that OsSerk2 is required for rice Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) and for normal development. Here we report the molecular characterization of OsSerk1. Overexpression of OsSerk1 results in a semi-dwarf phenotype whereas silencing of OsSerk1 results in a reduced angle of the lamina joint. OsSerk1 is not required for rice resistance to Xoo or Magnaporthe oryzae. Overexpression of OsSerk1 in OsSerk2-silenced lines complements phenotypes associated with brassinosteroid (BR) signaling defects, but not the disease resistance phenotype mediated by Xa21. In yeast, OsSERK1 interacts with itself forming homodimers, and also interacts with the kinase domains of OsSERK2 and BRI1, respectively. OsSERK1 is a functional protein kinase capable of auto-phosphorylation in vitro. We conclude that, whereas OsSERK2 regulates both rice development and immunity, OsSERK1 functions in rice development but not immunity to Xoo and M. oryzae.     

A patch of surface-exposed residues mediates negative regulation of immune signaling by tomato Pto kinase

Tomato (Lycopersicon esculentum) Pto kinase specifically recognizes the Pseudomonas effector proteins AvrPto and AvrPtoB, leading to induction of defense responses and hypersensitive cell death. Structural modeling of Pto combined with site-directed mutagenesis identified a patch of surface-exposed residues required for native regulation of signaling. Mutations in this area resulted in constitutive gain-of-function (CGF) forms of Pto that activated AvrPto-independent cell death via the cognate signaling pathway. The patch overlaps the peptide binding region of the kinase catalytic cleft and is part of a broader region required for interaction with bacterial effectors. We propose that the negative regulatory patch is normally occupied by a peptide that represses Pto signaling. Furthermore, we found that Pto kinase activity was required for Avr-dependent activation but dispensable for signaling by CGF forms of Pto. This suggests that Pto signals by a conformational change rather than phosphorylation of downstream substrates in the defense signaling pathway.     

The critical role of IL-1 receptor-associated kinase 4-mediated NF-κB activation in modified low-density lipoprotein-induced inflammatory gene expression and atherosclerosis

Exciting discoveries related to IL-1R/TLR signaling in the development of atherosclerosis plaque have triggered intense interest in the molecular mechanisms by which innate immune signaling modulates the onset and development of atherosclerosis. Previous studies have clearly shown the definitive role of proinflammatory cytokine IL-1 in the development of atherosclerosis. Recent studies have provided direct evidence supporting a link between innate immunity and atherogenesis. Although it is still controversial about whether infectious pathogens contribute to cardiovascular diseases, direct genetic evidence indicates the importance of IL-1R/TLR signaling in atherogenesis. In this study, we examined the role of IL-1R-associated kinase 4 (IRAK4) kinase activity in modified low-density lipoprotein (LDL)-mediated signaling using bone marrow-derived macrophage as well as an in vivo model of atherosclerosis. First, we found that the IRAK4 kinase activity was required for modified LDL-induced NF-κB activation and expression of a subset of proinflammatory genes but not for the activation of MAPKs in bone marrow-derived macrophage. IRAK4 kinase-inactive knockin (IRAK4KI) mice were bred onto ApoE(-/-) mice to generate IRAK4KI/ApoE(-/-) mice. Importantly, the aortic sinus lesion formation was impaired in IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Furthermore, proinflammatory cytokine production was reduced in the aortic sinus region of IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Taken together, our results indicate that the IRAK4 kinase plays an important role in modified LDL-mediated signaling and the development of atherosclerosis, suggesting that pharmacological inhibition of IRAK4 kinase activity might be a feasible approach in the development of antiatherosclerosis drugs.