Structural basis of MET receptor dimerization by the bacterial invasion protein InlB and the HGF/SF splice variant NK1

The structural basis of ligand-induced dimerization of the receptor tyrosine kinase MET by its natural ligand hepatocyte growth factor/scatter factor (HGF/SF) is not well understood. However, interesting insight into the molecular mechanism of MET dimerization has emerged from crystal structures of MET in complex with a bacterial agonist, the invasion protein internalin B (InlB) from pathogenic Listeria monocytogenes. MET activation by InlB promotes uptake of bacteria into host cells. Structural and biophysical data suggest that InlB is monomeric on its own but dimerizes upon binding to the membrane-anchored MET receptor promoting the formation of a signaling active 2:2 complex. The dimerization interface is small and unusually located on the convex side of the curved InlB leucine-rich repeat (LRR) domain. As InlB does not dimerize in solution, the dimerization site could only be identified by studying packing contacts of InlB in various crystal forms and had to be proven by scrutinizing its biological relevance in cellular assays. InlB dimerization is thus an example of a low-affinity contact that appears irrelevant in solution but becomes physiologically significant in the context of 2-dimensional diffusion restricted to the membrane plane. The resulting 2:2 InlB:MET complex has an InlB dimer at its center with one MET molecule bound peripherally to each InlB. This model of ligand-mediated MET dimerization may serve as a blue-print to understand MET activation by NK1, a naturally occurring HGF/SF splice variant and MET agonist. Crystal structures of NK1 repeatedly show a NK1 dimer, in which residues implicated in MET-binding are located on the outside. Thus, MET dimerization by NK1 may also be ligand-mediated with a NK1 dimer at the center of the 2:2 complex with one MET molecule bound peripherally to each NK1. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Targeting of the MET receptor tyrosine kinase by small molecule inhibitors leads to MET accumulation by impairing the receptor downregulation

The MET receptor tyrosine kinase is deregulated primarily via overexpression or point mutations in various human cancers and different strategies for MET inhibition are currently evaluated in clinical trials. We observed by Western blot analysis and by Flow cytometry that MET inhibition by different MET small molecule inhibitors surprisingly increases in a dose-dependent manner total MET levels in treated cells. Mechanistically, this inhibition-related MET accumulation was associated with reduced Tyr1003 phosphorylation and MET physical association with the CBL ubiquitin ligase with concomitant decrease in MET ubiquitination. These data may suggest careful consideration for design of anti-MET clinical protocols.     

Exogenously administered HGF activator augments liver regeneration through the production of biologically active HGF.

Hepatocyte growth factor (HGF) plays a crucial role in the recovery of injured liver. Liver functions are mostly impaired in patients with liver diseases including cirrhosis. However, a significant amount of inactive HGF precursor (proHGF) is reported in the plasma of these patients. proHGF is proteolytically converted to an active form (mature HGF) by HGF-activator. Thus conversion of proHGF into mature HGF presumably contributes to the recovery of liver functions. In this study, rats with a partial hepatectomy were used, as proHGF is accumulated in the remnant liver. Recombinant human HGF-activator was administered via the portal vein to investigate the effect on molecular forms of HGF and its biological signaling. rhHGF-activator promptly converted proHGF into mature HGF, reaching maximal levels at 5-10 min after the injection, while the decreased proHGF was quickly recovered to the initial levels in the liver. The HGF receptor/c-Met was found to be autophosphorylated in the liver treated with rhHGF-activator. Further, the proliferating cell nuclear antigen labeling index and the liver regeneration rate were significantly higher in rhHGF-activator group than in control animals. These results indicate that exogenously administered HGF-activator produces a biologically active HGF from its precursor form and increases the potential for liver regeneration in vivo.     

Involvement of HGF/MET signaling in appendicular muscle development in cartilaginous fish

In amniotes, limb muscle precursors de-epithelialize from the ventral dermomyotome and individually migrate into limb buds. In catsharks, Scyliorhinus, fin muscle precursors are also derived from the ventral dermomyotome, but shortly after de-epithelialization, they reaggregate within the pectoral fin bud and differentiate into fin muscles. Delamination of muscle precursors has been suggested to be controlled by hepatocyte growth factor (HGF) and its tyrosine kinase receptor (MET) in amniotes. Here, we explore the possibility that HGF/MET signaling regulates the delamination of appendicular muscle precursors in embryos of the catshark Scyliorhinus canicula. Our analysis reveals that Hgf is expressed in pectoral fin buds, whereas c-Met expression in fin muscle precursors is rapidly downregulated. We propose that alteration of the duration of c-Met expression in appendicular muscle precursors might underlie the evolution of individually migrating muscle precursors, which leads to the emergence of complex appendicular muscular systems in amniotes.     

Caspase cleavage releases a nuclear protein fragment that stimulates phospholipid scrambling at the plasma membrane

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Caspase-mediated cleavage of the signal-transducing IL-6 receptor subunit gp130

The present study characterizes the molecular mechanisms of CD95L-induced inhibition of IL-6 signaling, which is known to mediate hepatoprotective effects in response to various toxins. CD95L-induced caspase activation leads to degradation of gp130, thereby suppressing IL-6-induced phosphorylation of STAT3 (Tyr⁷⁰⁵) and of tyrosine phosphatase SHP2 (Tyr⁵⁸⁰). Degradation of gp130 protein in response to CD95L was largely prevented after inhibition of caspase 3 or 8. Introduction of a point mutation into a newly identified caspase cleavage site located within position 800–806 (DHVDGGD) of the cytoplasmic tail of gp130 leads to cleavage resistance of the respective receptor in an in vitro assay with recombinant active caspase 3. Correspondingly, the release of a C-terminal gp130-cleavage product of approximately 18 kDa was also inhibited after mutagenesis of this cleavage motif. In conclusion, this study demonstrates that caspase activation by CD95L antagonizes IL-6 signaling by a caspase-mediated cleavage of gp130 thereby probably counteracting hepatoprotective effects of IL-6.     

Engineering the NK1 fragment of hepatocyte growth factor/scatter factor as a MET receptor antagonist

The growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor MET, the tyrosine kinase encoded by the c-MET proto-oncogene, exert major roles in cancer invasion and metastasis and are key targets for therapy. NK1 is an alternative spliced variant of HGF/SF that consists of the N-terminal (N) and first kringle (K1) domains and has partial agonistic activity. NK1 crystallises as a head-to-tail dimer with an extensive inter-protomeric interface resulting from contacts between the two short interdomain linkers and reciprocal contacts between the N and K1 domains. Here we show that a subset of mutants at the NK1 dimer interface, such as the linker mutants Y124A or N127A or the kringle mutant V140A:I142A, bind the MET receptor with affinities comparable to wild-type NK1 but fail to assemble a dimeric, signalling competent NK1-MET complex. These NK1 variants have no detectable agonistic activity on, behave as bona fide receptor antagonists by blocking cell migration and DNA synthesis in target cells and have strong prospects as therapeutics for human cancer.     

Activation of the orphan receptor tyrosine kinase ALK by zinc

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development of the central and peripheral nervous system. The nature of the cognate ligand of this receptor in Vertebrates is still a matter of debate. During synaptic transmission the release of ionic zinc found in vesicles of certain glutamatergic and gabaergic terminals may act as a neuromodulator by binding to pre- or post-synaptic receptors. Recently, zinc has been shown to activate the receptor tyrosine kinase, TrkB, independently of neurotrophins. This activation occurs via increasing the Src family kinase activity. In the present study, we investigated whether the ALK activity could be modulated by extracellular zinc. We first showed that zinc alone rapidly activates ALK. This activation is dependent of ALK tyrosine kinase activity and dimerization of the receptor but is independent of Src family kinase activity. In contrast, addition of sodium pyrithione, a zinc ionophore, led to a further activation of ALK. This stronger activation is dependent of Src family kinase but independent of ALK activity and dimerization. In conclusion, zinc could constitute an endogenous ligand of ALK in vertebrates.     

Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation

IF (intermediate filament) proteins can be cleaved by caspases to generate proapoptotic fragments as shown for desmin. These fragments can also cause filament aggregation. The hypothesis is that disease-causing mutations in IF proteins and their subsequent characteristic histopathological aggregates could involve caspases. GFAP (glial fibrillary acidic protein), a closely related IF protein expressed mainly in astrocytes, is also a putative caspase substrate. Mutations in GFAP cause AxD (Alexander disease). The overexpression of wild-type or mutant GFAP promotes cytoplasmic aggregate formation, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD²²⁵ in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp²²⁵ produces two major cleavage products. While the C-GFAP (C-terminal GFAP) is unable to assemble into filaments, the N-GFAP (N-terminal GFAP) forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP into a human astrocytoma cell line induces the formation of cytoplasmic aggregates, which also disrupt the endogenous GFAP networks. In addition, we generated a neo-epitope antibody that recognizes caspase-cleaved but not the intact GFAP. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two mouse models of AxD. These findings suggest that caspase-mediated GFAP proteolysis may be a common event in the context of both the GFAP mutation and excess.     

The HGF/MET pathway as target for the treatment of multiple myeloma and B-cell lymphomas

Hepatocyte growth factor (HGF) and its receptor MET are essential during embryonic development and throughout postnatal life. However, aberrant MET activation, due to overexpression, mutations, or autocrine ligand production, contributes to the development and progression of a variety of human cancers, often being associated with poor clinical outcome and drug resistance. B cell malignancies arise from B cells that are clonally expanded at different stages of differentiation. Despite major therapeutic advances, most mature B cell malignancies remain incurable and biologically-oriented therapeutic strategies are urgently needed. This review addresses the role of the HGF/MET pathway during B cell development and discusses how its aberrant activation contributes to the development of B cell lymphoproliferative disorders, with particular emphasis on multiple myeloma and diffuse large B cell lymphoma. These insights, combined with the recent development of clinical-grade agents targeting the MET pathway, provide the rationale to envision the HGF/MET pathway as a new promising target for the treatment of B cell malignancies.     

Molecular evolution and domain structure of plasminogen-related growth factors (HGF/SF and HGF1/MSP).

Plasminogen-related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (HGF/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor-like/macrophage stimulating protein (HGF1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron-exon boundaries and kringle sequences indicate that HGF/SF, HGF1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N-terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of HGF/SF, HGF1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide-bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix-strand-helix motif proposed for the PAP domain of HGF/SF; these may be important for receptor interaction. Three-dimensional structures of the 4 kringle and the serine proteinase domains of HGF/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine-binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent HGF/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, HGF/SF, and HGF1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.     

HGF/SF-met signaling in the control of branching morphogenesis and invasion

Hepatocyte growth factor/Scatter factor (HGF/SF) is a multifunctional growth factor which can induce diverse biological events. In vitro, these include scattering, invasion, proliferation and branching morphogenesis. In vivo, HGF/SF is responsible for many processes during embryonic development and a variety of activities in adults, and many of these normal activities have been implicated in its role in tumorgenesis and metastasis. The c-Met receptor tyrosine kinase is the only known receptor for HGF/SF and mediates all HGF/SF induced biological activities. Upon HGF/SF stimulation, the c-Met receptor is tyrosine-phosphorylated which is followed by the recruitment of a group of signaling molecules and/or adaptor proteins to its cytoplasmic domain and its multiple docking sites. This action leads to the activation of several different signaling cascades that form a complete network of intra and extracellular responses. Different combinations of signaling pathways and signaling molecules and/or differences in magnitude of responses contribute to these diverse series of HGF/SF-Met induced activities and most certainly are influenced by cell type as well as different cellular environments. In this review, we focus on HGF/SF-induced branching morphogenesis and invasion, and bring together recent new findings which provide insight into how HGF/SF, via c-Met induces this response.     

Caspases and receptor cleavage

In addition to their established functions in programmed cell death, there is increasing evidence that caspases contribute to several other cellular processes beside of apoptosis. So-called "dependence receptors" represent a group of receptors, which derive from different protein families, but are functionally linked by their capability to regulate cell survival in presence of their respective ligands thereby preserving cellular homeostasis. In the absence of their ligands these receptors are cleaved by caspases thereby releasing pro-apoptotic receptor fragments (e.g. rearranged during transfection [RET]) or permitting the exposure of death domains, which were masked before through other receptor domains (e.g. deleted in colorectal carcinoma [DCC]). Apart from these, there are other plasma membrane receptors such as the epidermal growth factor receptor, which have been identified as substrates of caspases. In terms of signal-transduction, caspase-mediated cleavage of these receptors blocks ligand-induced activation of their intracellular signalling. It is hypothesized that this might be another mechanism, whereby caspases trigger cell toxicity through shut-down of survival signals.     

Comparison of Caspase Genes for the Induction of Apoptosis Following Gene Delivery

The polycation poly(ethylenimine) (PEI) was used to deliver the plasmids coding for various combinations of caspases to Cox-2 overexpressing cancer cell lines. It was found that the expression of the delivered genes, controlled by the Cox-2 promoter, correlated with the expression of the endogenous Cox-2 gene in each cell line in a relatively linear manner. Among the various caspase combination regimens, the combination of caspase 3 plus caspase 9 proved to be the most effective because of an apparent synergy between the two gene products, and produced phosphatidylserine flipping in addition to fragmentation of genomic DNA. Caspase 1 appeared to work independently of either caspases 3 or 9, as no synergistic effect was observed. Transfections with genes coding for granzyme B and caspase 8 yielded a lesser amount of cell death. The delivery of a combination of caspase genes could be readily moved to in vivo research of bladder and colon cancer treatments, and holds great applicability to a wide array of additional tumor types.     

Caspase activation throughout the first wave of spermatogenesis in the rat

Early in postnatal life, the first wave of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This may reflect an adjustment in the number of germ cells that can be adequately maintained by Sertoli cells. Two major pathways (intrinsic and extrinsic) are involved in the process of caspase activation and apoptosis in mammalian cells. The extrinsic pathway is characterized by the oligomerization of death receptors such as FAS or tumor necrosis factor, followed by the activation of caspase-8 and caspase-3. The intrinsic pathway involves the activation of procaspase-9, which in turn activates caspase-3. Extensive information is available concerning apoptotic inducers and their possible mechanisms in the adult rat. However, no data exist regarding the molecular and cellular mechanisms governing physiological cell death during puberty in the male rat. We have studied caspase activation throughout the first wave of spermatogenesis in the rat under physiological conditions, by combining the TUNEL procedure with the localization of active caspases in germ cells. We observed TUNEL-positive germ cells in rats of 5–40 days of age, the highest number being found in 25-day-old rats. TUNEL-positive and caspase-3-positive germ cells appeared as long chains of interconnected germ cells in 25-day-old rats. Caspase activation was assayed by either immunohistochemistry with antibodies against active caspase-3, -8, and -9, or by determining enzymatic activity in seminiferous tubules extracts. Both techniques showed activation of caspase-3, -8, and -9 in 25-day-old rats and low enzymatic activity at other ages. Confocal scanning laser microscopy indicated that active caspase-3, -8, and -9 co-localized with TUNEL-positive cells. Thus, caspase-3, -8, and -9 are active in apoptotic germ cells during the first wave of rat spermatogenesis. The extrinsic pathway of apoptosis may therefore play an important role in germ cell apoptosis during puberty in the rat.This work was financed by a research grant from FONDECYT (1040800) to R.D.M.     

Bidirectional regulation of insulin receptor autophosphorylation and kinase activity by peroxynitrite

Accumulating evidence suggests that enhanced peroxynitrite formation occurs during diabetes. This report describes the effect of peroxynitrite on insulin receptor (IR) function. Addition of peroxynitrite to purified IR resulted in concentration-dependent tyrosine nitration and thiol oxidation. Interestingly, the basal and insulin-stimulated IR autophosphorylation and tyrosine kinase activity were upregulated at low peroxynitrite concentrations, but downregulated at high peroxynitrite concentrations. Concomitantly, peroxynitrite dramatically reduced ¹²⁵I-insulin binding capacity and phosphotyrosine phosphatase activity of IR preparations. Moreover, SIN-1 administration decreased blood glucose levels in normal mice via upregulation of IR/IRS-1 tyrosine phosphorylation. In contrast, SIN-1 markedly increased blood glucose levels in diabetic mice concomitant with downregulation of IR/IRS-1 tyrosine phosphorylation. Taken together, these data provide new insights regarding how peroxynitrite influences IR function in vitro and in vivo, suggesting that peroxynitrite plays a dual role in regulation of IR autophosphorylation and tyrosine kinase activity, and SIN-1 has hyperglycemic effect in diabetic mice.     

Functional androgen receptor confers sensitization of androgen-independent prostate cancer cells to anticancer therapy via caspase activation

Therapeutic resistance remains an unresolved problem in the clinical management of human prostate cancer (PC). Despite initial positive response to androgen ablation therapy (AAT), virtually all PC patients will relapse due to acquisition of hormone refractory disease and selective outgrowth of tumor cells with multidrug resistance phenotype. We here provide the first experimental evidence that restoring a functional androgen receptor (AR) in the androgen-independent prostate cancer PC3 cells enhances their sensitivity to growth arrest and suppresses their colony-forming ability in response to paclitaxel and gamma-irradiation. Furthermore, functional AR increases the susceptibility of these cells to the apoptotic potentials of therapeutic agents, as evidenced by an increase in caspase activity, annexin V binding, and internucleosomal DNA fragmentation, by inducing caspase activation. The abrogation of the cytotoxic effects by 4-hydroxyflutamide suggests a crucial role for AR activation in enhancing the therapeutic sensitivity of these cells in a ligand-independent fashion. Our data thus demonstrate that a functional AR is a prerequisite for effective therapeutic response and that aberrant expression or blockade by AAT may trigger pathways leading to emergence of PC cells with therapeutic resistance phenotype. Since the mainstay of primary therapy for PC has been AAT by pharmaco-therapeutic or surgical means, this study thus provides a new frontier for revising the AAT therapeutic strategy in conjunction with radiation and/or chemotherapeutic agents.     

Molecular mechanisms of LL-37-induced receptor activation: An overview

The human cathelicidin peptide LL-37 plays a crucial role in the immune system on many levels, from the first line of defense in epithelial cells to restoring the tissue after infection. On host cells, the majority of the LL-37-induced effects are mediated via the direct or indirect activation of several structurally unrelated cell surface receptors or intracellular targets. How LL-37 is able to affect multiple receptors is currently not well understood. So far, the mechanistic details underlying receptor activation are poorly investigated and evidence for a conventional ligand/receptor interaction is scarce. Over the past few decades, a large number of studies have reported on the activation of a receptor and/or components of the downstream signal transduction pathway induced by LL-37. This review summarizes the current knowledge on molecular mechanisms underlying LL-37-induced receptor activation.     

二烯丙基二硫通过激活Caspase3和释放Cyt-c诱导Raji细胞凋亡

二烯丙基二硫（diallyl disulfide,DADS）作为天然植物大蒜中的提取物,能抑制多种肿瘤细胞生长,但其抑瘤的分子机制还不十分清楚。在该研究中,作者采用CCK-8（cell counting kit）技术检测发现,DADS能有效地抑制人淋巴瘤Raji细胞增殖,形态学观察、DNA琼脂糖凝胶电泳和流式细胞仪检测证实DADS呈时间和浓度依赖性诱导Raji细胞凋亡, DADS处理细胞24 h后, MCL1和Bcl-2蛋白表达下降,而Bax 和Bak蛋白表达水平无变化,Bid和Caspase3被激活,线粒体中Cyt-c释放增多,用Caspase 抑制剂Z-VAD-FMK能部分阻断DADS诱导人淋巴瘤Raji细胞凋亡, 提示DADS诱导的Raji细胞凋亡作用通过Bcl-2/MCL1-线粒体-caspase3通路介导。     

SPAP2, an Ig family receptor containing both ITIMs and ITAMs

This study reports cloning and characterization of SPAP2, a novel transmembrane protein. The extracellular portion of SPAP2 contains six immunoglobulin-like domains and its intracellular segment has two immunoreceptor tyrosine-based activation motifs (ITAMs) and two immunoreceptor tyrosine-based inhibition motifs (ITIMs). We also identified four alternatively spliced products. Sequence alignment with the genomic database revealed that the SPAP2 gene contains 16 exons and is localized at chromosome 1q21. PCR analyses demonstrated that SPAP2 mRNA is expressed in restricted human tissues including the kidney, salivary gland, adrenal gland, uterus, and bone marrow. Tyrosine-phosphorylated SPAP2 is specifically associated with SH2 domain-containing tyrosine kinases Syk and Zap70 and SH2 domain-containing tyrosine phosphatases SHP-1 and SHP-2. Site-specific mutagenesis studies revealed that tyrosyl residues 650 and 662 embedded in the ITIMs are responsible for the binding of Syk and Zap70 while tyrosyl residues 692 and 722 embedded in the ITIMs are involved in interactions with SHP-1 and SHP-2. Finally, recruitment of SHP-1 to the tyrosine-phosphorylated ITIMs led to a marked activation of the enzyme.