Proapoptotic function of the MET tyrosine kinase receptor through caspase cleavage

The MET tyrosine kinase, the receptor of hepatocyte growth factor-scatter factor (HGF/SF), is known to be essential for normal development and cell survival. We report that stress stimuli induce the caspase-mediated cleavage of MET in physiological cellular targets, such as epithelial cells, embryonic hepatocytes, and cortical neurons. Cleavage occurs at aspartic residue 1000 within the SVD site of the juxtamembrane region, independently of the crucial docking tyrosine residues Y1001 or Y1347 and Y1354. This cleavage generates an intracellular 40-kDa MET fragment containing the kinase domain. The p40 MET fragment itself causes apoptosis of MDCK epithelial cells and embryonic cortical neurons, whereas its kinase-dead version is impaired in proapoptotic activity. Finally, HGF/SF treatment does not favor MET cleavage and apoptosis, confirming the known survival role of ligand-activated MET. Our results show that stress stimuli convert the MET survival receptor into a proapoptotic factor.     

The shadow of death on the MET tyrosine kinase receptor

The MET tyrosine kinase receptor is a high-affinity receptor for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF-MET system is necessary for embryonic development, and aberrant MET signalling favours tumorigenesis and metastasis. MET is a prototype of tyrosine kinase receptor, which is able to counteract apoptosis through the initiation of a survival signal involving notably the PI3K-Akt pathway. Paradoxically, the MET receptor is also able to promote apoptosis when activated by HGF/SF or independently of ligand stimulation. The molecular mechanisms underlying this uncommon response have been recently investigated and revealed dual antiapoptotic or proapoptotic property of MET according to the cell type or stress conditions. Although the involvement of MET in the regulation of integrated biological responses mostly took into account its efficient antiapoptotic function, its proapoptotic responses could also be important for regulation of the survival/apoptosis balance and play a role during the development or tumour progression.     

The MET receptor tyrosine kinase in invasion and metastasis

Various cytokines and soluble growth factors upon interaction with their membrane receptors are responsible for inducing cellular proliferation, differentiation, movement, and protection from anoikis (a planned suicide activated by normal cells in absence of attachment to neighboring cells or extracellular matrix (EMC)). Among those soluble factors a major position is exerted by hepatocyte growth factor (HGF) together with its receptor MET and macrophage-stimulating protein (MSP) in cooperation with its receptor RON.     

Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.     

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.     

Phosphorylation of the MET receptor on juxtamembrane tyrosine residue 1001 inhibits its caspase-dependent cleavage

The MET tyrosine kinase is the hepatocyte growth factor/scatter factor (HGF/SF) receptor, which elicits multiple biological responses in epithelial cells, including cell survival. We previously demonstrated that in stress conditions, the MET receptor is cleaved by caspases within its juxtamembrane region, generating a pro-apoptotic intracellular fragment of 40 kDa. The caspase cleavage site at aspartic acid D1000 is adjacent to tyrosine Y1001, which when phosphorylated upon MET activation, is involved in CBL recruitment, allowing receptor ubiquitination and down regulation. Scanning mutagenesis of the MET juxtamembrane region led us to demonstrate that V999 and D1000 are essential for the caspase cleavage, while D1000 and Y1001 are essential for CBL recruitment. By examining whether overlapping of these sites leads to a functional interference, an inverse relationship was found between generation of p40 MET and phosphorylation of MET, with a direct involvement of phosphorylated Y1001 in protecting MET against its caspase cleavage. A molecular modeling analysis of caspase 3 interaction with the juxtamembrane region of MET confirmed that phosphorylation of this tyrosine is not compatible with its recognition by active caspase 3. These data demonstrate a direct protection mechanism of an activated phosphorylated MET receptor, against its caspase-dependent cleavage.     

Intracellular calcium regulates the tyrosine kinase receptor encoded by the MET oncogene.

Previous work (Gandino, L., Di Renzo, M. F., Giordano, S., Bussolino, F., and Comoglio, P.M. (1990) Oncogene 5, 721-725) has shown that the tyrosine kinase activity of the receptor encoded by the MET protooncogene is negatively modulated by protein kinase C (PKC). We now show that an increase of intracellular Ca2+ has a similar inhibitory effect in vivo, via a PKC-independent mechanism. In GTL-16 cells the p145MET kinase is overexpressed and constitutively phosphorylated on tyrosine. A rapid and reversible decrease of p145MET tyrosine phosphorylation was induced by treatment with the calcium ionophores A23187 or ionomycin. Experiments performed with the ionophores in absence of extracellular calcium showed that a rise in cytoplasmic Ca2+ concentration to 450 nM (due to release from intracellular stores) resulted in a similar effect. These Ca2+ concentrations had no effect on p145MET autophosphorylation in an in vitro kinase assay. This suggests that the effect of Ca2+ on p145MET tyrosine phosphorylation is not direct but may be mediated by Ca(2+)-activated proteins(s). Involvement of Ca(2+)-dependent tyrosine phosphatases was ruled out by experiments carried out in presence of Na2VO4. In vivo labeling with [32P]orthophosphate showed that the rise of intracellular Ca2+ induces serine phosphorylation of p145MET on a specific phosphopeptide. This suggests that Ca2+ negatively modulates p145MET kinase through the phosphorylation of a critical serine residue by a Ca(2+)-activated serine kinase distinct from PKC.     

Triptolide (PG-490) induces apoptosis of dendritic cells through sequential p38 MAP kinase phosphorylation and caspase 3 activation

Dendritic cells (DCs) are the most potent antigen-presenting cells that play crucial roles in the regulation of immune response. Triptolide, an active component purified from the medicinal plant Tripterygium wilfordii Hook F., has been demonstrated to act as a potent immunosuppressive drug capable of inhibiting T cell activation and proliferation. However, little is known about the effects of triptolide on DCs. The present study shows that triptolide does not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10ng/ml, as demonstrated by phosphatidylserine exposure, mitochondria potential decrease, and nuclear DNA condensation. Triptolide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that the anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.     

Interactome disassembly during apoptosis occurs independent of caspase cleavage

Protein–protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas‐mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome‐wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.     

Signal transduction of angiotensin II type 1 receptor through receptor tyrosine kinase

In cultured vascular smooth muscle cells, the angiotensin II (AngII) type-1 (AT(1)) receptor generates growth-promoting signals via the epidermal growth factor (EGF) receptor system. This 'transactivation' mechanism now appears to be utilized by a variety of G-protein-coupled receptors in many cells. The AngII-induced EGF receptor transactivation leads to activation of downstream signaling molecules including Ras, ERK, c-fos, Akt/protein kinase B, and p70 S6 kinase. We propose three possible mechanisms may be involved in the transactivation, (i) an upstream tyrosine kinase, (ii) reactive oxygen species, and (iii) a juxtacrine activation of the EGF receptor ligand. Whether the EGF receptor signal transduction induced by AngII plays an essential role in cardiovascular remodeling remains to be investigated.     

Anaplastic lymphoma kinase is a dependence receptor whose proapoptotic functions are activated by caspase cleavage

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, initially discovered as part of the NPM-ALK fusion protein, resulting from the t(2;5) translocation that is frequently associated with anaplastic large-cell lymphomas. The native ALK protein is normally expressed in the developing and, at a weaker level, adult nervous system. We recently demonstrated that the oncogenic, constitutively kinase-activated NPM-ALK protein was antiapoptotic when expressed in Jurkat lymphoblastic cells treated with cytotoxic drugs. In contrast, we now show that Jurkat cells overexpressing the wild-type ALK receptor are more sensitive to doxorubicin-induced apoptosis than parental cells. Moreover, the ALK protein is cleaved during apoptosis in a caspase-dependent manner. Mutation of aspartic residues to asparagine allowed us to map the caspase cleavage site in the juxtamembrane region of ALK. In order to assess the role of ALK in neural cell-derived tissue, we transiently expressed ALK in the 13.S.1.24 rat neuroblast immortalized cell line. ALK expression led to apoptotic cell death of the neuroblasts. ALK ligation by specific activating antibodies decreased ALK-facilitated apoptosis in both lymphoid and neuronal cell lines. Moreover, ALK transfection reduced the survival of primary cultures of cortical neurons. Thus, ALK has a proapoptotic activity in the absence of ligand, whereas it is antiapoptotic in the presence of its ligand and when the kinase is intrinsically activated. These properties place ALK in the growing family of dependence receptors.     

Caspase cleavage of the MET receptor generates an HGF interfering fragment

The MET tyrosine kinase receptor activated by its ligand HGF/SF, induces several cellular responses, including survival. Nonetheless, the MET receptor is cleaved in stress conditions by caspases within its intracellular region, generating a 40 kDa fragment, p40 MET, with pro-apoptotic properties. Here, we established that this cleavage splits the receptor at the juxtamembrane ESVD site, causing the concomitant generation of p100 MET, corresponding to the entire extracellular region of the MET receptor still spanning the membrane. This fragment is able to bind HGF/SF and to prevent HGF-dependent signaling downstream of full MET, demonstrating its function as a decoy receptor.     

The phosphorylation of tyrosine 332 is necessary for the caspase 3-dependent cleavage of PKCdelta and the regulation of cell apoptosis

Protein kinase C delta (PKCdelta plays a major role in the regulation of cell apoptosis and survival. PKCdelta is cleaved by caspase 3 to generate a constitutively active catalytic domain that mediates both its apoptotic and anti-apoptotic effects. The caspase cleavage site of PKCdelta in the hinge region is flanked by the two tyrosine residues, Y311 and Y332. Here, we examined the role of the phosphorylation of tyrosines 311 and 332 in the cleavage and apoptotic function of PKCdelta using the apoptotic stimuli, TRAIL and cisplatin. Tyrosine 332 was constitutively phosphorylated in the A172 and HeLa cells and was further phosphorylated by TRAIL and cisplatin. This phosphorylation was inhibited by the Src inhibitors, PP2 and SU6656, and by silencing of Src. Treatment of the A172 and HeLa cells with TRAIL induced cleavage of the WT PKCdelta and of the PKCdeltaY311F mutant, whereas a lower level of cleavage was observed in the PKCdeltaY332F mutant. Similarly, a smaller degree of cleavage of the PKCdeltaY332 mutant was observed in LNZ308 cells treated with cisplatin. Mutation of Y332F affected the apoptotic function of PKCdelta; overexpression of the PKCdeltaY332 mutant increased the apoptotic effect of TRAIL, whereas it decreased the apoptotic effect of cisplatin. Inhibition of Src decreased the cleavage of PKCdelta and modified the apoptotic responses of the cells to TRAIL and cisplatin, similar to effect of the PKCdeltaY332F mutant. These results demonstrate that the phosphorylation of tyrosine 332 by Src modulates the cleavage of PKCdelta and the sensitivity of glioma cells to TRAIL and cisplatin.     

Defective posttranslational processing activates the tyrosine kinase encoded by the MET proto-oncogene (hepatocyte growth factor receptor).

The MET proto-oncogene encodes a 190-kDa disulfide-linked heterodimeric receptor (p190 alpha beta) whose tyrosine kinase activity is triggered by the hepatocyte growth factor. The mature receptor is made of two subunits: an alpha chain of 50 kDa and a beta chain of 145 kDa, arising from proteolytic cleavage of a single-chain precursor of 170 kDa (pr170). In a colon carcinoma cell line (LoVo), the precursor is not cleaved and the Met protein is exposed at the cell surface as a single-chain polypeptide of 190 kDa (p190NC). The expression of the uncleaved Met protein is due to defective posttranslational processing, since in this cell line (i) the proteolytic cleavage site Lys-303-Arg-Lys-Lys-Arg-Ser-308 is present in the precursor, (ii) p190NC is sensitive to mild trypsin digestion of the cell surface, generating alpha and beta chains of the correct size, and (iii) the 205-kDa insulin receptor precursor is not cleaved as well. p190NC is a functional tyrosine kinase in vitro and is activated in vivo, as shown by constitutive autophosphorylation on tyrosine. The MET gene is neither amplified nor rearranged in LoVo cells. Overlapping cDNA clones selected from a library derived from LoVo mRNA were sequenced. No mutations were present in the MET-coding region. These data indicate that the tyrosine kinase encoded by the MET proto-oncogene can be activated as a consequence of a posttranslational defect.     

Activation of anaplastic lymphoma kinase receptor tyrosine kinase induces neuronal differentiation through the mitogen-activated protein kinase pathway

Anaplastic lymphoma kinase (ALK) is a novel neuronal orphan receptor tyrosine kinase that is essentially and transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. To determine whether ALK could play a role in neuronal differentiation, we established a model system that allowed us to mimic the normal activation of this receptor. We expressed, in PC12 cells, a chimeric protein in which the extracellular domain of the receptor was replaced by the mouse IgG 2b Fc domain. The Fc domain induced the dimerization and oligomerization of the chimeric protein leading to receptor phosphorylation and activation, thus mimicking the effect of ligand binding, whereas the wild type ALK remained as a monomeric nonphosphorylated protein. Expression of the chimera, but not that of the wild type ALK or of a kinase inactive form of the chimera, induced the differentiation of PC12 cells. Analysis of the signaling pathways involved in this process pointed to an essential role of the mitogen-activated protein kinase cascade. These results are consistent with a role for ALK in neuronal differentiation.     

Execution of macrophage apoptosis by Mycobacterium avium through apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase signaling and caspase 8 activation

Macrophage apoptosis is an important component of the innate immune defense machinery (against pathogenic mycobacteria) responsible for limiting bacillary viability. However, little is known about the mechanism of how apoptosis is executed in mycobacteria-infected macrophages. Apoptosis signal-regulating kinase 1 (ASK1) was activated in Mycobacterium avium-treated macrophages and in turn activated p38 mitogen-activated protein (MAP) kinase. M. avium-induced macrophage cell death could be blocked in cells transfected with a catalytically inactive mutant of ASK1 or with dominant negative p38 MAP kinase arguing in favor of a central role of ASK1/p38 MAP kinase signaling in apoptosis of macrophages challenged with M. avium. ASK1/p38 MAP kinase signaling was linked to the activation of caspase 8. At the same time, M. avium triggered caspase 8 activation, and cell death occurred in a Fas-associated death domain (FADD)-dependent manner. The death signal induced upon caspase 8 activation linked to mitochondrial death signaling through the formation of truncated Bid (t-Bid), its translocation to the mitochondria and release of cytochrome c. Caspase 8 inhibitor (z-IETD-FMK) could block the release of cytochrome c as well as the activation of caspases 9 and 3. The final steps of apoptosis probably involved caspases 9 and 3, since inhibitors of both caspases could block cell death. Of foremost interest in the present study was the finding that ASK1/p38 signaling was essential for caspase 8 activation linked to M. avium-induced death signaling. This work provides the first elucidation of a signaling pathway in which ASK1 plays a central role in innate immunity.     

The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation.

Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.