Proteolytic activity in the tumour microenvironment is an important factor in cancer development since it can also affect intracellular signalling pathways via positive feedback loops that result in ...either increased tumour growth or resistance to anticancer mechanisms. In this study, we demonstrated extracellular cathepsin L-mediated cleavage of epidermal growth factor receptor (EGFR) and identified the cleavage site in the extracellular domain after R224. To further evaluate the relevance of this cleavage, we cloned and expressed a truncated version of EGFR, starting at G225, in HeLa cells. We confirmed the constitutive activation of the truncated protein in the absence of ligand binding and determined possible changes in intracellular signalling. Furthermore, we determined the effect of truncated EGFR protein expression on HeLa cell viability and response to the EGFR inhibitors, tyrosine kinase inhibitor (TKI) erlotinib and monoclonal antibody (mAb) cetuximab. Our data reveal the nuclear localization and phosphorylation of EGFR and signal trancducer and activator of transcription 3 (STAT3) in cells that express the truncated EGFR protein and suggest that these phenomena cause resistance to EGFR inhibitors.
The biological activity of the soluble form of the Notch ligand (sNL) and requirement of the intracellular domain (ICD) of the Notch ligand have been debated. Here we show that soluble Delta1 (sD1) ...activates Notch2 (N2), but much more weakly than full‐length Delta1 (fD1). Furthermore, tracing the N2 molecule after sD1 stimulation revealed that sD1 has a defect in the cleavage releasing ICD of N2 (intracellular cleavage), although it triggers cleavage in the extracellular domain of N2. This represents the molecular basis of the lower activity of sD1 and suggests the presence of an unknown mechanism regulating activation of the intracellular cleavage. The fact that Delta1 lacking its ICD (D1ΔICD) exhibits the phenotype similar to that exhibited by sD1 indicates that the ICD of D1 (D1ICD) is involved in such an as yet unknown mechanism. Furthermore, the findings that D1ΔICD acts in a dominant‐negative fashion against fD1 and that the signal‐transducing activity of sD1 is enhanced by antibody‐mediated cross‐linking suggest that the multi merization of Delta1 mediated by D1ICD may be required for activation of the N2 intracellular cleavage.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Human lactase‐phlorizin hydrolase (LPH), a brush border membrane hydrolase of the small intestine. is synthesized as a precursor molecule that undergoes proteolytic cleavage to yield mature LPH ...(LPHβ) by a trypsin‐like protease (Naim et al., 1987, 1991). Arg868‐Ala869 has been previously proposed to be the putative cleavage site for this processing step. Site‐directed mutagenesis of this monobasic site does not lead to the generation of an uncleaved proLPH species, which strongly suggests the existence of an additional cleavage site. Further analyses of LPH synthesized in different cell lines lend support to this hypothesis. Biosynthetic labeling of human intestinal biopsy samples in the presence of trypsin reveals an LPHβ species that is slightly smaller than the intracellularly cleaved molecule. When the proLPH molecule is screened for potential cleavage sites, two dibasic pairs are revealed upstream of the N‐terminal end of brush border LPH at Lys851‐Arg852 and Arg830‐Lys831. Treatment of proLPH with trypsin for different periods of time supports the idea of at least two cleavage steps, whereby Arg868‐Ala869 represents the final cleavage site that generates LPHβfinal. We propose that the initial cleavage of proLPH takes place intracellularly at a site further away from Arg868‐Ala869, to generate LPHinitial; LPHβinitial is subsequently cleaved extracellularly in the gut lumen, presumably by trypsin, at Arg868‐Ala869 to mature brush border LPH (LPHβfinal).
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Human lactase-phlorizin hydrolase (LPH), a brush border membrane hydrolase of the small intestine, is synthesized as a precursor molecule that undergoes proteolytic cleavage to yield mature LPH ...(LPHbeta) by a trypsin-like protease (Naim et al., 1987, 1991). Arg868-Ala869 has been previously proposed to be the putative cleavage site for this processing step. Site-directed mutagenesis of this monobasic site does not lead to the generation of an uncleaved proLPH species, which strongly suggests the existence of an additional cleavage site. Further analyses of LPH synthesized in different cell lines lend support to this hypothesis. Biosynthetic labeling of human intestinal biopsy samples in the presence of trypsin reveals an LPHbeta species that is slightly smaller than the intracellularly cleaved molecule. When the proLPH molecule is screened for potential cleavage sites, two dibasic pairs are revealed upstream of the N-terminal end of brush border LPH at Lys851-Arg852 and Arg830-Lys831. Treatment of proLPH with trypsin for different periods of time supports the idea of at least two cleavage steps, whereby Arg868-Ala869 represents the final cleavage site that generates LPHbeta. We propose that the initial cleavage of proLPH takes place intracellularly at a site further away from Arg868-Ala869, to generate LPHbeta initial; LPHbeta is subsequently cleaved extracellularly in the gut lumen, presumably by trypsin, at Arg868-Ala869 to mature brush border LPH (LPHbeta initial).
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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