Core Fucosylation Regulates Epidermal Growth Factor Receptor-mediated Intracellular Signaling

Abstract

α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an α1,6-linkage to form core fucosylation in mammals. We recently found that disruption of the Fut8 gene induces severe growth retardation and early postnatal death. To investigate the molecular mechanism involved, we have established embryonic fibroblasts of Fut8+/+ and Fut8–/–, derived from wild-type and Fut8-null mice, respectively. Interestingly, the epidermal growth factor (EGF)-induced phosphorylation levels of the EGF receptor (EGFR) were substantially blocked in Fut8–/– cells, compared with Fut8+/+ cells, while there are no significant changes in the total activities of tyrosine phosphatase for phosphorylated EGFR between two cells. The inhibition of EGFR phosphorylation was completely restored by re-introduction of the Fut8 gene to Fut8–/– cells. Consistent with this, EGFR-mediated JNK or ERK activation was significantly suppressed in Fut8–/– cells. Finally, we found that the core fucosylation of N-glycans is required for the binding of the EGF to its receptor, whereas no effect was observed for the expression levels of EGFR on the cell surface. Collectively, these results strongly suggest that core fucosylation is essential for EGF receptor-mediated biological functions. α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an α1,6-linkage to form core fucosylation in mammals. We recently found that disruption of the Fut8 gene induces severe growth retardation and early postnatal death. To investigate the molecular mechanism involved, we have established embryonic fibroblasts of Fut8+/+ and Fut8–/–, derived from wild-type and Fut8-null mice, respectively. Interestingly, the epidermal growth factor (EGF)-induced phosphorylation levels of the EGF receptor (EGFR) were substantially blocked in Fut8–/– cells, compared with Fut8+/+ cells, while there are no significant changes in the total activities of tyrosine phosphatase for phosphorylated EGFR between two cells. The inhibition of EGFR phosphorylation was completely restored by re-introduction of the Fut8 gene to Fut8–/– cells. Consistent with this, EGFR-mediated JNK or ERK activation was significantly suppressed in Fut8–/– cells. Finally, we found that the core fucosylation of N-glycans is required for the binding of the EGF to its receptor, whereas no effect was observed for the expression levels of EGFR on the cell surface. Collectively, these results strongly suggest that core fucosylation is essential for EGF receptor-mediated biological functions. The remodeling of cell surface growth factor receptors by modification of their oligosaccharide structures is associated with certain functions and biological events (1Akiyama S.K. Yamada Yamada an in the and of glycoproteins have a core and their are by (Fut8) epidermal growth EGF growth epidermal growth EGF growth catalyzes the transfer of a from to the residue of and N-linked oligosaccharides in via α1,6-linkage to form core fucosylation in in The core N-glycans are in a of glycoproteins and are the of core fucosylation is in and the of The of core fucosylation of a for is to with from with and recently that the of the core fucose from the by to and is to have for in growth factor receptor to EGF and growth biological functions cell growth and cell The binding of these to the of EGFR induces activation of its tyrosine to the receptor and the phosphorylation of tyrosine in of The of EGFR and the remodeling of N-glycans on EGFR functions that the binding of EGF to EGFR was significantly by with EGF binding its tyrosine was by of certain that N-glycans are required for the the of a that a in the of and of N-linked oligosaccharides significantly the of EGF to to its receptor, EGFR and EGFR-mediated ERK phosphorylation in or N-linked oligosaccharides on EGFR to for receptor to the of core fucosylation in functions have investigate the functions of the core we Fut8-null of Fut8 induces severe growth early postnatal and changes in the We found in mice, a of core fucose to and a of the induces the expression and activation of to the observed in core the we that EGF receptors are that for the growth a in EGF receptor-mediated observed in Fut8-null and of the Fut8 gene to Fut8-null EGF and the receptor-mediated in embryonic fibroblasts derived from wild-type and Fut8-null were of Fut8 was a oligosaccharide a to were with and the cell was in of and The cell was for Fut8 were in and for The were and were a of were on and to The were with or were an were and with an the total and to cell or EGFR was to or and to and the were blocked with in with and with in for with were a and of was with the with and to of for of were in and for The was by of and with The was with of were to for and was by the surface was were with and with for the were with and in and the cell was with the The were and of EGFR was by an phosphatase Yamada of with EGF for were to and on was with or of Fut8+/+ or Fut8–/– cell the in for EGFR was with in were and for The cell were in and with on for with the were in of was a with were a of in and The was with and for the with was by for S.K. The were with and on of The was with a of Fut8 early postnatal Fut8-null changes in the and growth We have to the for these and found that of receptor is with the changes To the molecular mechanism of of retardation in Fut8–/– mice, we have established embryonic from Fut8+/+ and Fut8–/– of in Fut8–/– by of Fut8 the of of core fucosylation the Fut8 gene was Fut8–/– by the of a the Fut8 activities in Fut8–/– cells, with a that for Fut8+/+ and of Fut8 gene Fut8–/– in of Fut8 the levels of were with in Fut8+/+ of Fut8 Fut8 activities were a an The and Fut8 were and in Fut8+/+ and restored in Fut8–/– cells. re-introduction of Fut8 gene Fut8–/– the the of core N-glycans were by of the the core fucose in these cells. in molecular were strongly with in Fut8+/+ in Fut8–/– The in Fut8–/– was completely by the Fut8 gene to Fut8–/– cells, that the modification by core fucosylation on N-glycans is by the Fut8 of on Fut8–/– cells. of cell from Fut8–/–, and restored were to a of of in of binding with the with in oligosaccharide of cell surface was by The Fut8+/+ Fut8–/– restored the expression levels of core fucosylation on the cell surface were by a the the core structures of N-linked The binding of the was significantly in Fut8–/– cells, compared with Fut8+/+ and the in the Fut8 gene restored of EGFR in Fut8–/– and is a in cell cell and cell have that the remodeling of N-glycans on EGFR the of core fucosylation is required for the biological functions of EGFR was in core fucosylation was on EGFR in Fut8+/+ on EGFR from Fut8–/– cells. The re-introduction of the Fut8 gene restored the core fucosylation that was in Fut8–/– cells. significant in the expression levels of EGFR on cell surface were found by between the The binding of EGF to the of the EGFR induces the activation of its tyrosine to the phosphorylation of receptor tyrosine The levels of EGFR with EGF were in a in Fut8+/+ cells. Interestingly, the was significantly suppressed in Fut8–/– cells, whereas the was in the restored The tyrosine phosphorylation levels are by tyrosine and tyrosine To or tyrosine the levels of phosphorylation of we an phosphatase in and found that there are no significant changes in the total activities of tyrosine phosphatase for phosphorylated EGFR between Fut8-null and wild-type we found that levels of EGFR in Fut8-null were that in wild-type these results strongly suggest that the core fucosylation of EGFR its biological phosphorylation levels of EGFR EGF of the EGFR from Fut8–/–, and restored cells. EGFR was from cell with and to The were by and with restored restored cell surface and of EGFR from Fut8–/–, and restored cells. were and were with The were to were with EGF the for and in cell were with the were to and the were with and of levels of EGFR from was by the phosphatase The was with or of Fut8+/+ or Fut8–/– cell the for EGFR was with with were in The was by of and with EGFR was with of JNK and ERK in Fut8–/– is established that EGF the to cell To core fucosylation is for EGFR-mediated JNK and were by with and ERK JNK and ERK phosphorylation by were in cells, whereas activation of JNK or ERK was in of with EGF and Consistent with the phosphorylation levels of EGFR in the of JNK and ERK activation in was in the restored cells. these in to EGF between Fut8+/+ and Fut8–/– were of EGF the binding of EGF to its receptor is by a of core that EGFR activation the is that of phosphorylation of ERK by was observed in we significant changes in growth the that growth factor receptor-mediated cell growth of phosphorylated JNK and ERK in Fut8–/– cells. of Fut8–/–, or the restored were with EGF and growth factor the for and in cell were to The were with and of EGF to the mechanism by core fucosylation the EGF a binding of to EGFR in of was in the binding of to EGFR was in Fut8–/– compared with Fut8+/+ or the restored whereas levels of binding were found that and binding of EGFR were in Fut8+/+ are and for and and the restored are for and EGFR was in Fut8–/– these results suggest that the of N-glycans by core fucosylation on EGFR the binding EGF to is required and for for the of of binding between Fut8–/–, and the cells. binding of to these cells. The binding of to the was a a of with are the of The results were in two of the binding The are the of Fut8+/+ Fut8–/– restored structures of a of growth factor receptors and to to the and biological of the the the of Fut8 expression and its core on EGF in Fut8–/–, and the re-introduction of Fut8 gene to Fut8–/– was We found that the phosphorylation of EGFR was in Fut8–/– cells, compared with Fut8+/+ cells. Consistent with EGFR-mediated the phosphorylation of JNK or ERK was in Fut8–/– cells. in Fut8–/– were completely by re-introduction of the Fut8 gene to Fut8–/– cells. the levels of phosphorylation of EGFR were in mice, compared with wild-type results for the that core fucosylation an in EGFR-mediated biological of that the oligosaccharide of the EGFR is for its the of oligosaccharide between an inhibition of EGF binding to the cell surface and EGFR Consistent with this, the of in the binding of EGF to EGFR and receptor-mediated ERK in the of the of N-glycans to the expression levels of EGFR on cell surface. was that N-glycans of EGFR receptors by catalyzes the of an in the of EGFR to its expression levels on cell surface Collectively, these results suggest that is an in receptor-mediated no that core fucosylation EGFR functions have to N-glycans of EGFR from have and found to a of core N-glycans the the core levels the expression of Fut8 is for in the biological functions for the of core in are EGFR a in cell and The of functions in the of EGFR to EGF in Fut8–/– completely by re-introduction of the Fut8 core fucosylation EGFR expression on the cell that core fucosylation changes in is by the binding of EGF to its receptor is in Fut8–/– cells, compared with that in Fut8+/+ or restored that the binding of EGF to EGFR in and the of no significant changes in EGFR-mediated between with of EGF was and and Consistent with this, a that and binding of EGFR were in Fut8+/+ and restored cells. binding of EGFR was in Fut8–/– the for by the that core fucosylation the binding of is required and for The for the to is to that core fucosylation the biological functions of growth and molecular receptors cell and cell we found that was in We found that of Fut8 in the of in and and of cell and in we of the biological of the core fucosylation on EGFR and EGFR-mediated The the that N-glycans glycoproteins on cell the N-glycans have and the molecular of core cell growth and cell in Fut8–/– The remodeling of cell surface growth factor receptors by modification of their oligosaccharide structures is associated with certain functions and biological events (1Akiyama S.K. Yamada Yamada an in the and of glycoproteins have a core and their are by α1,6-Fucosyltransferase (Fut8) epidermal growth EGF growth epidermal growth EGF growth catalyzes the transfer of a from to the residue of and N-linked oligosaccharides in via α1,6-linkage to form core fucosylation in in The core N-glycans are in a of glycoproteins and are the of core fucosylation is in and the of The of core fucosylation of a for is to with from with and recently that the of the core fucose from the by to and is to have for in growth factor receptor to EGF and growth biological functions cell growth and cell The binding of these to the of EGFR induces activation of its tyrosine to the receptor and the phosphorylation of tyrosine in of The of EGFR and the remodeling of N-glycans on EGFR functions that the binding of EGF to EGFR was significantly by with EGF binding its tyrosine was by of certain that N-glycans are required for the the of a that a in the of and of N-linked oligosaccharides significantly the of EGF to to its receptor, EGFR and EGFR-mediated ERK phosphorylation in or N-linked oligosaccharides on EGFR to for receptor to the of core fucosylation in functions have To investigate the functions of the core we Fut8-null of Fut8 induces severe growth early postnatal and changes in the We found in mice, a of core fucose to and a of the induces the expression and activation of to the observed in core the we that EGF receptors are that for the growth a in EGF receptor-mediated observed in Fut8-null and of the Fut8 gene to Fut8-null EGF and the receptor-mediated in cells. embryonic fibroblasts derived from wild-type and Fut8-null were of Fut8 was a oligosaccharide a to were with and the cell was in of and The cell was for Fut8 were in and for The were and were a of were on and to The were with or were an were and with an the total and to cell or EGFR was to or and to and the were blocked with in with and with in for with were a and of was with the with and to of for of were in and for The was by of and with The was with of were to for and was by the surface was were with and with for the were with and in and the cell was with the The were and of EGFR was by an phosphatase Yamada of with EGF for were to and on was with or of Fut8+/+ or Fut8–/– cell the in for EGFR was with in were and for The cell were in and with on for with the were in of was a with were a of in and The was with and for the with was by for S.K. The were with and on of The was with a embryonic fibroblasts derived from wild-type and Fut8-null were Fut8 of Fut8 was a oligosaccharide a to were with and the cell was in of and The cell was for Fut8 were in and for The were and were a of were on and to The were with or were an were and with an the total and to cell or EGFR was to or and to and the were blocked with in with and with in for with were a and of was with the with and to of for of were in and for The was by of and with The was with of were to for and was by the surface was were with and with for the were with and in and the cell was with the The were and of EGFR was by an phosphatase Yamada of with EGF for were to and on was with or of Fut8+/+ or Fut8–/– cell the in for EGFR was with in were and for The cell were in and with on for with the were in of was a with EGF were a of in and The was with and for the with was by for S.K. The were with and on of The was with a of Fut8 early postnatal Fut8-null changes in the and growth We have to the for these and found that of receptor is with the changes To the molecular mechanism of of retardation in Fut8–/– mice, we have established embryonic from Fut8+/+ and Fut8–/– of in Fut8–/– by of Fut8 the of of core fucosylation the Fut8 gene was Fut8–/– by the of a the Fut8 activities in Fut8–/– cells, with a that for Fut8+/+ and of Fut8 gene Fut8–/– in of Fut8 the levels of were with in Fut8+/+ the the of core N-glycans were by of the the core fucose in these cells. in molecular were strongly with in Fut8+/+ in Fut8–/– The in Fut8–/– was completely by the Fut8 gene to Fut8–/– cells, that the modification by core fucosylation on N-glycans is by the Fut8 of on Fut8–/– cells. of cell from Fut8–/–, and restored were to a of of in of binding with the with in oligosaccharide of cell surface was by The Fut8+/+ Fut8–/– restored the expression levels of core fucosylation on the cell surface were by a the the core structures of N-linked The binding of the was significantly in Fut8–/– cells, compared with Fut8+/+ and the in the Fut8 gene restored of EGFR in Fut8–/– and is a in cell cell and cell have that the remodeling of N-glycans on EGFR the of core fucosylation is required for the biological functions of EGFR was in core fucosylation was on EGFR in Fut8+/+ on EGFR from Fut8–/– cells. The re-introduction of the Fut8 gene restored the core fucosylation that was in Fut8–/– cells. significant in the expression levels of EGFR on cell surface were found by between the The binding of EGF to the of the EGFR induces the activation of its tyrosine to the phosphorylation of receptor tyrosine The levels of EGFR with EGF were in a in Fut8+/+ cells. Interestingly, the was significantly suppressed in Fut8–/– cells, whereas the was in the restored The tyrosine phosphorylation levels are by tyrosine and tyrosine To or tyrosine the levels of phosphorylation of we an phosphatase in and found that there are no significant changes in the total activities of tyrosine phosphatase for phosphorylated EGFR between Fut8-null and wild-type we found that levels of EGFR in Fut8-null were that in wild-type these results strongly suggest that the core fucosylation of EGFR its biological phosphorylation levels of EGFR EGF of the EGFR from Fut8–/–, and restored cells. EGFR was from cell with and to The were by and with restored restored cell surface and of EGFR from Fut8–/–, and restored cells. were and were with The were to were with EGF the for and in cell were with the were to and the were with and of levels of EGFR from was by the phosphatase The was with or of Fut8+/+ or Fut8–/– cell the for EGFR was with with were in The was by of and with EGFR was with of JNK and ERK in Fut8–/– is established that EGF the to cell To core fucosylation is for EGFR-mediated JNK and were by with and ERK JNK and ERK phosphorylation by were in cells, whereas activation of JNK or ERK was in of with EGF and Consistent with the phosphorylation levels of EGFR in the of JNK and ERK activation in was in the restored cells. these in to EGF between Fut8+/+ and Fut8–/– were of EGF the binding of EGF to its receptor is by a of core that EGFR activation the is that of phosphorylation of ERK by was observed in we significant changes in growth the that growth factor receptor-mediated cell growth of phosphorylated JNK and ERK in Fut8–/– cells. of Fut8–/–, or the restored were with EGF and growth factor the for and in cell were to The were with and of EGF to the mechanism by core fucosylation the EGF a binding of to EGFR in of was in the binding of to EGFR was in Fut8–/– compared with Fut8+/+ or the restored whereas levels of binding were found that and binding of EGFR were in Fut8+/+ are and for and and the restored are for and EGFR was in Fut8–/– these results suggest that the of N-glycans by core fucosylation on EGFR the binding EGF to is required and for for the of of binding between Fut8–/–, and the cells. binding of to these cells. The binding of to the was a a of with are the of The results were in two of the binding The are the of Fut8+/+ Fut8–/– restored The of Fut8 early postnatal Fut8-null changes in the and growth We have to the for these and found that of receptor is with the changes To the molecular mechanism of of retardation in Fut8–/– mice, we have established embryonic from Fut8+/+ and Fut8–/– of in Fut8–/– by of Fut8 the of of core fucosylation the Fut8 gene was Fut8–/– by the of a the Fut8 activities in Fut8–/– cells, with a that for Fut8+/+ and of Fut8 gene Fut8–/– in of Fut8 the levels of were with in Fut8+/+ cells. the the of core N-glycans were by of the the core fucose in these cells. in molecular were strongly with in Fut8+/+ in Fut8–/– The in Fut8–/– was completely by the Fut8 gene to Fut8–/– cells, that the modification by core fucosylation on N-glycans is by the Fut8 the expression levels of core fucosylation on the cell surface were by a the the core structures of N-linked The binding of the was significantly in Fut8–/– cells, compared with Fut8+/+ and the in the Fut8 gene restored of EGFR in Fut8–/– and is a in cell cell and cell have that the remodeling of N-glycans on EGFR the of core fucosylation is required for the biological functions of EGFR was in core fucosylation was on EGFR in Fut8+/+ on EGFR from Fut8–/– cells. The re-introduction of the Fut8 gene restored the core fucosylation that was in Fut8–/– cells. significant in the expression levels of EGFR on cell surface were found by between the The binding of EGF to the of the EGFR induces the activation of its tyrosine to the phosphorylation of receptor tyrosine The levels of EGFR with EGF were in a in Fut8+/+ cells. Interestingly, the was significantly suppressed in Fut8–/– cells, whereas the was in the restored The tyrosine phosphorylation levels are by tyrosine and tyrosine To or tyrosine the levels of phosphorylation of we an phosphatase in and found that there are no significant changes in the total activities of tyrosine phosphatase for phosphorylated EGFR between Fut8-null and wild-type we found that levels of EGFR in Fut8-null were that in wild-type these results strongly suggest that the core fucosylation of EGFR its biological functions. of JNK and ERK in Fut8–/– is established that EGF the to cell To core fucosylation is for EGFR-mediated JNK and were by with and ERK JNK and ERK phosphorylation by were in cells, whereas activation of JNK or ERK was in of with EGF and Consistent with the phosphorylation levels of EGFR in the of JNK and ERK activation in was in the restored cells. these in to EGF between Fut8+/+ and Fut8–/– were of EGF the binding of EGF to its receptor is by a of core that EGFR activation the is that of phosphorylation of ERK by was observed in we significant changes in growth the that growth factor receptor-mediated cell growth of EGF to the mechanism by core fucosylation the EGF a binding of to EGFR in of was in the binding of to EGFR was in Fut8–/– compared with Fut8+/+ or the restored whereas levels of binding were found that and binding of EGFR were in Fut8+/+ are and for and and the restored are for and EGFR was in Fut8–/– these results suggest that the of N-glycans by core fucosylation on EGFR the binding EGF to is required and for for the of structures of a of growth factor receptors and to to the and biological of the the the of Fut8 expression and its core on EGF in Fut8–/–, and the re-introduction of Fut8 gene to Fut8–/– was We found that the phosphorylation of EGFR was in Fut8–/– cells, compared with Fut8+/+ cells. Consistent with EGFR-mediated the phosphorylation of JNK or ERK was in Fut8–/– cells. in Fut8–/– were completely by re-introduction of the Fut8 gene to Fut8–/– cells. the levels of phosphorylation of EGFR were in mice, compared with wild-type results for the that core fucosylation an in EGFR-mediated biological of that the oligosaccharide of the EGFR is for its the of oligosaccharide between an inhibition of EGF binding to the cell surface and EGFR Consistent with this, the of in the binding of EGF to EGFR and receptor-mediated ERK in the of the of N-glycans to the expression levels of EGFR on cell surface. was that N-glycans of EGFR receptors by catalyzes the of an in the of EGFR to its expression levels on cell surface Collectively, these results suggest that is an in receptor-mediated no that core fucosylation EGFR functions have to N-glycans of EGFR from have and found to a of core N-glycans the the core levels the expression of Fut8 is for in the biological functions for the of core in are EGFR a in cell and The of functions in the of EGFR to EGF in Fut8–/– completely by re-introduction of the Fut8 core fucosylation EGFR expression on the cell that core fucosylation changes in is by the binding of EGF to its receptor is in Fut8–/– cells, compared with that in Fut8+/+ or restored that the binding of EGF to EGFR in and the of no significant changes in EGFR-mediated between with of EGF was and and Consistent with this, a that and binding of EGFR were in Fut8+/+ and restored cells. binding of EGFR was in Fut8–/– the for by the that core fucosylation the binding of is required and for The for the to is to that core fucosylation the biological functions of growth and molecular receptors cell and cell we found that was in We found that of Fut8 in the of in and and of cell and in we of the biological of the core fucosylation on EGFR and EGFR-mediated The the that N-glycans glycoproteins on cell the N-glycans have and the molecular of core cell growth and cell in Fut8–/– structures of a of growth factor receptors and to to the and biological of the the the of Fut8 expression and its core on EGF in Fut8–/–, and the re-introduction of Fut8 gene to Fut8–/– was We found that the phosphorylation of EGFR was in Fut8–/– cells, compared with Fut8+/+ cells. Consistent with EGFR-mediated the phosphorylation of JNK or ERK was in Fut8–/– cells. in Fut8–/– were completely by re-introduction of the Fut8 gene to Fut8–/– cells. the levels of phosphorylation of EGFR were in mice, compared with wild-type results for the that core fucosylation an in EGFR-mediated biological functions. of that the oligosaccharide of the EGFR is for its the of oligosaccharide between an inhibition of EGF binding to the cell surface and EGFR Consistent with this, the of in the binding of EGF to EGFR and receptor-mediated ERK in the of the of N-glycans to the expression levels of EGFR on cell surface. was that N-glycans of EGFR receptors by catalyzes the of an in the of EGFR to its expression levels on cell surface Collectively, these results suggest that is an in receptor-mediated To no that core fucosylation EGFR functions have to N-glycans of EGFR from have and found to a of core N-glycans the the core levels the expression of Fut8 is for in the biological functions for the of core in are EGFR a in cell and The of functions in the of EGFR to EGF in Fut8–/– completely by re-introduction of the Fut8 core fucosylation EGFR expression on the cell that core fucosylation changes in is by the binding of EGF to its receptor is in Fut8–/– cells, compared with that in Fut8+/+ or restored cells. that the binding of EGF to EGFR in and the of no significant changes in EGFR-mediated between with of EGF was and and Consistent with this, a that and binding of EGFR were in Fut8+/+ and restored cells. binding of EGFR was in Fut8–/– the for by the that core fucosylation the binding of is required and for The for the to is to that core fucosylation the biological functions of growth and molecular receptors cell and cell we found that was in We found that of Fut8 in the of in and and of cell and in we of the biological of the core fucosylation on EGFR and EGFR-mediated The the that N-glycans glycoproteins on cell the N-glycans have and the molecular of core cell growth and cell in Fut8–/–