Phosphotyrosine Signaling Networks in Epidermal Growth Factor Receptor Overexpressing Squamous Carcinoma Cells

Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded μLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cγ (PLCγ), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6. Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded μLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cγ (PLCγ), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6. Overexpression and enhanced activation of the epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; HNSCC, head and neck squamous carcinoma; EGFR, epidermal growth factor receptor; IGF-1, insulin-like growth factor-1; FAK, focal adhesion kinase; HB-EGF, heparin-binding epidermal growth factor; SH2, Src homology 2 domain; PTB, phosphotyrosine binding domain; LPA, lysophosphatidic acid; TGFα, transforming growth factor α; IC50, half maximal inhibitory concentration; pY, phosphotyrosine; SCX, strong cation exchange; PLCγ, phospholipase Cγ; PI3-kinase, phosphatidyl inositol-3 kinase; GO, Gene Ontology database; OSI-774, erlotinib (Tarceva). receptor are frequently observed in human cancers (1.Yarden Y. The EGFR family and its ligands in human cancer. Signalling mechanisms and therapeutic kinase in cancer. growth factor for and and activation of the tyrosine activity with poor EGFR and Overexpression of EGF receptor a in of the head and and of EGF receptor shown and the of EGF receptor signaling in the of kinase in cancer. growth factor for and The EGF receptor family of receptor tyrosine kinases, EGFR, and to a of with in the and of internalization and degradation Y. the The EGF receptor a binding by a to tyrosine kinase and Y. of epidermal growth factor receptor and protein and activation of EGF The binding of ligands including EGF, TGFα, and in activation of the receptor tyrosine kinase activity and at multiple tyrosine located in the (1.Yarden Y. The EGFR family and its ligands in human cancer. Signalling mechanisms and therapeutic the as a for the binding of Src homology 2 and phosphotyrosine binding proteins, for example Grb2, and NSP2, which and cell signals. binding in additional interactions to signaling complexes to the by binding of cell through protein and the tyrosine phosphorylation of for and complex at The of EGF receptors on the of and through which receptor signaling example EGF receptors shown to in The EGF for and where in interactions with and the and internalization of the receptor the receptor in for the of through the in the of EGF The EGF for and both EGF receptor protein interactions as as the of receptor complexes the downstream In and shown between cell and in to EGF receptor which in shown to EGF activation of the phosphatidyl to the phosphorylation of the kinase The in human The to of activation and EGF receptor are of for epidermal growth factor receptor the example the insulin-like growth receptor which the in to EGF The of tyrosine kinases in to EGF receptor inhibition are for example of the Src which in the and by lysophosphatidic acid cell-cell and cell adhesion networks through the between growth factor receptors and adhesion the and be to cell to EGF receptor The of cells to growth and in the of adhesion to or cell-cell contacts in the of cell and in cell and in where and cell inhibition The EGF receptor and proteins cell adhesion and shown to and in a complex on receptor activity and cell adhesion and to cell growth cell and by and of focal adhesion The of was to 1) EGF receptor signaling networks cells EGF receptors and 2) signaling proteins and to inhibition of EGF receptor kinase The squamous carcinoma cell line HN5 of and human squamous in and in was used as a model to HN5 cells a of EGF receptor in of TGFα, and are to EGF receptor inhibition. in squamous cell of the head and neck in of and inhibition of EGF receptor signaling shown to growth in of epidermal growth factor tyrosine phosphorylation in human with of receptor inhibition in and in a EGF receptor kinase for EGF receptor kinase inhibition of The inhibition of HN5 cell to EGF receptor with of and receptor affinity were with multiple 1) to proteins and protein complexes associated with EGF receptor signaling and with kinase inhibition. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. of EGF receptor signaling in were The of proteins EGF receptor kinase inhibition by erlotinib of and cell by of epidermal growth factor receptor tyrosine and of EGF receptor kinase activity by addition of EGF was by protein immunoblot and peptide methods of complex protein isotope-coded affinity Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cγ (PLCγ), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF suggesting a complex balance between growth kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6. and were by to by to a of and in were used for to binding and and were in and to for at In receptor to the human epidermal growth factor was were and in was and for on a was by with of with and with of were at In was for with 2 HN5 cells of and human squamous in and in were in with HN5 cell were by and phosphatase to the of protein were The EGF receptor kinase erlotinib was to HN5 cells for or to cells were with for to were with to in of and of phosphatase for was by was by were by with for at to to were with and with HN5 cell for at with complexes were with of at proteins with and in The affinity of protein 2 complexity for protein of proteins or proteins on control and or control binding and the of protein as by were to was by of proteins to with and kinase EGFR kinase family 2 2 receptor and by affinity were and with were the either to complexity and strong cation The of multiple methods 1) the of protein and peptide with with and protein for was a cation with and with and at were by at a cell with a were the to the at a of was a with or and a acid with a of The was with to which a of was with and were on a by of and with a The was to in or in and where was to and were used to the of the In were in the and in with the of in in in were were identified and with and of both and and and and were to and the for and the were to the human was and and tyrosine of were which were as were for of the that protein of and of were proteins with or were in was by the peptide in and protein classes by affinity HN5 and binding 2 protein protein protein 2 control protein 2 protein protein cell protein protein protein growth factor receptor factor protein 2 SH2, protein protein growth factor receptor 3) protein protein kinase protein kinase kinase protein for for SH2, cell protein kinase SH2, kinase transforming and of SH2, protein substrate protein receptor tyrosine kinase, receptor 2 receptor receptor tyrosine kinase adhesion kinase 1) protein protein protein domain; to tyrosine kinase 2 adhesion kinase and SH2, substrate SH2, binding protein binding protein protein protein protein protein protein 2) factor factor factor protein protein protein SH2, protein 2 protein protein associated protein protein protein 2 family kinase, family complex protein kinase protein protein protein protein protein protein protein complex 2 in in a The was used to measure protein between for example EGF receptor with erlotinib or stimulation of receptor activity with affinity were in and with either or to the and to as described The between and peptide a measure to the protein the affinity proteins were identified and while the of was of were which were to peptide were used to and the between proteins and complexes identified by affinity and and affinity were reduced and in the were by a of increasing with a of protein and were reduced in and in with or for at were and in were by in with a of a of of acid and acid were used as were and were used as on peptide was by of the the at a of for proteins to and phosphorylation of tyrosine proteins and complexes were and the HN5 line as a model EGF receptor signaling associated with signaling in cell and HN5 receptors cell in EGF of human squamous carcinoma cells with and of EGF EGF receptor kinase activity to multiple were identified and to proteins, including or in The peptide and were and The proteins identified with or are in and the peptide of EGF receptor in and for are shown in in peptide and were and peptide were by were Gene Ontology where focal and proteins were receptor identified by and methods multiple and for and for growth factor receptor and/or in a In HN5 cells the EGF receptor was the by immunoblot which was to phosphorylated could be by inhibition of EGF receptor kinase The EGF receptor the peptide by by and and by by In model system, EGF receptor the protein by which associated proteins and were The classes of proteins observed and 3) were associated with cell adhesion and and cell and protein and degradation In affinity were where the EGF receptor was used for protein proteins in with identified by affinity selection, and are in known and both and EGF cells could be identified complex protein in both and were EGF receptor kinase activity was with phosphorylation of the sites at sites on the epidermal growth factor and sites of epidermal growth factor (EGF) receptor and are located in of a in EGF was observed complex peptide with and of was observed in both and and a that could be phosphorylated the of both sites was In phosphorylation of complexes with Src kinase identified additional EGFR and could cell inhibition of EGF receptor by erlotinib markedly of EGF receptor at sites Y845, and the tyrosine phosphorylation at and in both the EGF and control HN5 inhibition of EGF receptor kinase The inhibition of EGF receptor kinase activity total EGF receptor by identified by with and identified peptide in a receptor inhibition by erlotinib 2 reduced EGFR at and the phosphorylation of and phosphorylation of EGFR by Src family kinases by immunoblot of the affinity EGF receptor be at of at sites in and to complexity in both with phosphotyrosine binding signaling and with the affinity used for protein example erlotinib was by EGF stimulation to cell and affinity selection, of EGF receptor in the was observed both by and by immunoblot in the of EGF, between control and erlotinib was observed by in and multiple In the of EGF the EGF receptor to and erlotinib where tyrosine was on EGF receptor to the affinity with as the control with in EGF as Erk1/2, PLCγ, and with erlotinib in the of EGF with with the receptor tyrosine kinase, where of the receptor of pharmacological kinase of that phosphotyrosine methods are to the of protein phosphorylation and to the of of the phosphorylation of the HN5 cells by and immunoblot in which the EGF receptor kinase was either with erlotinib or with EGF protein kinase 2 family protein protein 2 substrate protein factor receptor 2 receptor tyrosine kinase adhesion kinase 1) 3) protein protein kinase protein kinase protein kinase protein transforming protein factor protein complex growth factor receptor kinase in a The of proteins and complexes the cell line HN5 were by immunoblot and by multiple of were were where EGF receptor kinase activity was by the EGFR kinase erlotinib for or with or for to and affinity In a EGF receptor was with EGF for or to In both the was used to of proteins the and control protein were with or with and to and of reduced or proteins were by and proteins were by are in The of proteins were by in erlotinib of or EGF of in EGF receptor binding sites for proteins and of cell through protein proteins were by erlotinib and/or EGF and were the by and the between the and phosphotyrosine binding proteins, which the proteins, was and were both down-regulated by where the control could be and The was for where to EGF the were The of in both and receptor affinity EGF receptor The activation of EGF receptor and PI-3 kinase to including EGF receptors to in for phospholipase and with the was reduced by EGF receptor that activation in and activation of as the family by and complexes in cell adhesion and cell-cell and by transient to EGF or to erlotinib for and were and The kinases and are to EGF receptor signaling through the kinase reduced by at where the was in The erlotinib in phosphorylation was with by immunoblot of EGF receptor activity by EGF The which with EGF receptor through the Grb2, as a by and of the and proteins were in the and was observed in the receptor The of in the phosphotyrosine was reduced by EGF receptor kinase inhibition and was by EGF and and of internalization and degradation complexes were including the both and the binding protein and and and and the to the of EGF receptor signaling to functional interactions to EGF phosphorylation of the epidermal growth factor receptor signaling of epidermal growth factor signaling and in human of protein phosphorylation the proteins the carcinoma cell line were by by and to The for for and with EGF receptor at multiple to the of in EGF receptor signaling, the of proteins between the the proteins identified by in cells to functional interactions to EGF were in the phosphotyrosine affinity proteins by stimulation of EGF receptor, Grb2, and were between the phosphotyrosine of cell proteins, by identified proteins, of which were to the receptor, and phosphorylation of the epidermal growth factor receptor signaling and to of cell proteins proteins with EGFR, PLCγ, PI-3 kinase and of proteins in total of epidermal growth factor signaling and in human between the and HN5 cell and the tyrosine protein kinases and protein kinases, multiple functional were the proteins by affinity the of EGF receptor in the model cell IGF-1 receptor and the proteins by affinity Yes, FAK, EphA2, and were observed by affinity a the of tyrosine kinases to the proteins and complexes and the of between example activation of EGF receptor, by or by shown to in the activation of through indirect phosphorylation of the Src the kinase activation In Src known to EGF receptor on and of between Src and epidermal growth factor signaling The activation of Src and kinases was by of EGF receptor was phosphorylated on Y845, a known Src phosphorylation Src phosphorylation at for kinase was observed by immunoblot and was by was phosphorylated on multiple Src family sites with the a known to Src and EGF receptor to by binding to its receptors of between Src and epidermal growth factor of EGF of epidermal growth factor receptor by lysophosphatidic acid and of tyrosine kinase activity of epidermal growth factor receptor in the lysophosphatidic protein kinase addition to HN5 cells and which could be by the EGF receptor kinase reduced phosphorylation of the EGF receptor at Y845, a phosphorylated by Src kinases, with a of EGF receptor and Src family and activation of epidermal growth factor receptors in cells by the of epidermal growth factor by for the of multiple human in family or could be by immunoblot of EGF receptor kinase inhibition a of EGF receptor to Src or of the EGF receptor and Src family kinases The EGF for and IGF-1 receptor was identified by HN5 cells and IGF-1 and phosphorylation of the phosphorylation of was by erlotinib its phosphorylation was EGF and was by or the of and could be through EGF receptor IGF-1 activation of the PI-3 kinase was to EGF receptor while erlotinib phosphorylation and in was observed adhesion proteins including substrate focal adhesion kinases and and were observed in and immunoblot proteins were shown to form interactions with EGF receptor by affinity and shown to with Focal adhesion kinase signaling activities and in the control of cell and as of a complex with for focal adhesion were observed comprising known at and at to the and The phosphorylation at a peptide of to be in the a located with multiple tyrosine by a for Src the tyrosine phosphorylation was unequivocally observed at sites and in the and was for the The tyrosine phosphorylation of the shown to be phosphorylated in by Src Src on tyrosine to of while direct phosphorylation of by EGF receptor The of focal adhesion complexes paxillin, and to be through phosphorylation and in a growth cell and by and of focal adhesion The of focal adhesion proteins was markedly of the EGF receptor by example in HN5 cells with EGF, was and and protein was by EGF and and and was a in focal adhesion complex phosphorylation EGF addition with in cells and that despite EGF phosphorylation of focal adhesion proteins, the phosphotyrosine of the complex was to the phosphatase activation and of focal adhesion complexes growth cell and by and of focal adhesion complexes were the and through and immunoblot were to of EGF receptor kinase inhibition for a of of focal adhesion proteins the phosphotyrosine and erlotinib reduced of by immunoblot was reduced in the in the receptor with a in tyrosine phosphorylation in EGF receptor which was by of paxillin, and the were reduced by EGF receptor inhibition while in the phosphorylation of ACK1, or and were In HN5 transient EGF receptor inhibition or indirectly reduced phosphorylation of cell adhesion The sustained activation of the EGF receptor tyrosine kinase, through or and shown to in and in of epidermal growth factor receptor in the and of by a human EGF receptor Overexpression of the human EGF receptor to The in a model of HNSCC, identified proteins predominantly by EGF receptor and Src family kinases, and proteins that form with proteins, comprising multiple functional classes and The affinity a of EGF receptor and cell adhesion signaling of EGF receptor activation, of with and of kinase inhibition. methods of the of proteins with a The of both EGF receptor kinase and EGF stimulation to and to the of used to including to affinity the of and the of in the phosphorylation both and could be despite the complexity of the peptide mixtures. In proteins a in and the of was by the combined of the to proteins, by to and measure protein was and were for both protein and for The in between was the in of between was the in peptide between was to the of complexity and the for between and be with additional by HN5 cells were shown to multiple proteins NSP2, Grb2, PI3-kinase, PLCγ, and to signaling complexes. activation of the and of receptor signaling by of as a substrate of the epidermal and growth factor of epidermal growth factor of a factor for with EGF receptors and as for of the and of the EGF receptor HN5 cells was observed on Y845, and and and and In addition of and Src to EGF receptor identified as additional tyrosine phosphorylation sites known sites be in binding and as and and as interactions sites of binding sites for and on receptor and the receptors for epidermal growth growth and growth by and for the of and for the of the and of EGF receptor internalization and the and both the and with EGF receptor either or through the and are for degradation of the receptor phosphorylation of the protein and with epidermal growth factor (EGF) receptor EGF of the to the epidermal growth factor Y. and of the epidermal growth factor with to the of internalization complexes to of for example in cells in by receptor tyrosine kinases, including EGF receptor, to the cell and The of receptor degradation are for the family EGF receptor are down-regulated and in to while EGF a of to the cell and enhanced transforming activity of receptors for signaling by The by EGF receptor kinase activity Y. and of the epidermal growth factor and the phosphorylation sites the sites of the epidermal growth factor receptor are for receptor kinase activity and of tyrosine in the and which the of total EGF receptor observed kinase inhibition of the Src family of tyrosine kinase shown to cell cell and and Src shown to with EGF receptor to of receptor binding of between Src and epidermal growth factor Src and with and be by EGF receptor activation of the and Src was shown to to phosphorylation and activation of in cell of kinases Src kinases growth in squamous cell carcinoma of the head and In HN5 the phosphorylation of EGF receptor on kinase in growth factor receptor and interactions in and the phosphorylation of Src on a for Src kinase activation, Src family kinases were in and to downstream In to Src and IGF-1 receptor, where phosphorylation of the kinase for kinase activation, phosphorylation of EGFR on kinase activity Y. tyrosine at the kinase for the transforming activity of human epidermal growth factor as a for downstream of on the epidermal growth factor receptor binding to the protein of EGF receptor kinase activity reduced Src family phosphorylation at Y845, with in cell activation of the of EGF receptor kinase direct on Src kinase activity of kinase by through epidermal growth factor receptor in Src family activation was in on EGF receptor kinase EGF receptor EGFR phosphorylation and phosphorylation of and the phosphotyrosine the Src family for to EGF receptor kinase inhibition. that while EGF receptor associated Src family activity be down-regulated in to receptor kinase while the total of Src family activity suggesting of Src between the IGF-1 receptor and EGF receptor was observed where EGFR kinase the IGF-1 stimulation of and phosphorylation in with where EGF receptor down-regulated phosphorylation The of insulin-like growth factor in human cells the epidermal growth factor receptor tyrosine IGF-1 stimulation reduced phosphorylation in EGF HN5 suggesting between the receptors for to of the The control of and cell-cell associated with cell cell and are to the of cell of focal adhesion and cell-cell complexes were identified by and anti-EGFr and FAK, Pyk2, and were by activation and inhibition of EGF receptor kinase EGF receptor complexes with the of and in the of phosphorylated EGF for cell its kinase activity and to cell of the EGF receptor by EGF addition to a in the of paxillin, and in the phosphotyrosine affinity be by the between EGF receptor activity and focal adhesion FAK, and growth cell and by and of focal adhesion by and that growth factor receptor activation a tyrosine to and focal adhesion cell the EGF receptor kinase was by a of focal adhesion was observed, for example for as by and for and by immunoblot was observed for where the phosphotyrosine affinity was by EGF receptor while with EGF receptor was The transient EGF receptor inhibition to phosphorylation of focal adhesion suggesting a balance between the phosphorylation of focal adhesion and tyrosine phosphatase The and are of the family of proteins and in the of and complexes in cell-cell of with and in phosphorylation of by EGF receptor shown to the complex to and the of the complex with epidermal growth factor with in and phosphorylation of human and binding to and and the of and to to the of squamous carcinoma Y. of squamous cell and carcinoma in the inhibition of EGF receptor kinase the in complexes were a in phosphorylation and/or of the of and the phosphotyrosine was of The as a receptor for the The and cell and with and which are by EGF receptor activation to signaling through kinase the of at in cell and carcinoma to be associated with and to and in and activation and growth signaling and activation of kinase to tyrosine phosphorylation of and activation of and and signaling both and EGF receptor shown to phosphorylation through associated Src family kinases and receptor complexes EGF receptor in a and to EGF receptor phosphorylation at Y845, and epidermal growth factor (EGF) receptor activation and and to phosphorylation of EGF receptor of or by EGF or erlotinib was observed, for complexes to EGF receptor as a signaling in a receptor additional mechanisms by EGF receptor as a signaling of its kinase methods for EGF and the of EGF receptor kinase activity on protein phosphorylation and complex were and complexes with known and poorly described were unequivocally identified by and affinity and interactions between EGF receptor, proteins, downstream signaling and kinases were observed that could be by EGF receptor kinase interactions complexes be by or by affinity by of of affinity to in protein for protein complex and the activation of and are the for EGF receptor kinase as a factor to Y. of EGF receptor and its as a EGF receptors as or The of and of a direct for the EGF receptor kinase in be by of phosphorylated and its known protein interactions between and interactions between EGF receptor, IGF-1 receptor, and signaling in downstream and PI-3 kinase and EGF receptor as a signaling for The of methods for peptide and and in and and and to the of tyrosine kinase signaling The of methods to the of EGF receptor and cell adhesion signaling of proteins complexes kinase activation and inhibition between cells and to EGF receptor for and for with and and and for with