IKK-i and TBK-1 are Enzymatically Distinct from the Homologous Enzyme IKK-2

Abstract

NF-κB is sequestered in the cytoplasm by the inhibitory IκB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IκBs leading to their degradation that results in NF-κB activation. IKK-1 and IKK-2 are two direct IκB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and their of We that and rhTBK-1 are to We by and that and rhTBK-1 are in the and that phosphorylation is and rhTBK-1 have the of IKK-2 the of in the of and IKKs are their their NF-κB is sequestered in the cytoplasm by the inhibitory IκB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IκBs leading to their degradation that results in NF-κB activation. IKK-1 and IKK-2 are two direct IκB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and their of We that and rhTBK-1 are to We by and that and rhTBK-1 are in the and that phosphorylation is and rhTBK-1 have the of IKK-2 the of in the of and IKKs are their their NF-κB that in the of of in and NF-κB is sequestered in the cytoplasm in by of the IκB of inhibitory the Stimulation of cells results in and degradation of NF-κB and of Two IκB and IKK-2 the in have and by is phosphorylation and by IKK-1 and IKK-2 are in human and to the in the of NF-κB by of agonists is that and IKK-2 is the IKK-1 and IKK-2 are in the and in the the and of IKK-1 and IKK-2 are by are the phosphorylation and by have that is NF-κB in to of and and IKK-2 two of IKK-1 and IKK-2 have IKK-i and and of of results in NF-κB IKK-i and to IKK-2 in the and in the the of IKK-i and IKK-2 of IKK-i and IKK-2 in in and IKK-i is in cells and and is and expressed, the of in the in in to agonists and IKK-i and in and and the IKK-2 and IKK-1 cells that have agonists is that the NF-κB have in and in their their and of and have purified to homogeneity and by of rhTBK-1 purified and have cloned, in and purified and rhTBK-1 to and their to and and We that and rhTBK-1 the to IKK-i and is the IKK-1 and that the of is rhTBK-1 and We that and and are their and that phosphorylation is We by that and rhTBK-1 are in the and that of to results in and is of and rhTBK-1 of of and that the and rhTBK-1 are and the of of to their in NF-κB activation. the of and rhTBK-1 that their the NF-κB of the the and and their and in the to of IKK-i and and and and to the the the to the of IKK-i the of the the of the of the of IKK-i and and of purified and to of and to the the the to the of the of the the of the of the of and and of purified and in the and identified in IKK-i and to by the in the to the the the to the in the and of in the the and the in the the and the are the the the to the and to that IKK-i IKK-i IKK-i IKK-i and and IKK-i in cells the the and to recombinant human and in by the of the and to cells of recombinant the of the and of in of to cells that in the and the cells of the recombinant of and cells of by and of recombinant proteins. cells of of to the cells and the and and the of of of cells IKK-i and and the to of in to the and to of and of IKK-i in to the to of by in to the to of in and of of cells to and of to the and to of and of in to the to of by to the to of and the and and and of and rhTBK-1 by in by the to and by IKK-i and and of rhTBK-1 of by to and in and recombinant and to the to the of the and the and in of the in and in the in to by the of and by of the of and to in the of of the the the identified by the of of the of the of the and to the of the to of the to of and IKK-2 and of IKK-i of of in of by the of of the to by and and in the of of the by the of of in is in of to of to and of to by the of of and results to the the in in and of is the of to the of of to the is of of purified of in the of the the of IKK-i and and to IKK-i and and to IKK-2 and have recently of IKK-1 and IKK-2 in their and and have purified to homogeneity and by the of and rhTBK-1 have the and have cloned, expressed, and purified and rhTBK-1 and their to of and and rhTBK-1 the in and purified to the by by and the of and rhTBK-1 by and rhTBK-1 and to kinases. in and IKK-1 and and IKK-i are IκB are to We the of and rhTBK-1 of the and of and and and rhTBK-1 the of and rhTBK-1 to and in of IKK-i and to that and IKK-1 and IKK-2 and are of have in of of and rhTBK-1 and to to to to in and rhTBK-1 are and is and and results the the is and and is and that is in that the of is the of the by of and the have that is the IKK-1 and IKK-2 are the of and is to is of and rhTBK-1 is of IKK-1 and IKK-2 are the and and and the that in IKK-i the is to the of phosphorylation of the in the of IKK-i and rhTBK-1 and are to that of and and that the in to of the to and the of of the results the in that the to that the of the and the the and rhTBK-1 to by to the of the identified the in and rhTBK-1 and rhTBK-1 and are by phosphorylation of their of and rhTBK-1 is phosphorylation in the and TBK-1. and and recombinant recombinant of of of the are of the identified of of the and in the of in of the the in the identified by the of the of phosphorylation by the to of the is and the the is identified to the of phosphorylation of IKK-i and in the of of IKK-i and in rhTBK-1 and rhTBK-1 cloned, expressed, and purified to of the purified the rhTBK-1 results are in the results by and that IKK-i and results to to the of and rhTBK-1 the rhTBK-1 and of the and rhTBK-1 the the of the is to the of results are to and in results of results in by of of the and of the IKK-i that purified and rhTBK-1 have and their kinases. of have the in the of and rhTBK-1 the have the of IKK-2 of the have their of of and and and of and and of rhTBK-1 rhTBK-1 and the of in and rhTBK-1 in cells have to of IKK-1 and IKK-2 and that the the in the of and rhTBK-1 We IKK-1 of and to in the and rhTBK-1 of the in and rhTBK-1 to the to and that of the to the and that the phosphorylation of and in phosphorylation of the to have of the to the phosphorylation of by and in the and of and to to in in the of the is that the phosphorylation of and rhTBK-1 to of to the by the and the the of the rhTBK-1 the and rhTBK-1 their in the in the by the in the to the to the the and that that and of the of and rhTBK-1 have that the in cells by IKK-2 in the and that the IKK-2 are and and that the IKK-2 is the to the the the are and and that the IKK-2 is and of the IKK-2 by is to by that is the and results that the of and rhTBK-1 are to and that of have that is of the in the of and in the of and rhTBK-1 is the that the is the two is the rhTBK-1 the and and rhTBK-1 and to results and that the and of and in the and and and rhTBK-1 the to the of the of and that the of and rhTBK-1 are and the of of and by of of and that their TBK-1. of by to the of and of rhTBK-1 by to the of and of and by of in in the have cloned, expressed, and purified and rhTBK-1 and their of and the to IKK-1 and IKK-i and are and rhTBK-1 of and rhTBK-1 are that of to their that the identified and rhTBK-1 is the of IKK-2 that of of IKK-2 in the NF-κB and IKK-i IKK-2 kinases. IKK-i and of and that the of to the in the of the is the of and rhTBK-1 is is in in the IKK-1 and IKK-2 have to IKK-i and the and in the of the of the of of IKK-i and to their in NF-κB activation. NF-κB that in the of of in and NF-κB is sequestered in the cytoplasm in by of the IκB of inhibitory the Stimulation of cells results in and degradation of NF-κB and of Two IκB and IKK-2 the in have and by is phosphorylation and by IKK-1 and IKK-2 are in human and to the in the of NF-κB by of agonists IκB recombinant human IκB recombinant human IKK-1 is that and IKK-2 is the IKK-1 and IKK-2 are in the and in the the and of IKK-1 and IKK-2 are by are the phosphorylation and by have that is NF-κB in to of and and IKK-2 two of IKK-1 and IKK-2 have IKK-i and and of of results in NF-κB IKK-i and to IKK-2 in the and in the the of IKK-i and IKK-2 of IKK-i and IKK-2 in in and IKK-i is in cells and and is and expressed, the of in the in in to agonists and IKK-i and in and and the IKK-2 and IKK-1 cells that have agonists is that the NF-κB have in and in their their and of and have purified to homogeneity and by of rhTBK-1 purified and have cloned, in and purified and rhTBK-1 to and their to and and We that and rhTBK-1 the to IKK-i and is the IKK-1 and that the of is rhTBK-1 and We that and and are their and that phosphorylation is We by that and rhTBK-1 are in the and that of to results in and is of and rhTBK-1 of of and that the and rhTBK-1 are and the of of to their in NF-κB activation. the of and rhTBK-1 that their the NF-κB of the the and and their and in the to of IKK-i and and and and to the the the to the of IKK-i the of the the of the of the of IKK-i and and of purified and to of and to the the the to the of the of the the of the of the of and and of purified and in the and identified in IKK-i and to by the in the to the the the to the in the and of in the the and the in the the and the are the the the to the and to that IKK-i IKK-i IKK-i IKK-i and and IKK-i in cells the the and to recombinant human and in by the of the and to cells of recombinant the of the and of in of to cells that in the and the cells of the recombinant of and cells of by and of recombinant proteins. cells of of to the cells and the and and the of of of cells IKK-i and and the to of in to the and to of and of IKK-i in to the to of by in to the to of in and of of cells to and of to the and to of and of in to the to of by to the to of and the and and and of and rhTBK-1 by in by the to and by IKK-i and and of rhTBK-1 of by to and in and recombinant and to the to the of the and the and in of the in and in the in to by the of and by of the of and to in the of of the the the identified by the of of the of the of the and to the of the to of the to of and IKK-2 and of IKK-i of of in of by the of of the to by and and in the of of the by the of of in is in of to of to and of to by the of of and results to the the in in and of is the of to the of of to the is of of purified of in the of the the of IKK-i and and to IKK-i and and to IKK-2 of the the and and their and in the to of IKK-i and and of the the and and their and in the to of IKK-i and and and and to the the the to the of IKK-i the of the the of the of the of IKK-i and and of purified and to of and to the the the to the of the of the the of the of the of and and of purified and in the and identified in IKK-i and to by the in the to the the the to the in the and of in the the and the in the the and the are the the the to the and to that IKK-i IKK-i IKK-i IKK-i and and IKK-i in cells the the and to recombinant human and in by the of the and to cells of recombinant the of the and of in of to cells that in the and the cells of the recombinant of and cells of by and of recombinant proteins. cells of of to the cells and the and to the the the to the of IKK-i the of the the of the of the of IKK-i and and of purified and to of and to the the the to the of the of the the of the of the of and and of purified and in the and identified in IKK-i and to by the in the to the the the to the in the and of in the the and the in the the and the are the the the to the and to that IKK-i IKK-i IKK-i IKK-i and and IKK-i in cells the the and to recombinant human and in by the of the and to cells of recombinant the of the and of in of to cells that in the and the cells of the recombinant of and cells of by and of recombinant proteins. cells of of to the cells and the and to the the the to the of IKK-i the of the the of the of the of IKK-i and and of purified and to and to the the the to the of IKK-i the of the the of the of the of IKK-i and and of purified and to of and to the the the to the of the of the the of the of the of and and of purified and in the and identified and to the the the to the of the of the the of the of the of and and of purified and in the and identified in IKK-i and to by the in the to the the the to the in the and of in the the and the in the the and the are the the the to the and to that IKK-i IKK-i IKK-i IKK-i and in IKK-i and to by the in the to the the the to the in the and of in the the and the in the the and the are the the the to the and to that IKK-i IKK-i IKK-i IKK-i and and IKK-i in cells the the and to recombinant human and in by the of the and to cells of recombinant the of the and of in of to cells that in the and the cells of the recombinant of and cells of by and of recombinant proteins. cells of of to the cells and the and IKK-i in cells the the and to recombinant human and in by the of the and to cells of recombinant the of the and of in of to cells that in the and the cells of the recombinant of and cells of by and of recombinant proteins. cells of of to the cells and the and and the of of of cells IKK-i and and the to of in to the and to of and of IKK-i in to the to of by in to the to of in and of of cells to and of to the and to of and of in to the to of by to the to of and the and and and and the of of of cells IKK-i and and the to of in to the and to of and of IKK-i in to the to of by in to the to of in and of of cells to and of to the and to of and of in to the to of by to the to of and the and and of of cells IKK-i and and the to of in to the and to of and of IKK-i in to the to of by in to the to of in and of cells IKK-i and and the to of in to the and to of and of IKK-i in to the to of by in to the to of in and of of cells to and of to the and to of and of in to the to of by to the to of and the and and of cells to and of to the and to of and of in to the to of by to the to of and the and and and of and rhTBK-1 by in by the to and by IKK-i and of and rhTBK-1 by in by the to and by IKK-i and and of rhTBK-1 of by to and in and recombinant and to the to the of the and the and in of the in and of rhTBK-1 of by to and in and recombinant and to the to the of the and the and in of the in and in the in to by the of and by of the of and to in the of of the the the identified by the of of the of the of the and to the of the to of the to of and IKK-i and in the in to by the of and by of the of and to in the of of the the the identified by the of of the of the of the and to the of the to of the to of and IKK-2 and of IKK-i of of in of by the of of the to by and and in the of of the by the of of in is in of to of to and of to by the of of and results to the the in in and of is the of to the of of to the is of of purified of in the of the the of IKK-i and and to IKK-i and and to IKK-2 IKK-2 and of IKK-i of of in of by the of of the to by and and in the of of the by the of of in is in of to of to and of to by the of of and results to the the in in and of is the of to the of of to the is of of purified of in the of the the of IKK-i and and to IKK-i and and to IKK-2 and have recently of IKK-1 and IKK-2 in their and and have purified to homogeneity and by the of and rhTBK-1 have the and have cloned, expressed, and purified and rhTBK-1 and their to of and and rhTBK-1 the in and purified to the by by and the of and rhTBK-1 by and rhTBK-1 and to kinases. in and IKK-1 and and IKK-i are IκB are to We the of and rhTBK-1 of the and of and and and rhTBK-1 the of and rhTBK-1 to and in of IKK-i and to that and IKK-1 and IKK-2 and are of have in of of and rhTBK-1 and to to to to in and rhTBK-1 are and is and and results the the is and and is and that is in that the of is the of the by of and the have that is the IKK-1 and IKK-2 are the of and is to is of and rhTBK-1 is of IKK-1 and IKK-2 are the and and and the that in IKK-i the is to the of phosphorylation of the in the of IKK-i and rhTBK-1 and are to that of and and that the in to of the to and the of of the results the in that the to that the of the and the the and rhTBK-1 to by to the of the identified the in and rhTBK-1 and rhTBK-1 and are by phosphorylation of their the of phosphorylation of IKK-i and in the of of IKK-i and in rhTBK-1 and rhTBK-1 cloned, expressed, and purified to of the purified the rhTBK-1 results are in the results by and that IKK-i and results to to the of and rhTBK-1 the rhTBK-1 and of the and rhTBK-1 the the of the is to the of results are to and in results of results in by of of the and of the IKK-i that purified and rhTBK-1 have and their kinases. of have the in the of and rhTBK-1 the have the of IKK-2 of the have their of of and and and of and and of rhTBK-1 rhTBK-1 and the of in and rhTBK-1 in cells have to of IKK-1 and IKK-2 and that the the in the of and rhTBK-1 We IKK-1 of and to in the and rhTBK-1 of the in and rhTBK-1 to the to and that of the to the and that the phosphorylation of and in phosphorylation of the to have of the to the phosphorylation of by and in the and of and to to in in the of the is that the phosphorylation of and rhTBK-1 to of to the by the and the the of the rhTBK-1 the and rhTBK-1 their in the in the by the in the to the to the the and that that and of the of and rhTBK-1 have that the in cells by IKK-2 in the and that the IKK-2 are and and that the IKK-2 is the to the the the are and and that the IKK-2 is and of the IKK-2 by is to by that is the and results that the of and rhTBK-1 are to and that of have that is of the in the of and in the of and rhTBK-1 is the that the is the two is the rhTBK-1 the and and rhTBK-1 and to results and that the and of and in the and and and rhTBK-1 the to the of the of and that the of and rhTBK-1 are and the of of and by of of and that their TBK-1. of by to the of and of rhTBK-1 by to the of and of and by of in in the have cloned, expressed, and purified and rhTBK-1 and their of and the to IKK-1 and IKK-i and are and rhTBK-1 of and rhTBK-1 are that of to their that the identified and rhTBK-1 is the of IKK-2 that of of IKK-2 in the NF-κB and IKK-i IKK-2 kinases. IKK-i and of and that the of to the in the of the is the of and rhTBK-1 is is in in the IKK-1 and IKK-2 have to IKK-i and the and in the of the of the of of IKK-i and to their in NF-κB activation. IKK-i and have recently of IKK-1 and IKK-2 in their and and have purified to homogeneity and by the of and rhTBK-1 have the and have cloned, expressed, and purified and rhTBK-1 and their to of and and rhTBK-1 the in and purified to the by by and the of and rhTBK-1 by and rhTBK-1 and to kinases. in and IKK-1 and and IKK-i are IκB are to We the of and rhTBK-1 of the and of and and and rhTBK-1 the of and rhTBK-1 to and in of IKK-i and to that and IKK-1 and IKK-2 and are of have in of and rhTBK-1 are and is and and results the the is and and is and that is in that the of is the of the by of and the have that is the IKK-1 and IKK-2 are the of and is to is of and rhTBK-1 is of IKK-1 and IKK-2 are the and and and the that in IKK-i the is to the of phosphorylation of the in the of IKK-i and rhTBK-1 and are to that of and and that the in to of the to and the of of the results the in that the to that the of the and the the and rhTBK-1 to by to the of the identified the in and rhTBK-1 and rhTBK-1 and are by phosphorylation of their the of phosphorylation of IKK-i and in the of of IKK-i and in rhTBK-1 and rhTBK-1 cloned, expressed, and purified to of the purified the rhTBK-1 results are in the results by and that IKK-i and results to to the of and rhTBK-1 the rhTBK-1 and of the and rhTBK-1 the the of the is to the of results are to and in results of results in by of of the and of the IKK-i that purified and rhTBK-1 have and their kinases. of have the in the of and rhTBK-1 the have the of IKK-2 of the have their kinases. and rhTBK-1 in cells have to of IKK-1 and IKK-2 and that the the in the of and rhTBK-1 We IKK-1 of and to in the and rhTBK-1 of the in and rhTBK-1 to the to and that of the to the and that the phosphorylation of and in phosphorylation of the to have of the to the phosphorylation of by and in the and in the of the is that the phosphorylation of and rhTBK-1 to of to the by the and the the of the rhTBK-1 the and rhTBK-1 their in the in the by the in the to the to the the and that that and of the of and rhTBK-1 have that the in cells by IKK-2 in the and that the IKK-2 are and and that the IKK-2 is the to the the the are and and that the IKK-2 is and of the IKK-2 by is to by that is the and results that the of and rhTBK-1 are to and that of We have that is of the in the of and in the of and rhTBK-1 is the that the is the two is the rhTBK-1 the and and rhTBK-1 and to results and that the and of and in the and and and rhTBK-1 the to the of the of and that the of and rhTBK-1 are and the of in the have cloned, expressed, and purified and rhTBK-1 and their of and the to IKK-1 and IKK-i and are and rhTBK-1 of and rhTBK-1 are that of to their that the identified and rhTBK-1 is the of IKK-2 that of of IKK-2 in the NF-κB and IKK-i IKK-2 kinases. IKK-i and of and that the of to the in the of the is the of and rhTBK-1 is is in in the IKK-1 and IKK-2 have to IKK-i and the and in the of the of the of of IKK-i and to their in NF-κB activation. We and of the the of in We and of and We in the of