A Novel Non-catalytic Mechanism Employed by the C-terminal Src-homologous Kinase to Inhibit Src-family Kinase Activity

Although C-terminal Src kinase (CSK)-homologous kinase (CHK) is generally believed to inactivate Src-family tyrosine kinases (SFKs) by phosphorylating their consensus C-terminal regulatory tyrosine (TyrT), exactly how CHK inactivates SFKs is not fully understood. Herein, we report that in addition to phosphorylating TyrT, CHK can inhibit SFKs by a novel non-catalytic mechanism. First, CHK directly binds to the SFK members Hck, Lyn, and Src to form stable protein complexes. The complex formation is mediated by a non-catalytic TyrT-independent mechanism because it occurs even in the absence of ATP or when TyrT of Hck is replaced by phenylalanine. Second, the non-catalytic CHK-SFK interaction alone is sufficient to inactivate SFKs by inhibiting the catalytic activity of autophosphorylated SFKs. Third, CHK and Src co-localize to specific plasma membrane microdomains of rat brain cells, suggesting that CHK is in close proximity to Src such that it can effectively inactivate Src in vivo. Fourth, native CHK·Src complex exists in rat brain, and recombinant CHK·Hck complex exists in transfected HEK293T cells, implying that CHK forms stable complexes with SFKs in vivo. Taken together, our findings suggest that CHK inactivates SFKs (i) by phosphorylating their TyrT and (ii) by this novel TyrT-independent mechanism involving direct binding of CHK to SFKs. It has been documented that autophosphorylated SFKs can still be active, in some cases even when their TyrT is phosphorylated. Thus, the ability of the TyrT-independent mechanism to suppress the activity of both non-phosphorylated and autophosphorylated SFKs represents a fail-safe measure employed by CHK to down-regulate SFK signaling under all circumstances. Although C-terminal Src kinase (CSK)-homologous kinase (CHK) is generally believed to inactivate Src-family tyrosine kinases (SFKs) by phosphorylating their consensus C-terminal regulatory tyrosine (TyrT), exactly how CHK inactivates SFKs is not fully understood. Herein, we report that in addition to phosphorylating TyrT, CHK can inhibit SFKs by a novel non-catalytic mechanism. First, CHK directly binds to the SFK members Hck, Lyn, and Src to form stable protein complexes. The complex formation is mediated by a non-catalytic TyrT-independent mechanism because it occurs even in the absence of ATP or when TyrT of Hck is replaced by phenylalanine. Second, the non-catalytic CHK-SFK interaction alone is sufficient to inactivate SFKs by inhibiting the catalytic activity of autophosphorylated SFKs. Third, CHK and Src co-localize to specific plasma membrane microdomains of rat brain cells, suggesting that CHK is in close proximity to Src such that it can effectively inactivate Src in vivo. Fourth, native CHK·Src complex exists in rat brain, and recombinant CHK·Hck complex exists in transfected HEK293T cells, implying that CHK forms stable complexes with SFKs in vivo. Taken together, our findings suggest that CHK inactivates SFKs (i) by phosphorylating their TyrT and (ii) by this novel TyrT-independent mechanism involving direct binding of CHK to SFKs. It has been documented that autophosphorylated SFKs can still be active, in some cases even when their TyrT is phosphorylated. Thus, the ability of the TyrT-independent mechanism to suppress the activity of both non-phosphorylated and autophosphorylated SFKs represents a fail-safe measure employed by CHK to down-regulate SFK signaling under all circumstances. Src-family kinases (SFKs) 1The abbreviations used are: SFK, Src-family tyrosine kinase; CSK, C-terminal Src kinase; CHK, CSK-homologous kinase; MES, 4-morpholineethanesulfonic acid; HEK cells, human embryonic kidney cells. are non-receptor protein-tyrosine kinases that participate in many cellular functions ranging from cell growth and proliferation to memory and learning (1Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed The kinase activity of SFKs is by by their interaction with cellular the regulatory the are of a consensus tyrosine exists in by their of the and Biol. PubMed this all with The in Hck, Lyn, and Src are in to the of the form of the Hck and Biol. PubMed Biol. PubMed Biol. PubMed and human Biol. PubMed the Hck the novel in the to the consensus in the kinase to and the C-terminal regulatory tyrosine to the the consensus and the C-terminal regulatory tyrosine to and Src and TyrT to and in the kinase and of a consensus regulatory tyrosine the exists in by their of the and Biol. PubMed this all with The in Hck, Lyn, and Src are in to the of the form of the Hck and Biol. PubMed Biol. PubMed Biol. PubMed and human Biol. PubMed the Hck the novel in the to the consensus in the kinase to and the C-terminal regulatory tyrosine to the the consensus and the C-terminal regulatory tyrosine to and Src and TyrT to and Biol. PubMed PubMed of to of SFKs (1Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Biol. PubMed Biol. PubMed The of the autophosphorylated kinase of the Src-family kinase that the the kinase by with the in the catalytic PubMed that the Src-family Hck a novel exists in by their of the and Biol. PubMed this all with The in Hck, Lyn, and Src are in to the of the form of the Hck and Biol. PubMed Biol. PubMed Biol. PubMed and human Biol. PubMed the Hck the novel in the to the consensus in the kinase to and the C-terminal regulatory tyrosine to the the consensus and the C-terminal regulatory tyrosine to and Src and TyrT to and in the and that of Hck to Hck Biol. PubMed the of by is not to the of and Hck TyrT in PubMed of 1997; PubMed and Hck 1997; PubMed that the of the kinase is by involving binding of (i) the to the and (ii) the kinase to the The consensus TyrT of SFKs is to be by regulatory tyrosine C-terminal Src kinase and CSK-homologous kinase (CHK) exists in by their of the and Biol. PubMed this all with The in Hck, Lyn, and Src are in to the of the form of the Hck and Biol. PubMed Biol. PubMed Biol. PubMed and human Biol. PubMed the Hck the novel in the to the consensus in the kinase to and the C-terminal regulatory tyrosine to the the consensus and the C-terminal regulatory tyrosine to and Src and TyrT to and Biol. PubMed PubMed PubMed PubMed is in all the of CHK is it is in and cells. that inactivates SFKs by phosphorylating their Although are of suggesting that CHK can TyrT of SFK Lyn, and PubMed PubMed SFK Hck, Lyn, and Src we the mechanism by CHK inactivates SFKs. that in addition to of SFKs by phosphorylating their TyrT, CHK inactivate SFKs by a novel mechanism. this mechanism CHK directly binds to SFKs to form stable and this binding alone is sufficient to inactivate SFKs. the our in we to CHK and inactivate Hck in transfected HEK293T cells. of our the transfected HEK293T that CHK·Hck complex formation and of Hck even when TyrT of Hck replaced by phenylalanine. The that CHK·Hck complex formation and Hck by this novel mechanism are not mediated by binding of TyrT of Hck to the of this novel occurs by a non-catalytic mechanism. the of our we that CHK and Src co-localize to specific microdomains of rat brain plasma membrane and that CHK and Src form stable protein in rat we that the interaction can suppress SFK activity of the of of Thus, by binding to and the activity of both the form and the fully autophosphorylated form of CHK is of a fail-safe mechanism to down-regulate SFK signaling under all circumstances. Hck and Biol. PubMed Biol. PubMed The by and by Biol. PubMed The a protein the C-terminal to of The and Hck and PubMed Biol. PubMed The from the of rat CHK by and PubMed 1997; PubMed a of and tyrosine that is a tyrosine from The and Biol. PubMed PubMed Biol. PubMed Biol. PubMed PubMed PubMed 1997; PubMed 1997; PubMed PubMed PubMed PubMed Biol. PubMed 1997; PubMed PubMed Cell Biol. PubMed and PubMed of to by the of are in from and to protein with the by and and of CHK from the by with and It used a that and and the The used and the are and the and are in The the Biol. PubMed the used The used and the to is in the The or to that the the used to with to the recombinant CHK and to of CHK and in of CHK by The cell in and The to a with and The the of the CHK protein a to the with a in CHK a in with The and to a of The with The from the with in CHK and by in the CHK The recombinant of of CHK·Src from in of The The and to the in of from in The to the and The to the plasma membrane and The membrane membrane microdomains by and with a from Cell Biol. PubMed and PubMed the membrane in of in MES, and The a and with a of and in the from the of the and to the and membrane the membrane in to the and the The from with of in The to The to the the to the of both the of CHK and Src by of CHK and SFK C-terminal used to CHK the kinase of of CHK, kinase and CHK used a to the to of the CHK activity PubMed The are in used a of CHK to the of CHK in TyrT The kinase CHK and of The of the by and the to The protein to and the SFK C-terminal the of the Src-family kinase is PubMed the of this with the of Hck C-terminal regulatory we the of CHK the and under The and by of the of by CHK and of CHK and of The in the of (i) ATP and kinase or (ii) the with that a of this of sufficient to and to the by of the to CHK to with the The The with of in and the with The and The with the by protein the and CHK the of to the of CHK to to with the of by and with the CHK with Hck, Lyn, or Src to a SFKs from the The binding to a to protein The and the and the binding to protein with a rat the in that CHK, CSK, and Src are in the brain with to protein in to from the of CHK·Hck complex and kinase activity of Hck and in HEK293T cells. Hck, and CHK alone or in HEK293T cells. a HEK293T transfected with the that the of protein of the formation of stable CHK·Hck and complexes in HEK293T by Hck and from the cell the The of CHK and the of Hck in the by the of of CHK and the kinase activity the of and in Hck and of the transfected Hck and kinase activity The specific of Hck and of of The of Hck and by and to that of Hck and The are with the of to of Hck and TyrT Biol. PubMed of the that of CHK with Hck to a and in and of with CHK in and in and the ability of CHK to and inactivate Hck with of CHK or in a of binding of Hck to CHK or by the binding to the protein of the with the of CHK in the by the kinase activity of Hck by the inactivates and forms a stable complex with autophosphorylated Hck in a of autophosphorylated with Hck by the of CHK autophosphorylated Hck Hck with CHK and the Hck catalytic activity by the of stable complex formation CHK and autophosphorylated CHK from the and the of Hck in the by and The of CHK in It is that the of used in this is that of the The of used to the of in all inactivates by phosphorylating TyrT and a stable with of CHK in a of the kinase activity by the the of all and the of by of with of The of is by The represents the of from and of stable complex formation CHK and The in the absence and of CHK to and by The of CHK in the by inactivates Src by phosphorylating TyrT and a stable CHK·Src Src with of CHK in a of the Src kinase activity by the the of Src all and the of Src by of Src in the absence and of CHK The the of and been Biol. PubMed The of is by of stable complex formation CHK and The Src in the absence and of CHK to The of Src in the complex with of by the of CHK in the the of CHK to SFKs and The PubMed used to the ability of CHK to inactivate Hck, Lyn, and SFK Hck, Lyn, and in a of of CHK, kinase and this SFKs of TyrT of by a SFKs in the absence of CHK under activity to the kinase activity of the a specific and many SFKs Biol. PubMed Biol. PubMed a PubMed and CHK not it used the this The and the addition of kinase and The to SFK kinase activity from the with the Biol. PubMed The of SFK kinase activity in the CHK that CHK the the of the SFK PubMed The is activity in activity in of the of SFK used to and the all and SFK Hck, Lyn, or in a kinase and CHK in The of the the by The to the SFKs and the used to and Hck, Lyn, and Src with CHK under in the The by and to a and the to the SFKs and The and by the Biol. PubMed The to and and the in our Biol. PubMed Biol. PubMed and used of by Lyn, occurs both Biol. PubMed and a novel by and It is that the of in the to of CHK, Hck, and in HEK293T in with the of Hck, CHK, and the or Biol. PubMed The and The used to HEK293T the with and by The of cellular in the cell by the PubMed Hck and in the of the HEK293T The catalytic activity of Hck and in HEK293T cell of by kinase employed the because it has been documented a specific and Hck Biol. PubMed 1997; PubMed The from by a Biol. PubMed of CHK and to Hck autophosphorylated with ATP and kinase in by the in of of the autophosphorylated Hck (i) the of CHK the activity of autophosphorylated Hck and (ii) the formation of a stable complex CHK and autophosphorylated autophosphorylated Hck in CHK Hck activity The kinase to The of a of Hck and a in and TyrT the of Hck to Hck to CHK the CHK and the to the Src-family Hck in CHK in and by and the of recombinant CHK with protein-tyrosine kinase activity the activity by the and of CHK with in the The that the recombinant CHK and that Hck by CHK and not by in the CHK protein-tyrosine activity and activity of the ATP in the Hck in in the that the of Hck activity not a of of Hck by it to of The and of the CHK are in CHK TyrT of Hck by a that CHK the consensus TyrT the of we the in of kinase the CHK the of tyrosine to Hck in the of tyrosine in CHK this Hck TyrT suggesting that CHK can inactivate Hck and SFKs by phosphorylating the consensus the of Hck by CHK, we the of CHK of and of CHK of the SFK C-terminal and in of the SFK C-terminal to of the the and of of CHK and to the of the SFK C-terminal the to a in the of TyrT by the from to in in of the a not that CHK of Hck TyrT not the CHK and Hck a of TyrT by CHK in addition to the binding of TyrT of to the CHK TyrT by CHK is by the the of this we used to the to form a stable that CHK in the and absence of ATP and that the complex stable to with a the of in the the of with CHK The ability of CHK to even in the absence of that the complex formation mediated by non-catalytic the it not mediated by the binding of TyrT of to the of the the of CHK and and in to a in the of TyrT by CHK, the of the from the to CHK in CHK in the the Hck in the direct binding of CHK to Hck and in CHK Hck in and the with the of and with TyrT of and is the mechanism of Hck activity Biol. PubMed we how CHK the of Hck in the of of CHK with the of Hck the CHK of the catalytic activity of Hck by the Hck the in CHK in a in the of Hck the of Hck is by and TyrT by CHK, we used to the of the CHK employed to of Hck all and in the of and of and that the of Hck with the in the of of and the CHK of the Hck activity of the Hck activity and to that of the of Hck in the absence of the of the TyrT Hck we the of the by the absence of CHK, Hck by and a of of of Hck in The of of CHK to a in both the catalytic activity and the of Hck and and of the Hck in the of CHK that the of of Hck all in of of Hck, that the of TyrT be of of such a of TyrT CHK this of Hck that TyrT by CHK to the Hck we CHK a with Hck and Hck in HEK293T TyrT of Hck by in that Hck by CHK by a novel non-catalytic mechanism involving the formation of a stable CHK·Hck complex that TyrT Hck and CHK form a stable complex in cells, the in HEK293T by Hck from the cell of the transfected cells, and the of CHK in the by in CHK with Hck, that CHK and Hck a stable protein complex in cells. CHK TyrT of Hck, it is to that binding of the TyrT of Hck to the of CHK to the CHK·Hck complex the of this we CHK and the TyrT replaced by in HEK293T and CHK and the Hck form a stable in the of TyrT by the Hck still the ability to form a stable complex with CHK in HEK293T cells, that the CHK·Hck complex formation not a tyrosine this The that the CHK·Hck complex formation mediated by TyrT-independent non-catalytic the of CHK and of Hck and in HEK293T in of CHK a in Hck that of the recombinant Hck in HEK293T in the it TyrT by the Biol. PubMed the of in Hck activity by CHK is because the of Hck that CHK Hck and that CHK suppress Hck and it from is not to by TyrT by the CSK, it the Hck of CHK, in a in and and a in the catalytic activity of the Hck the that CHK can inactivate Hck by a mechanism TyrT be by CHK form a stable complex with CHK in HEK293T we that the of is a of the binding of CHK to the Hck The of CHK to and the binding and of Hck by CHK is of TyrT it is to that the can be when a CHK tyrosine kinase activity is used in the the of this the in CHK is to the ATP binding of the of many protein the of by the recombinant CHK not to CHK, is to form a stable complex with the in HEK293T of has the protein kinase activity and the and of in HEK293T The suggest that the CHK ability to and inactivate SFKs. it is that the of the CHK and such the recombinant to inactivate in HEK293T cells. this we in and the recombinant CHK and we the of CHK and by The of CHK and not the CHK the of and it to of that the not of to in HEK293T and the CHK the the ability of CHK to both and Hck in Taken together, the in and suggest that the or the of the CHK interaction with and of CHK and Although that CHK can inactivate Hck by it CHK can inactivate the fully autophosphorylated form of is to many and in of growth to and of the Src-family The fully autophosphorylated form of the Src kinases the cell growth from the the of our this this Hck to by with and The by the with the to the and the of the autophosphorylated Hck kinase activity in the absence and of of CHK the used the to CHK the autophosphorylated in autophosphorylated Hck with CHK, that CHK can form a complex with the autophosphorylated the binding effectively inactivates the autophosphorylated Hck, with of the autophosphorylated Hck when with CHK The that CHK can and inactivate autophosphorylated CHK of and Src with TyrT and the of and CHK·Src the mechanism employed by CHK to inactivate Hck is to CHK of we in CHK can TyrT of and Src and form stable complexes with in CHK effectively inactivates protein kinase CHK that are that of CHK is of of kinase to the of CHK Hck CHK a in the of CHK a in the in of in the of CHK is to a in of the by and to the of CHK and of the that CHK a in it a in TyrT that can in in the kinase a novel addition to CHK a in that both TyrT and the of are employed by CHK to inactivate It is that CHK of kinase activity and of the that this CHK of of of is by TyrT the of TyrT is to be of of It is from this of TyrT by CHK that TyrT alone fully the CHK ability to suppress the of CHK that of of activity that the binding of CHK to to form the is with CHK a stable complex of in the complex by that CHK can to even when is autophosphorylated that CHK can of our that CHK can inactivate in both by phosphorylating TyrT and by the non-catalytic TyrT-independent mechanism that formation of the stable to Hck and Lyn, Src is by CHK of Src with of CHK to a in the of and in the Src of a by the Src TyrT, to a the of CHK a in TyrT Thus, the of CHK effectively inactivates Src TyrT The that CHK the non-catalytic mechanism to inactivate of the that CHK forms a stable complex with of the CHK·Src complex with the that CHK is of binding to Src even when Src is Thus, to CHK of Hck and Lyn, CHK is of Src both by phosphorylating TyrT and by the non-catalytic mechanism that binding of CHK to Src to form the stable CHK·Src CHK and Src to of the and CHK·Src in with of SFKs by CHK in to their the are to the plasma CHK, to the plasma is to in the of CHK, Hck, Lyn, and Src are CHK to and inactivate the SFKs and in that of SFKs by CHK their such that their are Thus, it is to the CHK and SFKs co-localize to specific microdomains of the plasma CHK and Src are in brain 1997; PubMed we their and the CHK·Src protein complexes in rat in both Src and CHK are in the plasma membrane of rat brain cells. by the membrane the and plasma membrane of the membrane that both CHK and Src co-localize to both microdomains of CHK and Src in membrane microdomains their and in CHK of is in with the by that CHK to the membrane The in that CHK can and inactivate Hck when are in HEK293T cells. it is still that the CHK·Hck complex formation is from their this we to that native stable complexes in or kinases are in Src is in the not in the suggesting that CHK and Src form stable protein complexes in brain cells. is in rat brain and PubMed that Src in we Src or to the CHK·Src complexes in vivo. in Src is not in the we to in the of brain not It is documented that CHK can down-regulate the activity of SFKs in Biol. PubMed 1997; PubMed PubMed Biol. 1997; PubMed Biol. PubMed CHK of Src and with of cell growth Biol. PubMed of the by CHK and CSK, CHK to the mechanism in CHK inactivates SFKs by phosphorylating their consensus C-terminal regulatory tyrosine this we report that CHK can TyrT of SFK Hck, Lyn, and and that CHK can suppress their catalytic we both in and in transfected HEK293T that CHK can suppress SFK activity by a novel mechanism that not TyrT novel mechanism direct binding of CHK to SFKs and of their kinase activity that binding and of SFK activity are of TyrT, CHK still and effectively inactivate the the TyrT with phenylalanine. we that CHK and Src co-localize to specific microdomains in rat brain plasma membrane and that native CHK·Src protein complexes in rat brain cells. the of our in of CHK and Src their and in CHK of Src and binding to Src by the TyrT-independent mechanism. is the of CHK ability to inactivate SFKs by this novel non-catalytic SFKs are to be and autophosphorylated in PubMed SFKs to even when their TyrT is PubMed and Biol. that human binding or of the kinase Hck by even when TyrT and to the that TyrT and the interaction not be sufficient to inhibit SFK activity in vivo. Thus, this novel non-catalytic both inactivates the autophosphorylated Hck and Hck from represents a fail-safe cellular mechanism to suppress SFK signaling under all circumstances. the of by CHK in phosphorylating TyrT of SFKs and in phosphorylating SFKs in is CHK Hck to a of of of Hck even when CHK in a Src is a in of CHK to be a even TyrT to a of of of when CHK in a suggest that CHK to be or the of cellular to in phosphorylating TyrT of SFKs. It is that CHK binds Src and of and it Src the Src CHK can still and suppress Src of and CHK binding Src to this PubMed report that Src can that CHK binding Src and this in Src how CHK binding Src kinase activity the it Src is Although stable CHK·Src protein complexes in rat brain, we to stable suggesting that the complexes are the CHK·Src complexes in rat brain or that the complexes are of in brain cells. PubMed to the formation of a stable complex in when both kinases the of of CHK and in their and in their kinase it is that both CHK and to SFKs. to the of the stable CHK and we that direct binding of CHK to Hck when in the of CHK to the of protein kinases PubMed 1997; PubMed 1997; PubMed in of the kinase is to (i) it binds to the and of and (ii) it forms a with the in the of the kinase that complexes even in the absence of ATP that of with ATP in CHK to SFKs. that formation or of the SFK binding and are of with of the kinase formation of the CHK and it is to that the of the a of this we to the of to how the the CHK ability to and inactivate The cell of the of a in the is and in the is are the the of the kinase the and are the of the kinase their and the and with to the kinase suggest that such Thus, the of the by the the of CHK that the or the formation of the SFK binding and PubMed a of in and in the of the kinase of of a binding of SFK It is that this is used by CHK to and inactivate SFKs by the non-catalytic mechanism. this is close to in that a the the of binding the in the in this we suggest the the mechanism of CHK of SFKs SFKs a the interaction of the C-terminal with the and the interaction of the kinase with the the of the kinase form in in that the SFK to the signaling complex it binds binding to the the kinase from the The in the of of SFKs are (i) of the from TyrT by a to the form of the kinase and (ii) to the kinase to fully active, autophosphorylated form CHK in the is to the signaling complex Biol. PubMed CHK in close proximity to the CHK binds the active, autophosphorylated SFK and catalytic CHK can the form of the SFK, the SFK from by the binding is by that CHK of The TyrT the and this the and of the SFK to to their CHK and the SFK from the in addition to of SFKs by the CHK-SFK binding represents a and mechanism of SFK and of SFKs been with the of many of and PubMed PubMed this our that CHK can inactivate SFKs by a novel non-catalytic mechanism that the formation of a complex is because that the CHK ability to and suppress SFK activity are the of to the in both CHK and SFKs that their stable the of such and the and the of of rat brain membrane microdomains by with

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.