Signaling Properties of a Non-metazoan Src Kinase and the Evolutionary History of Src Negative Regulation

Abstract

Choanoflagellates, unicellular organisms that are closely related to metazoans, possess cell adhesion and signaling proteins previously thought to be unique to animals, suggesting that these components may have played roles in the evolution of metazoan multicellularity. We have cloned, expressed, and purified the nonreceptor tyrosine kinase MbSrc1 from the choanoflagellate Monosiga brevicollis. The kinase has the same domain arrangement as mammalian Src kinases, and we find that the individual Src homology 3 (SH3), SH2, and catalytic domains have similar functions to their mammalian counterparts. to mammalian the and catalytic domains of MbSrc1 to be We and the of kinase and that the of to the same in the mammalian Src the metazoan and may have played in the of signaling in Choanoflagellates, unicellular organisms that are closely related to metazoans, possess cell adhesion and signaling proteins previously thought to be unique to animals, suggesting that these components may have played roles in the evolution of metazoan multicellularity. We have cloned, expressed, and purified the nonreceptor tyrosine kinase MbSrc1 from the choanoflagellate Monosiga brevicollis. The kinase has the same domain arrangement as mammalian Src kinases, and we find that the individual Src homology 3 (SH3), SH2, and catalytic domains have similar functions to their mammalian counterparts. to mammalian the and catalytic domains of MbSrc1 to be We and the of kinase and that the of to the same in the mammalian Src the metazoan and may have played in the of signaling in of organisms the that and that of cell signaling and adhesion components are in of unicellular organisms that are closely related to The of these proteins in that the of and that may have as the evolution of of the choanoflagellate Monosiga tyrosine kinase from the of of the kinase and kinase of cell the of proteins in to Src kinase of the and Src homology and of tyrosine and Src homology and of tyrosine and domains of mammalian Src Src are in and are in the of cell and and of Src Src to cell and as the of the from the of Src in Src possess domain that have SH2, and kinase catalytic The tyrosine that the tyrosine kinase the and the domain that Src in the The to Src in may have in Src the Src in the and in of Src from the choanoflagellate Monosiga that of and Src The of we of choanoflagellate from brevicollis. their and are related the and are from of Src from and of the of Src and the of Src the of the of the and choanoflagellate Src that the individual domains of MbSrc1 Src from and the in MbSrc1 in mammalian of the tyrosine of MbSrc1 from to the of The of in mammalian Src in the evolution of and may have to the of metazoan in the to and the the MbSrc1 from MbSrc1 the and of MbSrc1 in mammalian the and of The MbSrc1 domain and domain in the and of from the and the and of mammalian the and of and from the and the and and the The the in the Src Src The the in the and proteins and domains and in and purified as the proteins from in MbSrc1 in the in and MbSrc1 of of of MbSrc1 purified as previously MbSrc1 in and in the of and The purified and of of MbSrc1 the kinase Src kinase of and the of as the to The and and as The and the of MbSrc1 in the of and as previously tyrosine of MbSrc1 and in kinase The to and MbSrc1 from the and to and the of and the of and and in The and in of the and in in and and and MbSrc1 domain in the cell and in the and the The to the and the MbSrc1 The from the of and in in of to the of in and The cell and in the and in of cell to the The proteins and proteins of to as from MbSrc1 in and in as from of cell and of the the as MbSrc1 from MbSrc1 from The domain of MbSrc1 that of mammalian Src the to SH2, and tyrosine kinase catalytic domains The are in The tyrosine in mammalian Src are in of MbSrc1 to the the of of The of in MbSrc1 mammalian Src kinases, of of the in that are are in MbSrc1 the the to in in MbSrc1 mammalian Src kinases, in the kinase to the domain and in The of the kinase in and MbSrc1 are in are in the MbSrc1 these have the to as the in MbSrc1 and MbSrc1 has of the of mammalian Src and of MbSrc1 and domains of mammalian Src kinase are in and have in the to these roles are in the of in and in that the of the domain of these roles the of the domains to in these are in the MbSrc1 we the domains as proteins in and purified We the and proteins and from mammalian that the domain to proteins in the The domain to that the Src and domains to the MbSrc1 domain the of the the mammalian domain from the of Src the of the MbSrc1 domain to to the MbSrc1 and domains are cell to the as The proteins and proteins The of and the of the MbSrc1 domain to domain and of The from to the of the MbSrc1 and domains their MbSrc1 to the of similar to the of the to the domain of mammalian the MbSrc1 domain of to the from in the of domain to the of the that the MbSrc1 and domains the of their mammalian and that the of the MbSrc1 from the of the domain to the of kinase of mammalian Src the of and to We the and purified the MbSrc1 catalytic mammalian Src We MbSrc1 tyrosine and MbSrc1 the from of that the and the the Src kinase of the kinase kinase MbSrc1 the of mammalian suggesting that MbSrc1 be of The Src to that the domain of MbSrc1 of of purified MbSrc1 the The and kinase MbSrc1 the Src in the of the the MbSrc1 mammalian Src kinases, the and domains the and Src kinase of Src the same the and of purified MbSrc1 the domain the The of and to the Src kinase that of MbSrc1 the that the of MbSrc1 purified from We MbSrc1 tyrosine and MbSrc1 of and to in tyrosine kinase that mammalian Src kinases, to that the MbSrc1 purified from and that the to in of MbSrc1 of MbSrc1 of in The of MbSrc1 MbSrc1 and MbSrc1 the and to their the and domains of Src in be in are We MbSrc1 to the domain of similar to in the in the to to domain MbSrc1 of to the the and in the domain of MbSrc1 the to The the that the the tyrosine kinase and domains in the of of MbSrc1 of the to are of and of the of mammalian Src in we the from brevicollis. We from the of tyrosine kinase and to the from SH2, and kinase catalytic domains and the mammalian has the of that of Src of the are to and of of in and in brevicollis. in of these and that may have mammalian Src of from The the domain arrangement of and of and The domain the domain and the catalytic domain in that are Src that are and and the as in and purified the We the tyrosine kinase the of mammalian of of the of MbSrc1 The and the and the of and and MbSrc1 and as we in the of MbSrc1 we of MbSrc1 and purified from of MbSrc1 in the of to of as We the of MbSrc1 to be these We to that the tyrosine of MbSrc1 and that the the the MbSrc1 the of that of the MbSrc1 MbSrc1 in the of as and to MbSrc1 and to The the The of the from the MbSrc1 the from of from the of the from MbSrc1 in The and of are The in the and that the the to the of we MbSrc1 mammalian We the of MbSrc1 in mammalian of MbSrc1 of mammalian Src kinase mammalian to the same and of to the of to MbSrc1 the the of the of the mammalian of MbSrc1 in in the of The of and MbSrc1 and and in and of MbSrc1 in of MbSrc1 and the kinase in Src MbSrc1 tyrosine kinase in these as of cell of the tyrosine in kinase of the tyrosine of mammalian to of the The of and MbSrc1 in these similar to the of We that MbSrc1 the in mammalian Src and of the the the of MbSrc1 of MbSrc1 in mammalian of of MbSrc1 and The and and MbSrc1 and in and from in and the in the of in MbSrc1 the of in we of be to the of MbSrc1 in the to to as we previously We in that the of the MbSrc1 to the we the of the MbSrc1 We the to the domain as the the the of to MbSrc1 MbSrc1 mammalian Src in Src of and of of the of MbSrc1 to the the in of mammalian the in the that of the MbSrc1 to MbSrc1 in from the and the in and to the the of of MbSrc1 in the of to the and that these are in the the and kinase domains may be to that to the of the of that the and domains in to the in the of we of MbSrc1 in be the domain the that the domain of the of these has to and related of these MbSrc1 in to be MbSrc1 the and that to kinase kinase We in MbSrc1 to the kinase the that the and that the kinase the that are in the of of the of and and that of the of and of the the that may have the from unicellular to metazoan may be from their have the to that the evolution of and of choanoflagellate and metazoan and the The of tyrosine and signaling components and in that these proteins the of of the signaling in domain and and that the components the and in the evolution of we have the and signaling of choanoflagellate and mammalian tyrosine We have to Src from and signaling domains of MbSrc1 SH2, and are similar to the domains in mammalian Src and that the of MbSrc1 from that of mammalian Src kinases, the in MbSrc1 The of in kinase be the and of MbSrc1 in are in MbSrc1 mammalian Src in and that MbSrc1 the of metazoan Src and in and MbSrc1 are the domain of MbSrc1 similar to as that the be their proteins to the domain of MbSrc1 the to mammalian Src kinases, the the and the to to the kinase and domains the of the kinase and domains in MbSrc1 may be of and are the of mammalian Src that the of MbSrc1 in and the of the of MbSrc1 and and The of be in to of the the domain the in that the in of in The of in may in to the as the of The of MbSrc1 are to the of the in brevicollis. of MbSrc1 in the of to to their in that the in mammalian Src in the of are in of Src from and are the that of Src may the to multicellularity. of organisms the that and that of cell signaling and adhesion components are in of unicellular organisms that are closely related to The of these proteins in that the of and that may have as the evolution of The of the choanoflagellate Monosiga tyrosine kinase from the of of the kinase and kinase of cell the of proteins in to Src kinase of the and Src homology and of tyrosine and Src homology and of tyrosine and domains of mammalian Src Src are in and are in the of cell and and of Src Src to cell and as the of the from the of Src in Src possess domain that have SH2, and kinase catalytic The tyrosine that the tyrosine kinase the and the domain that Src in the The to Src in may have in Src the Src in the and in of Src from the choanoflagellate Monosiga that of and Src The of we of choanoflagellate from brevicollis. their and are related the and are from of Src from and of the of Src and the of Src the of the of the and choanoflagellate Src that the individual domains of MbSrc1 Src from and the in MbSrc1 in mammalian of the tyrosine of MbSrc1 from to the of The of in mammalian Src in the evolution of and may have to the of metazoan in the to and the the MbSrc1 from MbSrc1 the and of MbSrc1 in mammalian the and of The MbSrc1 domain and domain in the and of from the and the and of mammalian the and of and from the and the and and the The the in the Src Src The the in the and proteins and domains and in and purified as the proteins from in MbSrc1 in the in and MbSrc1 of of of MbSrc1 purified as previously MbSrc1 in and in the of and The purified and of of MbSrc1 the kinase Src kinase of and the of as the to The and and as The and the of MbSrc1 in the of and as previously tyrosine of MbSrc1 and in kinase The to and MbSrc1 from the and to and the of and the of and and in The and in of the and in in and and and MbSrc1 domain in the cell and in the and the The to the and the MbSrc1 The from the of and in in of to the of in and The cell and in the and in of cell to the The proteins and proteins of to as from MbSrc1 in and in as from of cell and of the the as in the to and the the MbSrc1 from MbSrc1 the and of MbSrc1 in mammalian the and of The MbSrc1 domain and domain in the and of from the and the and of mammalian the and of and from the and the and and the The the in the Src Src The the in the and proteins and domains and in and purified as the proteins from in MbSrc1 in the in and MbSrc1 of of of MbSrc1 purified as previously MbSrc1 in and in the of and The purified and of of MbSrc1 the kinase Src kinase of and the of as the to The and and as The and the of MbSrc1 in the of and as previously tyrosine of MbSrc1 and in kinase The to and MbSrc1 from the and to and the of and the of and and in The and in of the and in in and and and MbSrc1 domain in the cell and in the and the The to the and the MbSrc1 The from the of and in in of to the of in and The cell and in the and in of cell to the The proteins and proteins of to as from MbSrc1 in and in as from of cell and of the the as MbSrc1 from MbSrc1 from The domain of MbSrc1 that of mammalian Src the to SH2, and tyrosine kinase catalytic domains The are in The tyrosine in mammalian Src are in of MbSrc1 to the the of of The of in MbSrc1 mammalian Src kinases, of of the in that are are in MbSrc1 the the to in in MbSrc1 mammalian Src kinases, in the kinase to the domain and in The of the kinase in and MbSrc1 are in are in the MbSrc1 these have the to as the in MbSrc1 and MbSrc1 has of the of mammalian Src and of MbSrc1 and domains of mammalian Src kinase are in and have in the to these roles are in the of in and in that the of the domain of these roles the of the domains to in these are in the MbSrc1 we the domains as proteins in and purified We the and proteins and from mammalian that the domain to proteins in the The domain to that the Src and domains to the MbSrc1 domain the of the the mammalian domain from the of Src the of the MbSrc1 domain to to the to the of the MbSrc1 and domains their MbSrc1 to the of similar to the of the to the domain of mammalian the MbSrc1 domain of to the from in the of domain to the of the that the MbSrc1 and domains the of their mammalian and that the of the MbSrc1 from the of the domain to the of kinase of mammalian Src the of and to We the and purified the MbSrc1 catalytic mammalian Src We MbSrc1 tyrosine and MbSrc1 the from of that the and the the Src kinase of the kinase kinase MbSrc1 the of mammalian suggesting that MbSrc1 be of The Src to that the domain of MbSrc1 of of purified MbSrc1 the The and kinase MbSrc1 the Src in the of the the MbSrc1 mammalian Src kinases, the and domains the and Src kinase of Src the same the and of purified MbSrc1 the domain the The of and to the Src kinase that of MbSrc1 the that the of MbSrc1 purified from We MbSrc1 tyrosine and MbSrc1 of and to in tyrosine kinase that mammalian Src kinases, to that the MbSrc1 purified from and that the to in of MbSrc1 of MbSrc1 of in The of MbSrc1 MbSrc1 and MbSrc1 the and to their the and domains of Src in be in are We MbSrc1 to the domain of similar to in the in the to to domain MbSrc1 of to the the and in the domain of MbSrc1 the to The the that the the tyrosine kinase and domains in the of of MbSrc1 of the to are of and of the of mammalian Src in we the from brevicollis. We from the of tyrosine kinase and to the from SH2, and kinase catalytic domains and the mammalian has the of that of Src of the are to and of of in and in brevicollis. in of these and that may have mammalian Src of from The the domain arrangement of and of and The domain the domain and the catalytic domain in that are Src that are and and the as in and purified the We the tyrosine kinase the of mammalian of of the of MbSrc1 The and the and the of and and MbSrc1 and as we in the of MbSrc1 we of MbSrc1 and purified from of MbSrc1 in the of to of as We the of MbSrc1 to be these We to that the tyrosine of MbSrc1 and that the the the MbSrc1 the of that of the MbSrc1 MbSrc1 in the of as and to MbSrc1 and to The the The of the from the MbSrc1 the from of from the of the from MbSrc1 in The and of are The in the and that the the to the of we MbSrc1 mammalian We the of MbSrc1 in mammalian of MbSrc1 of mammalian Src kinase mammalian to the same and of to the of to MbSrc1 the the of the of the mammalian of MbSrc1 in in the of The of and MbSrc1 and and in and of MbSrc1 in of MbSrc1 and the kinase in Src MbSrc1 tyrosine kinase in these as of cell of the tyrosine in kinase of the tyrosine of mammalian to of the The of and MbSrc1 in these similar to the of We that MbSrc1 the in mammalian Src and of the the the of MbSrc1 of MbSrc1 in mammalian of of MbSrc1 and The and and MbSrc1 and in and from in and the in the of in MbSrc1 the of in we of be to the of MbSrc1 in the to to as we previously We in that the of the MbSrc1 to the we the of the MbSrc1 We the to the domain as the the the of to MbSrc1 MbSrc1 mammalian Src in Src of and of of the of MbSrc1 to the the in of mammalian the in the that of the MbSrc1 to MbSrc1 in from the and the in and to the the of of MbSrc1 in the of to the and that these are in the the and kinase domains may be to that to the of the of that the and domains in to the in the of we of MbSrc1 in be the domain the that the domain of the of these has to and related of these MbSrc1 in to be MbSrc1 the and that to kinase kinase We in MbSrc1 to the kinase the that the and that the kinase the that are in the of of the of and and that of the of and of the MbSrc1 from MbSrc1 from The domain of MbSrc1 that of mammalian Src the to SH2, and tyrosine kinase catalytic domains The are in The tyrosine in mammalian Src are in of MbSrc1 to the the of of The of in MbSrc1 mammalian Src kinases, of of the in that are are in MbSrc1 the the to in in MbSrc1 mammalian Src kinases, in the kinase to the domain and in The of the kinase in and MbSrc1 are in are in the MbSrc1 these have the to as the in MbSrc1 and MbSrc1 has of the of mammalian Src The and of MbSrc1 and domains of mammalian Src kinase are in and have in the to these roles are in the of in and in that the of the domain of these roles the of the domains to in these are in the MbSrc1 we the domains as proteins in and purified We the and proteins and from mammalian that the domain to proteins in the The domain to that the Src and domains to the MbSrc1 domain the of the the mammalian domain from the of Src the of the MbSrc1 domain to to the We to the of the MbSrc1 and domains their MbSrc1 to the of similar to the of the to the domain of mammalian the MbSrc1 domain of to the from in the of domain to the of the that the MbSrc1 and domains the of their mammalian and that the of the MbSrc1 from the of the domain to the of kinase of mammalian Src the of and to We the and purified the MbSrc1 catalytic mammalian Src We MbSrc1 tyrosine and MbSrc1 the from of that the and the the Src kinase of the kinase kinase MbSrc1 the of mammalian suggesting that MbSrc1 be of The Src to that the domain of MbSrc1 of mammalian Src kinases, the and domains the and Src kinase of Src the same the and of purified MbSrc1 the domain the The of and to the Src kinase that of MbSrc1 We the that the of MbSrc1 purified from We MbSrc1 tyrosine and MbSrc1 of and to in tyrosine kinase that mammalian Src kinases, to that the MbSrc1 purified from and that the to in of MbSrc1 to their the and domains of Src in be in are We MbSrc1 to the domain of similar to in the in the to to domain MbSrc1 of to the the and in the domain of MbSrc1 the to The the that the the tyrosine kinase and domains in the of and of the of mammalian Src in we the from brevicollis. We from the of tyrosine kinase and to the from SH2, and kinase catalytic domains and the mammalian has the of that of Src of the are to and of of in and in brevicollis. in of these and that may have mammalian Src We as in and purified the We the tyrosine kinase the of mammalian MbSrc1 and as we in the of MbSrc1 we of MbSrc1 and purified from of MbSrc1 in the of to of as We the of MbSrc1 to be these We to that the tyrosine of MbSrc1 and that the the the MbSrc1 the of that the of we MbSrc1 mammalian We the of MbSrc1 in mammalian of MbSrc1 of mammalian Src kinase mammalian to the same and of to the of to MbSrc1 the the of the of the mammalian of MbSrc1 in of MbSrc1 and the kinase in Src MbSrc1 tyrosine kinase in these as of cell of the tyrosine in kinase of the tyrosine of mammalian to of the The of and MbSrc1 in these similar to the of We that MbSrc1 the in mammalian Src and of the the the of MbSrc1 of in MbSrc1 the of in we of be to the of MbSrc1 in the to to as we previously We in that the of the MbSrc1 to the we the of the MbSrc1 We the to the domain as the the We the of to MbSrc1 MbSrc1 mammalian Src in Src of and of of the of MbSrc1 to the the in of mammalian the in the that of the MbSrc1 to MbSrc1 We to the the of of MbSrc1 in the of to the and that these are in the the and kinase domains may be to that to the of the of that the and domains in to the in the of we of MbSrc1 in be the domain the that the domain of the of these has to and related of these MbSrc1 in to be MbSrc1 the and that to kinase kinase We in MbSrc1 to the kinase the that the and that the kinase the that are in the of the that may have the from unicellular to metazoan may be from their have the to that the evolution of and of choanoflagellate and metazoan and the The of tyrosine and signaling components and in that these proteins the of of the signaling in domain and and that the components the and in the evolution of we have the and signaling of choanoflagellate and mammalian tyrosine We have to Src from and signaling domains of MbSrc1 SH2, and are similar to the domains in mammalian Src and that the of MbSrc1 from that of mammalian Src kinases, the in MbSrc1 The of in kinase be the and of MbSrc1 in are in MbSrc1 mammalian Src in and that MbSrc1 the of metazoan Src and in and MbSrc1 are the domain of MbSrc1 similar to as that the be their proteins to the domain of MbSrc1 the to mammalian Src kinases, the the and the to to the kinase and domains the of the kinase and domains in MbSrc1 may be of and are the of mammalian Src that the of MbSrc1 in and the of the of MbSrc1 and and The of be in to of the the domain the in that the in of in The of in may in to the as the of The of MbSrc1 are to the of the in brevicollis. of MbSrc1 in the of to to their in that the in mammalian Src in the of are in of Src from and are the that of Src may the to multicellularity. the that may have the from unicellular to metazoan may be from their have the to that the evolution of and of choanoflagellate and metazoan and the The of tyrosine and signaling components and in that these proteins the of of the signaling in domain and and that the components the and in the evolution of we have the and signaling of choanoflagellate and mammalian tyrosine We have to Src from and The signaling domains of MbSrc1 SH2, and are similar to the domains in mammalian Src and that the of MbSrc1 from that of mammalian Src kinases, the in MbSrc1 The of in kinase be the and of MbSrc1 in are in MbSrc1 mammalian Src in and that MbSrc1 the of metazoan Src The and in and MbSrc1 are the domain of MbSrc1 similar to as that the be their proteins to the domain of MbSrc1 the to mammalian Src kinases, the the and the to to the kinase and domains the of the kinase and domains in MbSrc1 may be of and are the of mammalian Src that the of MbSrc1 in and the of the of MbSrc1 and and The of be in to of the the domain the in that the in of in The of in may in to the as the of The of MbSrc1 are to the of the in brevicollis. of MbSrc1 in the of to to their in that the in mammalian Src in the of are in of Src from and are the that of Src may the to multicellularity. We of and of the We and and and of MbSrc1