Susan L. Young

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans. The genome sequence of the marine choanoflagellate Monosiga brevicollis has now been determined. Choanoflagellates are a mainly sessile group of protozoa resembling the 'feeding cells' of sponges, and are considered to be the closest living unicellular relatives of multicellular animals. Comparison of the M. brevicollis sequence with metazoan genomes suggests that the last unicellular ancestor of animals had intron-rich genes, some encoding protein domains characteristically associated with cell adhesion and the extracellular matrix in animals. This organism is strictly unicellular, but other choanoflagellates form colonies and may provide clues as to the origin of cell signalling and other systems in early metazoans.

Tyrosine kinase signaling has long been considered a hallmark of intercellular communication, unique to multicellular animals. Our genomic analysis of the unicellular choanoflagellate Monosiga brevicollis discovers a remarkable count of 128 tyrosine kinases, 38 tyrosine phosphatases, and 123 phosphotyrosine (pTyr)-binding SH2 proteins, all higher counts than seen in any metazoan. This elaborate signaling network shows little orthology to metazoan counterparts yet displays many innovations reminiscent of metazoans. These include extracellular domains structurally related to those of metazoan receptor kinases, alternative methods for membrane anchoring and phosphotyrosine interaction in cytoplasmic kinases, and domain combinations that link kinases to small GTPase signaling and transcription. These proteins also display a wealth of combinations of known signaling domains. This uniquely divergent and elaborate signaling network illuminates the early evolution of pTyr signaling, explores innovative ways to traverse the cellular signaling circuitry, and shows extensive convergent evolution, highlighting pervasive constraints on pTyr signaling.

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. 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The origin of metazoans required the evolution of mechanisms for maintaining differentiated cell types within a multicellular individual, in part through spatially differentiated patterns of gene transcription. The unicellular ancestor of metazoans was presumably capable of regulating gene expression temporally in response to changing environmental conditions, and spatial cell differentiation in metazoans may represent a co-option of preexisting regulatory mechanisms. Myc is a critical regulator of cell growth, proliferation, and death that is found in all metazoans but absent in other multicellular lineages, including fungi and plants. Homologs of Myc and its binding partner, Max, exist in two of the closest living relatives of animals, the choanoflagellate Monosiga brevicollis (Mb) and Capsaspora owczarzaki, a unicellular opisthokont that is closely related to metazoans and choanoflagellates. We find that Myc and Max from M. brevicollis heterodimerize and bind to both canonical and noncanonical E-boxes, the DNA-binding sites through which metazoan Myc proteins act. Moreover, in M. brevicollis, MbMyc protein can be detected in nuclear and flagellar regions. Like metazoan Max proteins, MbMax can form homodimers that bind to E-boxes. However, cross-species dimerization between Mb and human Myc and Max proteins was not observed, suggesting that the binding interface has diverged. Our results reveal that the Myc/Max network arose before the divergence of the choanoflagellate and metazoan lineages. Furthermore, core features of metazoan Myc function, including heterodimerization with Max, binding to E-box sequences in DNA, and localization to the nucleus, predate the origin of metazoans.

Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered Gbetagamma freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic alpha-helix (PM motif) are not sufficient for Ste5 action. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388-518) that is essential for its membrane recruitment and function. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Ste5(R407S K411S) is expressed normally, retains Gbetagamma and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. R407S K411S loss-of-function mutations abrogate the constitutive activity of gain-of-function Ste5 alleles, including one (P44L) that increases membrane affinity of the PM motif. Thus, the PH domain is essential for stable membrane recruitment of Ste5, and this association is critical for initiation of downstream signaling because it allows Ste5-bound Ste11 (MAPKKK) to be activated by membrane-bound Ste20 (MAPKKKK).