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. In contrast to mammalian c-Src, the SH2 and catalytic domains of MbSrc1 do not appear to be functionally coupled. We cloned and expressed the M. brevicollis homolog of c-Src C-terminal kinase (MbCsk) and showed that it phosphorylates the C terminus of MbSrc1, yet this phosphorylation does not inhibit MbSrc to the same degree seen in the mammalian Src/ Csk pair. Thus, Src autoinhibition likely evolved more recently within the metazoan lineage, and it may have played a role in the establishment of intercellular signaling in metazoans.A defining feature of multicellular organisms is the cellular machinery that enables cell-cell interaction and communication. Recent analyses show that an unexpected diversity of cell signaling and adhesion components are present in choanoflagellates, a group of unicellular organisms that are closely related to metazoans (1, 2). The presence of these proteins in choanoflagellates demonstrates that they evolved before the origin of animals and suggests that they may have served as preadaptations for the evolution of multicellularity (3).The genome of the choanoflagellate Monosiga brevicollis contains 128 tyrosine kinase genes.3 Expressed sequence tags (ESTs) generated from an M. brevicollis cDNA library demonstrated the expression of members of the receptor-tyrosine kinase and nonreceptor-tyrosine kinase families. Anti-phosphotyrosine Western blotting of M. brevicollis cell lysates confirmed the phosphorylation of cellular proteins in response to treatment with nutrients (1). M. brevicollis contains four Src kinase homologs (designated MbSrc1-4) (1), each of which contains the diagnostic SH3 4 and SH2 domains of mammalian Src kinases. Src family nonreceptor-tyrosine kinases are widely expressed in animals and are involved in the regulation of many processes including cell growth and differentiation, adhesion, and motility (4 -6). Mutation of Src or elevated Src expression or activity can lead to cell transformation and metastasis (7, 8) (Src was initially identified as the cellular homolog of the transforming gene from Rous sarcoma virus). For this reason the normal activity of Src in animal cells is tightly regulated. Src kinases possess a conserved domain architecture that is critical for enzymatic regulation; they have an N-terminal myristoylation sequence followed by SH3, SH2, and kinase catalytic domains. The C terminus possesses a tyrosine (Tyr-530) that is phosphorylated by the tyrosine...

Functional transition of Pak proto-oncogene during early evolution of metazoans

“…Functional analyses of SFK orthologs from the choanoflagellate Monosiga ovata and from the primitive multicellular animal Ephydatia fluviatilis showed that Src orthologs in M. ovata (MoSrc) are only partially regulated by Csk and have transforming activity, whereas some Src orthologs in E. fluviatilis (EfSrc) are strictly regulated in a manner similar to mammalian SFKs (Segawa et al, 2006). Similar results were also reported for SFK orthologs in another choanoflagellate, M. brevicollis (Li et al, 2008). These observations suggest that there might be a critical transition in the regulatory system of proto-oncogenes during the early evolution of metazoans.…”

Ancient origin of the integrin-mediated adhesion and signaling machinery

“…S7). Choanoflagellates do have c-Src, but they act in a different context than that of integrin adhesion, as recently demonstrated experimentally in M. brevicollis (44). Moreover, lineage-specific diversifications (independent of those occurring in metazoans) of both integrin α and β have occurred within the C. owczarzaki lineage.…”

Modular evolution of phosphorylation-based signalling systems

“…This timeline is supported by the observations that the single-cell choanoflagellate Monosiga brevicollis has an ancestral c-Src TK (MbSrc) with a Tyr527 site, and also has a Csk kinase, but that phosphorylation of Tyr527 in MbSrc by MbCsk does not significantly affect MbSrc TK activity. This indicates that at this early stage of pTyr signalling the Src SH2 and kinase domains were not functionally coupled for allosteric regulation [38]. A similar type of evolutionary process may have given rise to the mammalian Fes cytoplasmic TK, but in this case the intramolecular interaction between the SH2 and kinase domains has an exclusively positive effect on kinase activity.…”