Regulation of Src and Csk Nonreceptor Tyrosine Kinases in the Filasterean <italic>Ministeria vibrans</italic>

The development of the phosphotyrosine-based signaling system predated the evolution of multicellular animals. Single-celled choanoflagellates, the closest living relatives to metazoans, possess numerous tyrosine kinases, including Src family nonreceptor tyrosine kinases. Choanoflagellates also have Csk (C-terminal Src kinase), the enzyme that regulates Src in metazoans; however, choanoflagellate Csk kinases fail to repress the cognate Src. Here, we have cloned and characterized Src and Csk kinases from Ministeria vibrans, a filasterean (the sister group to metazoans and choanoflagellates). The two Src kinases (MvSrc1 and MvSrc2) are enzymatically active Src kinases, although they have low activity toward mammalian cellular proteins. Unexpectedly, MvSrc2 has significant Ser/Thr kinase activity. The Csk homologue (MvCsk) is enzymatically inactive and fails to repress MvSrc activity. We suggest that the low activity of MvCsk is due to sequences in the SH2− kinase interface, and we show that a point mutation in this region partially restores MvCsk activity. The inactivity of filasterean Csk kinases is consistent with a model in which the stringent regulation of Src family kinases arose more recently in evolution, after the split between choanoflagellates and multicellular animals. M animals evolved from unicellular ancestors more than 600 million years ago. This critical transition required the presence of systems for cellular adhesion, cell−cell communication, and intracellular signal transduction. The cellular components that mediate these processes have surprisingly deep phylogenetic roots; much of the machinery is present in three distinct unicellular lineages (choanoflagellates, filastereans, and ichthyosporeans) that are closely related to metazoans. Thus, many of the genomic innovations that were required for multicellularity were already present in the immediate ancestors of metazoans. In animal cells, tyrosine phosphorylation controls vital processes such as cell growth, differentiation, and survival. The components of phosphotyrosine (pTyr)-based signal transduction (receptor and nonreceptor tyrosine kinases, pTyr-binding domains, and tyrosine phosphatases) are abundant in choanoflagellates, the closest living relatives to metazoans. The genome of the choanoflagellate Monosiga brevicollis encodes a diverse set of tyrosine kinases (TKs), including homologues of the mammalian Src, Csk, Abl, and Tec nonreceptor kinases. The M. brevicollis Src family kinase MbSrc1 has enzymatic properties similar to those of its mammalian counterpart and can functionally replace c-Src in mammalian cells. On the other hand, the regulatory properties of the two Src kinases are different. Whereas mammalian c-Src is inhibited by phosphorylation on a Cterminal tyrosine (Y527) by Csk, phosphorylation of MbSrc1 at the equivalent site by the M. brevicollis Csk does not significantly inhibit activity. Similar results were observed in the choanoflagellate Monosiga ovata. This lack of Cskmediated Src regulation is more pronounced in the filasterean Capsaspora owczarzaki, which represents a sister group to metazoans and choanoflagellates. In Capsaspora, the Csk homologue (CoCsk) does not have any detectable tyrosine kinase activity and is incapable of phosphorylating or inhibiting the two Capsaspora Src kinases (CoSrc1 and CoSrc2). The previous results for M. brevicollis and C. owczarzaki suggest that, while Src and Csk TKs evolved prior to the divergence of filastereans from choanoflagellates and metazoans, the function of Csk as a negative regulator of Src evolved afterward. To understand the early evolution of the Src−Csk signaling pair, it is necessary to analyze kinases from additional organisms. Apart from Capsaspora, the only other known filasterean species is Ministeria vibrans, a free-living marine protist (Figure 1A). A polymerase chain reaction (PCR)based survey in Mi. vibrans identified 15 genes encoding tyrosine kinases (seven receptor TKs and eight nonreceptor TKs). The nonreceptor tyrosine kinases were further subdivided into six families, most of them representing groups common to both Capsaspora and mammalians (Src, Csk, Abl, Received: December 11, 2013 Revised: January 27, 2014 Published: February 12, 2014 Article pubs.acs.org/biochemistry © 2014 American Chemical Society 1320 dx.doi.org/10.1021/bi4016499 | Biochemistry 2014, 53, 1320−1329 This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.