posted on 2012-10-16, 00:00authored byKira P. Schultheiss, Hiroshi Suga, Iñaki Ruiz-Trillo, W. Todd Miller
Phosphotyrosine-based signaling plays a vital role in
cellular
communication in multicellular organisms. Unexpectedly, unicellular
choanoflagellates (the closest phylogenetic group to metazoans) possess
numbers of tyrosine kinases that are comparable to those in complex
metazoans. Here, we have characterized tyrosine kinases from the filasterean Capsaspora owczarzaki, a unicellular protist representing
the sister group to choanoflagellates and metazoans. Two Src-like
tyrosine kinases have been identified in C. owczarzaki (CoSrc1 and CoSrc2), both of which have the arrangement of SH3,
SH2, and catalytic domains seen in mammalian Src kinases. In Capsaspora cells, CoSrc1 and CoSrc2 localize to punctate
structures in filopodia that may represent primordial focal adhesions.
We have cloned, expressed, and purified both enzymes. CoSrc1 and CoSrc2
are active tyrosine kinases. Mammalian Src kinases are normally regulated
in a reciprocal fashion by autophosphorylation in the activation loop
(which increases activity) and by Csk-mediated phosphorylation of
the C-terminal tail (which inhibits activity). Similar to mammalian
Src kinases, the enzymatic activities of CoSrc1 and CoSrc2 are increased
by autophosphorylation in the activation loop. We have identified
a Csk-like kinase (CoCsk) in the genome of C. owczarzaki. We cloned, expressed, and purified CoCsk and found that it has
no measurable tyrosine kinase activity. Furthermore, CoCsk does not
phosphorylate or regulate CoSrc1 or CoSrc2 in cells or in vitro, and
CoSrc1 and CoSrc2 are active in Capsaspora cell lysates.
Thus, the function of Csk as a negative regulator of Src family kinases
appears to have arisen with the emergence of metazoans.