Tyrosine kinase evolution
Choanoflagellates are believed to be the closest living unicellular relatives of metazoans. The sequencing of the genome of the choanoflagellate Monosiga brevicollis has highlighted the presence of a number of signaling molecules that were previously thought to be unique to multicellular animals. In particular, the machinery necessary for phosphotyrosine-based signal transduction (tyrosine kinases, tyrosine phosphatases, and SH2 domains) is present in M. brevicollis. This suggests that the evolution of tyrosine kinases predated their widespread use in cellular signaling in metazoans. A comparison of M. brevicollis and metazoan tyrosine kinases can therefore reveal the structural and functional features that arose more recently in the metazoan lineage. In particular, our group has focused on the evolution of autoinhibition and substrate targeting in the M. brevicollis Src family kinases.
A striking feature of the M. brevicollis genome is the large number of receptor and nonreceptor tyrosine kinases as compared with metazoans. Many of the kinases contain combinations of domains that are not seen in any metazoan. For example, 10 of the 15 HMTKs (HM-motif tyrosine kinases) contain one or more PTB domains, FYTK contains a inositol lipid-binding FYVE domain, and Src4 contains a lipid-binding C2 domain; none of these domains is found in combination with a tyrosine kinase catalytic domain in metazoans. These variations in the domain architecture highlight the role of domain shuffling in the evolution of signal transduction pathways. A goal of our research is to understand the importance of these novel domain combinations in M. brevicollis kinases.
For more details, see:
N. King, M.J. Westbrook, S.L. Young, A. Kuo, M. Abedin, J. Chapman, S. Fairclough, U. Hellsten, Y. Isogai, I. Letunic, M. Marr, D. Pincus, N. Putnam, A. Rokas, K.J. Wright, R. Zuzow, W. Dirks, M. Good, D. Goodstein, D. Lemons, W. Li, J. Lyons, A. Morris, S. Nichols, D.J. Richter, A. Salamov, JGI Sequencing, P. Bork, W.A. Lim, G. Manning, W.T. Miller, W. McGinnis, H. Shapiro, R. Tjian, I.V. Grigoriev, and D. Rohhsar (2008). The genome of the choanoflagellate Monosiga brevicollis and the origins of metazoans. Nature 451, 783-788.
W. Li, S.L. Young, N. King, and W.T. Miller (2008). Signaling properties of a non-metazoan Src kinase and the evolutionary history of Src negative regulation. J. Biol. Chem. 283, 15491-15501.
G. Manning, S.L. Young, W.T. Miller, and Y. Zhai (2008). The protist, Monosiga brevicollis, has a tyrosine kinase signaling network more elaborate and diverse than found in any metazoan. Proc. Natl. Acad. Sci. USA. 105, 9674-9679.
W. Li, S. Scarlata, and W.T. Miller (2009). Evidence for convergent evolution in the signaling properties of a choanoflagellate tyrosine kinase. Biochemistry 48, 5180-5186.
V. Prieto-Echague, P.M. Chan, B.P. Craddock, E. Manser, and W.T. Miller (2011). PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis. PLoS ONE 6, e19296.
W.T. Miller (2012). Tyrosine kinase signaling and the emergence of multicellularity. Biochim Biophys Acta 1823, 1053-7.
Reengineering tyrosine kinases and their substrates
In collaboration with Wendell Lim's group at UCSF, we generated novel tyrosine kinases which recapitulated the signaling properties of natural Src family kinases (SFKs). In one construct, we replaced the SH2 domain of the SFK Hck with a PDZ domain to redirect the enzyme's substrate specificity. In additional constructs, we replaced the entire regulatory apparatus of Hck with a PDZ domain and C-terminal PDZ ligand sequences. The resulting artificial PDZ-Hck kinases displayed three salient features of modular signaling proteins: (i) their substrate specificity was governed by the PDZ domain; (ii) they displayed autoregulatory properties similar to natural SFKs; and (iii) they were versatile, and could be used to rewire two separate signaling pathways. Our data highlight the modularity and evolvability of signaling proteins, and suggest that the targeting function of modular domains is most amenable to manipulation. For more details, see Yadav et al., Biochemistry 2009.
We have also used a bottom-up synthetic strategy to determine the importance of the arrangement, spacing, and identity of the phosphorylation sites in the adaptor protein Cas. This work was carried out in collaboration with Dr. Kiyotaka Shiba (Cancer Institute, Tokyo, Japan). By polymerizing short DNA sequences encoding two phosphorylation motifs, we created a panel of Cas mutants in which the entire substrate domain was replaced by synthetic domains containing random numbers and arrangements of the motifs. Most of these synthetic Cas variants were recognized and phosphorylated by Src in vitro and in intact mammalian cells. The random polymer mutants also restored migration activity to Cas knockout cells; even artificial proteins containing a single motif retained some biological function. Our results suggest that the arrangement of Cas motifs is not critical for signaling. This method could be used to identify the minimal functional units in other signaling proteins. For more details, see Patwardhan et al., ACS Chemical Biology 2009, 4: 751-758.