Abstract
Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.
Highlights
The machinery necessary for phosphotyrosine-based signaling in metazoans includes ‘‘writer’’ domains, ‘‘readers’’ (SH2 and PTB domains), and ‘‘erasers’’ [1,2,3]
Because choanoflagellates are considered to be the closest living unicellular relatives of metazoans [5,6,7], the Monosiga brevicollis genome affords an important glimpse into the early evolution of tyrosine kinases and phosphatases. In addition to their catalytic domains, metazoan nonreceptor tyrosine kinases (NRTKs) possess noncatalytic domains that are important in kinase function [8,9,10]
We report that the enzyme is active, and that the PTB domain binds to peptides that contain phosphotyrosine
Summary
The machinery necessary for phosphotyrosine-based signaling in metazoans includes ‘‘writer’’ domains (tyrosine kinases), ‘‘readers’’ (SH2 and PTB domains), and ‘‘erasers’’ (tyrosine phosphatases) [1,2,3]. Because choanoflagellates are considered to be the closest living unicellular relatives of metazoans [5,6,7], the Monosiga brevicollis genome affords an important glimpse into the early evolution of tyrosine kinases and phosphatases. In addition to their catalytic domains, metazoan nonreceptor tyrosine kinases (NRTKs) possess noncatalytic domains that are important in kinase function [8,9,10]. Among the unique domain combinations are kinases containing C2, FYVE, and PTB domains These observations underscore the importance of domain shuffling in the emergence of tyrosine kinase signaling. Studies on two Src-related kinases from Monosiga brevicollis (MbSrc and MbSrc4) have suggested that the substrate targeting function of the SH3 and SH2 domains evolved earlier than the ability of the domains to engage in autoinhibitory interactions [13,14]
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