Abstract
TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitro kinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the "activation loop" of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.
Highlights
The expression of TESK1 protein is almost in parallel with its mRNA expression in rat spermatogenic cells [2]
Lines—We previously showed that TESK1 mRNA was predominantly expressed in mouse and rat testicular germ cells [1, 2]
We reported that the 3.6-kb TESK1 mRNA was predominantly expressed in rat testicular germ cells at stages of pachytene spermatocytes to round spermatids [1, 2]
Summary
The expression of TESK1 protein is almost in parallel with its mRNA expression in rat spermatogenic cells [2]. We have obtained evidence that TESK1 is a dual specificity protein kinase, capable of phosphorylating both serine/ threonine and tyrosine residues in its sequence and in exogenous substrates. Dual Specificity Kinase Activity of TESK1—To characterize autophosphorylation and substrate phosphorylation activities of TESK1, GST fusion protein of the N-terminal protein kinase domain of TESK1 (GST-PK) was expressed in E. coli and purified by glutathione-Sepharose (see Fig. 2A).
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