Saccharomyces cerevisiae Yak1p protein kinase autophosphorylates on tyrosine residues and phosphorylates myelin basic protein on a C-terminal serine residue

The serine/threonine protein kinase, Yak1p, functions as a negative regulat or of the cell cycle in Saccharomyces cerevisiae, acting downstream of the cAMP-dependent protein kinase. In the present work we report that overexpre ssion of haemagglutinin-tagged full-lengthYak1p and an N-terminally truncat ed form (residues 148-807) lead to growth arrest in PKA compromised yak1 nu ll yeast cells. Both forms of recombinant Yak1p kinase were catalytically a ctive and preferred myelin basic protein (MBP) as a substrate over several other proteins. Phosphopeptide analysis of bovine MBP by tandem MS revealed two major Yak1p phosphorylation sites, Thr-97 and Ser-164. Peptides contai ning each site were obtained and tested as Yak1p substrates. Both forms of Yak1p phosphorylated a peptide containing the Ser-164 residue with far more efficient kinetics than MBP. The maximal velocity (V-max) values of the fu ll-length Yak1p reaction were 110 +/- 21 (Ser-164) and 8.7 +/- 1.7 (MBP), a nd those of N-terminally truncated Yak1p were 560.7 +/- 74.8 (Ser-164) and 34.4 +/- 2.2 (MBP) pmol/min per mg of protein. Although neither form of Yak 1p was able to phosphorylate two generic protein tyrosine kinase substrates , both were phosphorylated on tyrosine residues in vivo and underwent tyros ine autophosphorylation when reacted with ATP in vitro. Tandem MS showed th at Tyr-530 was phosphorylated both in vivo and in vitro after reaction with ATP. Pre-treatment with protein tyrosine phosphatase 1B removed all of Yak 1p phosphotyrosine content and drastically reduced Yak1p activity against e xogenous substrates, suggesting that the phosphotyrosine content of the enz yme is essential for its catalytic activity. Although the N-terminally trun cated Yak1p was expressed at a lower level than the full-length protein, it s catalytic activity and phosphotyrosine content were significantly higher than those of the full-length enzyme. Taken together, our results suggest t hat Yak1p is a dual specificity protein kinase which autophosphorylates on Tyr-530 and phosphorylates exogenous substrates on Ser/Thr residues.