Dataset Information

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

ABSTRACT: The serine/threonine protein kinase, Yak1p, functions as a negative regulator of the cell cycle in Saccharomyces cerevisiae, acting downstream of the cAMP-dependent protein kinase. In the present work we report that overexpression of haemagglutinin-tagged full-lengthYak1p and an N-terminally truncated form (residues 148-807) lead to growth arrest in PKA compromised yak1 null yeast cells. Both forms of recombinant Yak1p kinase were catalytically active 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 containing 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 full-length Yak1p reaction were 110+/-21 (Ser-164) and 8.7+/-1.7 (MBP), and 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 Yak1p was able to phosphorylate two generic protein tyrosine kinase substrates, both were phosphorylated on tyrosine residues in vivo and underwent tyrosine autophosphorylation when reacted with ATP in vitro. Tandem MS showed that Tyr-530 was phosphorylated both in vivo and in vitro after reaction with ATP. Pre-treatment with protein tyrosine phosphatase 1B removed all of Yak1p phosphotyrosine content and drastically reduced Yak1p activity against exogenous substrates, suggesting that the phosphotyrosine content of the enzyme is essential for its catalytic activity. Although the N-terminally truncated Yak1p was expressed at a lower level than the full-length protein, its catalytic activity and phosphotyrosine content were significantly higher than those of the full-length enzyme. Taken together, our results suggest that Yak1p is a dual specificity protein kinase which autophosphorylates on Tyr-530 and phosphorylates exogenous substrates on Ser/Thr residues.

SUBMITTER: Kassis S

PROVIDER: S-EPMC1221062 | biostudies-other | 2000 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser 916, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser 1412, and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets