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

2000 ◽  
Vol 348 (2) ◽  
pp. 263-272 ◽  
Author(s):  
Shouki KASSIS ◽  
Tiffany MELHUISH ◽  
Roland S. ANNAN ◽  
Susan L. CHEN ◽  
John C. LEE ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Myelin Basic Protein ◽  
Basic Protein ◽  
Full Length ◽  
Tandem Ms ◽  
Tyrosine Residues

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 (Vmax) 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.

In vitro and in vivo phosphorylation of myelin basic protein by cerebral protein kinase

Nature ◽  
1974 ◽  
Vol 249 (5453) ◽  
pp. 150-151 ◽  
Author(s):  
EISHICHI MIYAMOTO ◽  
SHIRO KAKIUCHI ◽  
YASUO KAKIMOTO
Keyword(s):  
Protein Kinase ◽  
Myelin Basic Protein ◽  
Basic Protein

scholarly journals Catalytic Roles of Yeast GSK3β/Shaggy Homolog Rim11p in Meiotic Activation

Genetics ◽  
1999 ◽  
Vol 153 (3) ◽  
pp. 1145-1152
Author(s):  
Krishnamurthy Malathi ◽  
Yang Xiao ◽  
Aaron P Mitchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Complex Formation ◽  
Mutant Proteins ◽  
Tyrosine Residues ◽  
Low Level ◽  
Separable Functions

Abstract In Saccharomyces cerevisiae, many meiotic genes are activated by a heteromeric transcription factor composed of Ime1p and Ume6p. Ime1p-Ume6p complex formation depends upon the protein kinase Rim11p, which interacts with and phosphorylates both Ime1p and Ume6p in vitro. Rim11p may promote complex formation through its phosphorylation of Ime1p and Ume6p or simply through its interaction with both proteins. Here, we characterize mutant Ime1p derivatives that interact with Rim11p but are not phosphorylated in vitro. These mutant proteins are also defective in interaction with Ume6p. These results argue that Ime1p must be phosphorylated to interact with Ume6p. Our genetic observations suggest that Ime1p tyrosine residues are among the Rim11p phosphoacceptors, and we find that Ime1p reacts with an anti-phosphotyrosine antibody. Ime1p and Rim11p have been thought to act only through Ume6p, but we find that Ime1p and Rim11p promote meiosis at a very low level in the absence of Ume6p. A nonphosphorylatable mutant Ime1p derivative promotes sporulation through this Ume6p-independent pathway, as does a mutant Rim11p derivative that fails to interact with Ime1p. Therefore, Ime1p and Rim11p have two genetically separable functions in the sporulation program. However, catalytic activity of Rim11p is required for sporulation in the presence or absence of Ume6p.

scholarly journals Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin

2004 ◽  
Vol 377 (2) ◽  
pp. 395-405 ◽  
Author(s):  
Raffaele LOPREIATO ◽  
Sonia FACCHIN ◽  
Geppo SARTORI ◽  
Giorgio ARRIGONI ◽  
Stefano CASONATO ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Hybrid Approach ◽  
Inosine Monophosphate Dehydrogenase ◽  
Gene Encoding ◽  
Putative Protease ◽  
Structural Homologues ◽  
Novel Protein

The Saccharomyces cerevisiae piD261/Bud32 protein and its structural homologues, which are present along the Archaea–Eukarya lineage, constitute a novel protein kinase family (the piD261 family) distantly related in sequence to the eukaryotic protein kinase superfamily. It has been demonstrated that the yeast protein displays Ser/Thr phosphotransferase activity in vitro and contains all the invariant residues of the family. This novel protein kinase appears to play an important cellular role as deletion in yeast of the gene encoding piD261/Bud32 results in the alteration of fundamental processes such as cell growth and sporulation. In this work we show that the phosphotransferase activity of Bud32 is relevant to its functionality in vivo, but is not the unique role of the protein, since mutants which have lost catalytic activity but not native conformation can partially complement the disruption of the gene encoding piD261/Bud32. A two-hybrid approach has led to the identification of several proteins interacting with Bud32; in particular a glutaredoxin (Grx4), a putative glycoprotease (Ykr038/Kae1) and proteins of the Imd (inosine monophosphate dehydrogenase) family seem most plausible interactors. We further demonstrate that Grx4 directly interacts with Bud32 and that it is phosphorylated in vitro by Bud32 at Ser-134. The functional significance of the interaction between Bud32 and the putative protease Ykr038/Kae1 is supported by its evolutionary conservation.

scholarly journals Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

2011 ◽  
Vol 300 (5) ◽  
pp. C989-C997 ◽  
Author(s):  
Pimthanya Wanichawan ◽  
William E. Louch ◽  
Kristin H. Hortemo ◽  
Bjørg Austbø ◽  
Per Kristian Lunde ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Full Length ◽  
Kinase A

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.

scholarly journals The Gal3p-Gal80p-Gal4p Transcription Switch of Yeast: Gal3p Destabilizes the Gal80p-Gal4p Complex in Response to Galactose and ATP

1999 ◽  
Vol 19 (11) ◽  
pp. 7828-7840 ◽  
Author(s):  
Alok Kumar Sil ◽  
Samina Alam ◽  
Ping Xin ◽  
Ly Ma ◽  
Melissa Morgan ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Genetic Interaction ◽  
Transcription Activation ◽  
Full Length ◽  
Signal Transducer ◽  
Binding Peptide ◽  
Two Hybrid

ABSTRACT The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation ofGAL regulon genes. In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p. We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP. We have also found that retention of Gal80p by GSTG4AD (amino acids [aa] 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP. Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose. These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex. The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p. Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881.

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