Phosphorylation of the MAPKKK Regulator Ste50p in Saccharomyces cerevisiae: a Casein Kinase I Phosphorylation Site Is Required for Proper Mating Function

ABSTRACT The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.

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Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2870 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2870-2881 ◽

Cited By ~ 83

Author(s):

L C Robinson ◽

M M Menold ◽

S Garrett ◽

M R Culbertson

Keyword(s):

Protein Kinase ◽

Eukaryotic Cell ◽

Casein Kinase ◽

Temperature Sensitive ◽

Protein Kinase Activity ◽

Restrictive Temperature ◽

Loss Of Function ◽

Casein Kinase I ◽

Yeast Saccharomyces Cerevisiae

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

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Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

Blood ◽

10.1182/blood.v87.12.5287.bloodjournal87125287 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5287-5296 ◽

Cited By ~ 45

Author(s):

YL Zu ◽

Y Ai ◽

A Gilchrist ◽

ME Labadia ◽

RI Sha'afi ◽

...

Keyword(s):

Protein Kinase ◽

Enzymatic Activity ◽

Map Kinase ◽

Phorbol Ester ◽

Map Kinases ◽

Phosphorylation Site ◽

Human Neutrophils ◽

Inhibitory Peptide

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.

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

Biochemical Journal ◽

10.1042/bj20030638 ◽

2004 ◽

Vol 377 (2) ◽

pp. 395-405 ◽

Cited By ~ 45

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.

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Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies

Biochemical Journal ◽

10.1042/bj20080599 ◽

2008 ◽

Vol 413 (3) ◽

pp. 429-436 ◽

Cited By ~ 134

Author(s):

Yan Zeng ◽

Heidi Sankala ◽

Xiaoxiao Zhang ◽

Paul R. Graves

Keyword(s):

Protein Kinase ◽

P38 Mapk ◽

Kinase Inhibitor ◽

Mapk Pathway ◽

Phosphorylation Site ◽

Mitogen Activated Protein Kinase ◽

Processing Bodies ◽

Mitogen Activated Protein

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.

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A casein kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP-1B during neuroblastoma cell differentiation.

The Journal of Cell Biology ◽

10.1083/jcb.106.6.2057 ◽

1988 ◽

Vol 106 (6) ◽

pp. 2057-2065 ◽

Cited By ~ 109

Author(s):

J Díaz-Nido ◽

L Serrano ◽

E Méndez ◽

J Avila

Keyword(s):

Protein Kinase ◽

Neuroblastoma Cells ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Related Activity ◽

Dependent Protein Kinase ◽

Protein Map ◽

Dependent Protein

A neuroblastoma protein related to the brain microtubule-associated protein, MAP-1B, as determined by immunoprecipitation and coassembly with brain microtubules, becomes phosphorylated when N2A mouse neuroblastoma cells are induced to generate microtubule-containing neurites. To characterize the protein kinases that may be involved in this in vivo phosphorylation of MAP-1B, we have studied its in vitro phosphorylation. In brain microtubule protein, MAP-1B appears to be phosphorylated in vitro by an endogenous casein kinase II-like activity which also phosphorylates the related protein MAP-1A but scarcely phosphorylates MAP-2. A similar kinase activity has been detected in cell-free extracts of differentiating N2A cells. Using brain MAP preparations devoid of endogenous kinase activities and different purified protein kinases, we have found that MAP-1B is barely phosphorylated by cAMP-dependent protein kinase, Ca/calmodulin-dependent protein kinase, or Ca/phospholipid-dependent protein kinase whereas MAP-1B is one of the preferred substrates, together with MAP-1A, for casein kinase II. Brain MAP-1B phosphorylated in vitro by casein kinase II efficiently coassembles with microtubule proteins in the same way as in vivo phosphorylated MAP-1B from neuroblastoma cells. Furthermore, the phosphopeptide patterns of brain MAP-1B phosphorylated in vitro by either purified casein kinase II or an extract obtained from differentiating neuroblastoma cells are identical to each other and similar to that of in vivo phosphorylated neuroblastoma MAP-1B. Thus, we suggest that the observed phosphorylation of a protein identified as MAP-1B during neurite outgrowth is mainly due to the activation of a casein kinase II-related activity in differentiating neuroblastoma cells. This kinase activity, previously implicated in beta-tubulin phosphorylation (Serrano, L., J. Díaz-Nido, F. Wandosell, and J. Avila, 1987. J. Cell Biol. 105: 1731-1739), may consequently have an important role in posttranslational modifications of microtubule proteins required for neuronal differentiation.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00196.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. C989-C997 ◽

Cited By ~ 17

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.

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Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1594-1602.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1594-1602

Author(s):

A J Rossomando ◽

P Dent ◽

T W Sturgill ◽

D R Marshak

Keyword(s):

Protein Kinase ◽

Phosphorylation Site ◽

Mitogen Activated Protein Kinase ◽

Dual Specificity ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Protein Kinase Cascade ◽

Protein Kinase Kinase

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

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Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2870-2881.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2870-2881

Author(s):

L C Robinson ◽

M M Menold ◽

S Garrett ◽

M R Culbertson

Keyword(s):

Protein Kinase ◽

Eukaryotic Cell ◽

Casein Kinase ◽

Temperature Sensitive ◽

Protein Kinase Activity ◽

Restrictive Temperature ◽

Loss Of Function ◽

Casein Kinase I ◽

Yeast Saccharomyces Cerevisiae

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Inhibition of v-Mos kinase activity by protein kinase A.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.800 ◽

1996 ◽

Vol 16 (3) ◽

pp. 800-809 ◽

Cited By ~ 8

Author(s):

Y Yang ◽

C H Herrmann ◽

R B Arlinghaus ◽

B Singh

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Sodium Dodecyl ◽

Phosphorylation Site ◽

Catalytic Subunit ◽

In Vitro Mutagenesis ◽

Activity Increase ◽

Pka Catalytic Subunit

We investigated the effect of cyclic AMP-dependent protein kinase (PKA ) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37v-mos in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Combined tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies indicated that Ser-56 is the major in vivo phosphorylation site on v-Mos. In vivo phosphorylation at Ser-56 correlated with slower migration of the v-Mos protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, even though Ser-56 was phosphorylated by PKA, this phosphorylation was not involved in the inhibition of v-Mos kinase. The alanine-for-serine substitution at residue 56 did not affect the ability of v-Mos to autophosphorylate in vitro or, more importantly, to activate MEK1 in transformed NIH 3T3 cells. We identified Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. From our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation in mice could be explained at least in part by its inhibition of Mos kinase.

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Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein

Biochemical Journal ◽

10.1042/bj20051642 ◽

2006 ◽

Vol 396 (1) ◽

pp. 117-126 ◽

Cited By ~ 25

Author(s):

Paula Portela ◽

Silvia Moreno ◽

Silvia Rossi

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Pyruvate Kinase ◽

Phosphorylation Site ◽

Bovine Heart ◽

Specificity Constant ◽

Fructose 1,6 Bisphosphate ◽

Kinase A

Pyk1 (pyruvate kinase 1) from Saccharomyces cerevisiae was characterized as a substrate for PKA (protein kinase A) from bovine heart and yeast. By designing Pyk1 synthetic peptides containing potential PKA sequence targets (Ser22, Thr94 and Thr478) we determined that the peptide S22 was a substrate for PKA in vitro, with a Ksp* (specificity constant) 10-fold and 3-fold higher than Kemptide for bovine heart and yeast PKA respectively. In vitro phosphorylation of the Pyk1 S22A mutant protein was decreased by as much as 90% when compared with wild-type Pyk1 and the Pyk1 T94A mutant. The Ksp* values for Pyk1 and Pyk1 T94A were the same, indicating that both proteins are phosphorylated at the same site by PKA. Two-dimensional PAGE of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation prevented the formation of one of the Pyk1 isoforms. We conclude that in yeast the major PKA phosphorylation site of Pyk1 is Ser22.Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more active in the absence of FBP (fructose 1,6-bisphosphate). The specificity of yeast and mammalian PKA towards the S22 peptide and towards whole Pyk1 protein was measured and compared. The Ksp* for the S22 peptide is higher than that for Pyk1, indicating that the peptide modelled on Pyk1 is a much better substrate than Pyk1, regardless of which tissue was used as the source of PKA. However, the Km of Pyk1 protein is lower than that of the better substrate, the S22 peptide, indicating that ground-state substrate binding is not the major determinant of substrate specificity for PKA.

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