scholarly journals Expressed in the Yeast Saccharomyces cerevisiae, Human ERK5 Is a Client of the Hsp90 Chaperone That Complements Loss of the Slt2p (Mpk1p) Cell Integrity Stress-Activated Protein Kinase

2006 ◽  
Vol 5 (11) ◽  
pp. 1914-1924 ◽  
Author(s):  
Andrew W. Truman ◽  
Stefan H. Millson ◽  
James M. Nuttall ◽  
Victoria King ◽  
Mehdi Mollapour ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Map Kinase ◽  
Protein Kinases ◽  
Strong Association ◽  
Active Form ◽  
Cell Integrity ◽  
Binding Studies ◽  
Hsp90 Inhibition ◽  
Binding Surface ◽  
Hsp90 Chaperone

ABSTRACT ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2Δ Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2Δ yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5.

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C

1993 ◽  
Vol 13 (5) ◽  
pp. 3076-3083
Author(s):  
K Irie ◽  
M Takase ◽  
K S Lee ◽  
D E Levin ◽  
H Araki ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinases ◽  
Cell Lysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Kinase C ◽  
Mitogen Activated Protein

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.

scholarly journals Tor2 Directly Phosphorylates the AGC Kinase Ypk2 To Regulate Actin Polarization

2005 ◽  
Vol 25 (16) ◽  
pp. 7239-7248 ◽  
Author(s):  
Yoshiaki Kamada ◽  
Yuko Fujioka ◽  
Nobuo N. Suzuki ◽  
Fuyuhiko Inagaki ◽  
Stephan Wullschleger ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Point Mutation ◽  
Essential Role ◽  
Cell Integrity ◽  
Autoinhibitory Domain ◽  
Multicopy Suppressors

ABSTRACT The target of rapamycin (TOR) protein kinases, Tor1 and Tor2, form two distinct complexes (TOR complex 1 and 2) in the yeast Saccharomyces cerevisiae. TOR complex 2 (TORC2) contains Tor2 but not Tor1 and controls polarity of the actin cytoskeleton via the Rho1/Pkc1/MAPK cell integrity cascade. Substrates of TORC2 and how TORC2 regulates the cell integrity pathway are not well understood. Screening for multicopy suppressors of tor2, we obtained a plasmid expressing an N-terminally truncated Ypk2 protein kinase. This truncation appears to partially disrupt an autoinhibitory domain in Ypk2, and a point mutation in this region (Ypk2D239A) conferred upon full-length Ypk2 the ability to rescue growth of cells compromised in TORC2, but not TORC1, function. YPK2 D239A also suppressed the lethality of tor2Δ cells, suggesting that Ypks play an essential role in TORC2 signaling. Ypk2 is phosphorylated directly by Tor2 in vitro, and Ypk2 activity is largely reduced in tor2Δ cells. In contrast, Ypk2D239A has increased and TOR2-independent activity in vivo. Thus, we propose that Ypk protein kinases are direct and essential targets of TORC2, coupling TORC2 to the cell integrity cascade.

scholarly journals A Two-Hybrid Screen of the Yeast Proteome for Hsp90 Interactors Uncovers a Novel Hsp90 Chaperone Requirement in the Activity of a Stress-Activated Mitogen-Activated Protein Kinase, Slt2p (Mpk1p)

2005 ◽  
Vol 4 (5) ◽  
pp. 849-860 ◽  
Author(s):  
Stefan H. Millson ◽  
Andrew W. Truman ◽  
Victoria King ◽  
Chrisostomos Prodromou ◽  
Laurence H. Pearl ◽  
...  
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
Active Form ◽  
Mitogen Activated Protein Kinase ◽  
Strong Interactions ◽  
Phenotypic Analysis ◽  
Mitogen Activated Protein ◽  
Hsp90 Chaperone ◽  
Two Hybrid

ABSTRACT The Hsp90 chaperone cycle catalyzes the final activation step of several important eukaryotic proteins (Hsp90 “clients”). Although largely a functional form of Hsp90, an Hsp90-Gal4p DNA binding domain fusion (Hsp90-BD) displays no strong interactions in the yeast two-hybrid system, consistent with a general transience of most Hsp90-client associations. Strong in vivo interactions are though detected when the E33A mutation is introduced into this bait, a mutation that should arrest Hsp90-client complexes at a stage where the client is stabilized, yet prevented from attaining its active form. This E33A mutation stabilized the two-hybrid interactions of the Hsp90-BD fusion with ∼3% of the Saccharomyces cerevisiae proteome in a screen of the 6,000 yeast proteins expressed as fusions to the Gal4p activation domain (AD). Among the detected interactors were the two stress-activated mitogen-activated protein (MAP) kinases of yeast, Hog1p and Slt2p (Mpk1p). Column retention experiments using wild-type and mutant forms of Hsp90 and Slt2p MAP kinase, as well as quantitative measurements of the effects of stress on the two-hybrid interaction of mutant Hsp90-BD and AD-Slt2p fusions, revealed that Hsp90 binds exclusively to the dually Thr/Tyr-phosphorylated, stress-activated form of Slt2p [(Y-P,T-P)Slt2p] and also to the MAP kinase domain within this (Y-P,T-P)Slt2p. Phenotypic analysis of a yeast mutant that expresses a mutant Hsp90 (T22Ihsp82) revealed that Hsp90 function is essential for this (Y-P,T-P)Slt2p to activate one of its downstream targets, the Rlm1p transcription factor. The interaction between Hsp90 and (Y-P,T-P)Slt2p, characterized in this study, is probably essential in this Hsp90 facilitation of the Rlm1p activation by Slt2p.

scholarly journals The purification and some properties of the Mg2+-activated cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae

1996 ◽  
Vol 315 (2) ◽  
pp. 393-399 ◽  
Author(s):  
Francis M. DICKINSON
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Conformational Change ◽  
Aldehyde Dehydrogenase ◽  
Active Form ◽  
Enzyme Reaction ◽  
Protein Fluorescence ◽  
Binding Studies ◽  
Absolute Requirement ◽  
Activity Form ◽  
Presence And Absence

A purification procedure has been developed for the cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae that yields homogeneous enzyme. The enzyme seems to be a tetramer of identical 58 kDa subunits. The enzyme reaction is strongly stimulated by Mg2+ at low NADP+ concentrations but there is no absolute requirement for bivalent cations. The kinetics of the reaction have been studied in the presence and absence of MgCl2. NADP+ binding studies of the quenching of protein fluorescence in the presence and absence of MgCl2 show that the effect of Mg2+ is to increase the affinity of the enzyme for NADP+ by approx. 100-fold. NADP+ binding causes a slow conformational change in the enzyme and converts the enzyme from the inactive or low-activity form in which it is isolated into the fully active form. This conformational change seems to explain the marked lag-phases seen in enzyme assays. The enzyme is strongly inhibited by disulfiram and pyridoxal 5-phosphate.

scholarly journals Coordination of the mating and cell integrity mitogen-activated protein kinase pathways in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (11) ◽  
pp. 6517-6525 ◽  
Author(s):  
B M Buehrer ◽  
B Errede
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Cell Integrity ◽  
Mating Partner ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Mating Pathway ◽  
Transcriptional Output

Mating pheromone stimulates a mitogen-activated protein (MAP) kinase activation pathway in Saccharomyces cerevisiae that induces cells to differentiate and form projections oriented toward the gradient of pheromone secreted by a mating partner. The polarized growth of mating projections involves new cell wall synthesis, a process that relies on activation of the cell integrity MAP kinase, Mpk1. In this report, we show that Mpk1 activation during pheromone induction requires the transcriptional output of the mating pathway and protein synthesis. Consequently, Mpk1 activation occurs subsequent to the activation of the mating pathway MAP kinase cascade. Additionally, Spa2 and Bni1, a formin family member, are two coil-coil-related proteins that are involved in the timing and other aspects of mating projection formation. Both proteins also affect the timing and extent of Mpk1 activation. This correlation suggests that projection formation comprises part of the pheromone-induced signal that coordinates Mpk1 activation with mating differentiation. Stimulation of Mpk1 activity occurs through the cell integrity phosphorylation cascade and depends on Pkc1 and the redundant MAP/Erk kinases (MEKs), Mkk1 and Mkk2. Surprisingly, Mpk1 activation by pheromone was only partially impaired in cells lacking the MEK kinase Bck1. This Bck1-independent mechanism reveals the existence of an alternative activator of Mkk1/Mkk2 in some strain backgrounds that at least functions under pheromone-induced conditions.

scholarly journals MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

1993 ◽  
Vol 13 (5) ◽  
pp. 3076-3083 ◽  
Author(s):  
K Irie ◽  
M Takase ◽  
K S Lee ◽  
D E Levin ◽  
H Araki ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinases ◽  
Cell Lysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Kinase C ◽  
Mitogen Activated Protein

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.

Saccharomyces cerevisiae SSD1-VConfers Longevity by a Sir2p-Independent Mechanism

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1661-1672 ◽  
Author(s):  
Matt Kaeberlein ◽  
Alex A Andalis ◽  
Gregory B Liszt ◽  
Gerald R Fink ◽  
Leonard Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Life Span ◽  
Cell Cycle Progression ◽  
Polymorphic Locus ◽  
Cell Integrity ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Maximal Life Span ◽  
Independent Mechanism ◽  
Yeast Aging

AbstractThe SSD1 gene of Saccharomyces cerevisiae is a polymorphic locus that affects diverse cellular processes including cell integrity, cell cycle progression, and growth at high temperature. We show here that the SSD1-V allele is necessary for cells to achieve extremely long life span. Furthermore, addition of SSD1-V to cells can increase longevity independently of SIR2, although SIR2 is necessary for SSD1-V cells to attain maximal life span. Past studies of yeast aging have been performed in short-lived ssd1-d strain backgrounds. We propose that SSD1-V defines a previously undescribed pathway affecting cellular longevity and suggest that future studies on longevity-promoting genes should be carried out in long-lived SSD1-V strains.

Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 309-316 ◽  
Author(s):  
Carsten Krischek ◽  
Burkhard Meinecke
Keyword(s):  
Map Kinase ◽  
Protein Kinases ◽  
Chromatin Condensation ◽  
Specific Inhibitor ◽  
Histone H1 ◽  
Dependent Manner ◽  
Free Medium ◽  
Concentration Dependent Manner ◽  
Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C

1993 ◽  
Vol 13 (5) ◽  
pp. 3067-3075 ◽  
Author(s):  
K S Lee ◽  
K Irie ◽  
Y Gotoh ◽  
Y Watanabe ◽  
H Araki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinases ◽  
Map Kinases ◽  
Pheromone Response ◽  
Pheromone Response Pathway ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.

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