scholarly journals Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A.

1993 ◽  
Vol 13 (9) ◽  
pp. 5567-5581 ◽  
Author(s):  
M J Blacketer ◽  
C M Koehler ◽  
S G Coats ◽  
A M Myers ◽  
P Madaule

The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.

1993 ◽  
Vol 13 (9) ◽  
pp. 5567-5581
Author(s):  
M J Blacketer ◽  
C M Koehler ◽  
S G Coats ◽  
A M Myers ◽  
P Madaule

The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.


2007 ◽  
Vol 104 (8) ◽  
pp. 2979-2984 ◽  
Author(s):  
J.-H. Ahn ◽  
T. McAvoy ◽  
S. V. Rakhilin ◽  
A. Nishi ◽  
P. Greengard ◽  
...  

2020 ◽  
Vol 21 (23) ◽  
pp. 8939
Author(s):  
Stephanie Makhoul ◽  
Elena Kumm ◽  
Pengyu Zhang ◽  
Ulrich Walter ◽  
Kerstin Jurk

Distinct membrane receptors activate platelets by Src-family-kinase (SFK)-, immunoreceptor-tyrosine-based-activation-motif (ITAM)-dependent stimulation of spleen tyrosine kinase (Syk). Recently, we reported that platelet activation via glycoprotein (GP) VI or GPIbα stimulated the well-established Syk tyrosine (Y)-phosphorylation, but also stoichiometric, transient protein kinase C (PKC)-mediated Syk serine(S)297 phosphorylation in the regulatory interdomain-B, suggesting possible feedback inhibition. The transient nature of Syk S297 phosphorylation indicated the presence of an unknown Syk pS297 protein phosphatase. In this study, we hypothesize that the S-protein phosphatase 2A (PP2A) is responsible for Syk pS297 dephosphorylation, thereby affecting Syk Y-phosphorylation and activity in human washed platelets. Using immunoblotting, we show that specific inhibition of PP2A by okadaic acid (OA) alone leads to stoichiometric Syk S297 phosphorylation, as analyzed by Zn2+-Phos-tag gels, without affecting Syk Y-phosphorylation. Pharmacological inhibition of Syk by PRT060318 or PKC by GF109203X only minimally reduced OA-induced Syk S297 phosphorylation. PP2A inhibition by OA preceding GPVI-mediated platelet activation induced by convulxin extended Syk S297 phosphorylation but inhibited Syk Y-phosphorylation. Our data demonstrate a novel biochemical and functional link between the S-protein phosphatase PP2A and the Y-protein kinase Syk in human platelets, and suggest that PP2A represents a potential enhancer of GPVI-mediated Syk activity caused by Syk pS297 dephosphorylation.


1997 ◽  
Vol 17 (2) ◽  
pp. 620-626 ◽  
Author(s):  
Y Wang ◽  
D J Burke

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.


2008 ◽  
Vol 82 (7) ◽  
pp. 3612-3623 ◽  
Author(s):  
Yikun Li ◽  
Huijun Wei ◽  
Tung-Chin Hsieh ◽  
David C. Pallas

ABSTRACT The adenovirus early region 4 open reading frame 4 (E4orf4) protein specifically induces p53-independent cell death of transformed but not normal human cells, suggesting that elucidation of its mechanism may provide important new avenues for cancer therapy. Wild-type E4orf4 and mutants that retain cancer cell toxicity also induce growth inhibition in Saccharomyces cerevisiae, which provides a genetically tractable system for studying E4orf4 function. Interaction with the protein phosphatase 2A (PP2A) B regulatory subunit is required for E4orf4's effects, suggesting that E4orf4 may function by regulating B subunit-containing heterotrimeric PP2A holoenzymes (PP2ABAC), which consist of a B subunit complexed with the PP2A structural (A) and catalytic (C) subunits. However, it is not known whether E4orf4-induced growth inhibition requires interaction with the PP2A C subunit or whether E4orf4 might have PP2A B subunit-dependent effects that are independent of PP2ABAC holoenzyme formation. To test these possibilities in S. cerevisiae, we disrupted the stable formation of PP2ABAC heterotrimers and thus E4orf4/C subunit association by PP2A C subunit point mutations or by deletion of the gene for the PP2A methyltransferase, Ppm1p, and assayed for effects on E4orf4-induced growth inhibition. Our results support a model in which E4orf4 mediates growth inhibition and cell killing both through PP2ABAC heterotrimers and through a B regulatory subunit-dependent pathway(s) that is independent of stable complex formation with the PP2A C subunit. They also indicate that Ppm1p has a function other than regulating the assembly of PP2A heterotrimers and suggest that selective PP2A trimer inhibitors and PP6 inhibitors may be useful as adjuvant anticancer therapies.


2005 ◽  
Vol 4 (6) ◽  
pp. 1029-1040 ◽  
Author(s):  
Matthew S. Gentry ◽  
Yikun Li ◽  
Huijun Wei ◽  
Farhana F. Syed ◽  
Sameer H. Patel ◽  
...  

ABSTRACT Protein phosphatase 2A (PP2A) catalytic subunit can be covalently modified at its carboxy terminus by phosphorylation or carboxymethylation. Determining the effects of these covalent modifications on the relative amounts and functions of different PP2A heterotrimers is essential to understanding how these modifications regulate PP2A-controlled cellular processes. In this study we have validated and used a novel in vivo assay for assessing PP2A heterotrimer formation in Saccharomyces cerevisiae: the measurement of heterotrimer-dependent localization of green fluorescent protein-PP2A subunits. This assay relies on the fact that the correct cellular localization of PP2A requires that it be fully assembled. Thus, reduced localization would occur as the result of the inability to assemble a stable heterotrimer. Using this assay, we determined the effects of PP2A C-subunit phosphorylation mimic mutations and reduction or loss of PP2A methylation on the formation and localization of PP2AB/Cdc55p and PP2AB ′ /Rts1p heterotrimers. Collectively, our findings demonstrate that phosphorylation and methylation of the PP2A catalytic subunit can influence its function both by regulating the total amount of specific PP2A heterotrimers within a cell and by altering the relative proportions of PP2AB/Cdc55p and PP2AB ′ /Rts1p heterotrimers up to 10-fold. Thus, these posttranslational modifications allow flexible, yet highly coordinated, regulation of PP2A-dependent signaling pathways that in turn modulate cell growth and function.


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