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Saccharomyces cerevisiae gene YMR291W/TDA1 mediates the in vivo

phosphorylation of hexokinase isoenzyme 2 at serine-15

Karina Kettner a,,1, Udo Krause b,1, Sophie Mosler b, Claudia Bodenstein a, Thomas M. Kriegel a,2,Gerhard Rdel b,2

a Institut fr Physiologische Chemie, Technische Universitt Dresden, Dresden, Germanyb Institut fr Genetik, Technische Universitt Dresden, Dresden, Germany

a r t i c l e i n f o

Article history:

Received 14 December 2011

Revised 11 January 2012

Accepted 16 January 2012

Available online 28 January 2012

Edited by Judit Ovdi

Keywords:

Hexokinase

Hxk2

Phosphorylation

Protein kinase

YMR291W

TDA1

Saccharomyces cerevisiae

a b s t r a c t

Hxk2 is the predominant hexokinase ofSaccharomyces cerevisiaeduring growth on glucose. In addi-

tion to its role in glycolysis, the enzyme is involved in glucose sensing and regulation of gene expres-

sion. Glucose limitation causes the phosphorylation of Hxk2 at serine-15 which affects the nucleo-

cytoplasmic distribution and dimer stability of the enzyme. In order to identify the responsible

kinase, we screened selected protein kinase single-gene deletion mutants by high resolution clear

native PAGE. Deletion ofYMR291W/TDA1 resulted in the absence of the Hxk2 phosphomonomer,

indicating an indispensable role of the corresponding protein in Hxk2 phosphorylation.

2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Hexokinases represent a class of multifunctional enzymes

which, beyond their contribution to the uptake and initiation of

metabolism of glucose, fructose and mannose, are also involved

in glucose signalling in yeast, plants and humans [13]. Several

hexokinases have been shown to translocate to the nucleus to

mediate glucose repression of transcription [4,2,5] or to participate

in mechanisms finally leading to glucose homeostasis[1,6]. The

Saccharomyces cerevisiae genome encodes three hexose kinases:

the two differentially regulated hexokinase isoenzymes Hxk1 andHxk2, which phosphorylate both aldo- and keto-sugars, and the

glucokinase Glk1, which is specific for aldohexoses. Hxk2, the pre-

dominant hexose kinase during growth on glucose [7], is presumed

to be involved in metabolic signalling indicating glucose limitation

[8,9] and plays a prominent role in glucose repression of several

Mig1-regulated genes [10,11,2].

The regulatory functions of Hxk2 are reflected by its dynamic

distribution between cytosol and nucleus which is affected by glu-

cose availability and the transcriptional repressor Mig1[12]. Hxk2

translocation into the nucleus and interaction with Mig1 require

serine-311 of Mig1 [13] and the lysine-7 methionine-16 se-

quence motif of Hxk2 [12,14,2,15]. Because serine-15 of Hxk2 is

phosphorylated in vivo under low-glucose growth conditions

(0.1% glucose)[8], it was suggested that the phosphorylation state

of this amino acid affects glucose signalling [16]and the nucleo-cytoplasmic distribution of Hxk2[14]. Under high-glucose growth

conditions (2% glucose), Hxk2 interacts with Mig1 and Snf1 to gen-

erate a repressor complex[12,2]thereby preventing the Snf1 pro-

tein kinase-mediated phosphorylation of Mig1 at serine-311[13].

Disintegration of the repressor complex under glucose limitation

involves Hxk2 phosphorylation at serine-15[8], subsequent phos-

phorylation of Mig1 at serine-311 by Snf1, and exit of both Hxk2

and Mig1 from the nucleus[13].

The regulatory role of Hxk2 phosphorylation at serine-15 is

supported by the identification of Hxk2 as a substrate of the nucle-

ar exporter Xpo1 and the observation that serine-15 phosphoryla-

tion promotes Hxk2 nuclear export by facilitating its association

0014-5793/$36.00 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.doi:10.1016/j.febslet.2012.01.030

Abbreviations:hrCNE, high resolution clear native electrophoresis; S. cerevisiae,

Saccharomyces cerevisiae; Hxk2, S. cerevisiaehexokinase isoenzyme 2; WT, wild type Corresponding author. Address: Institut fr Physiologische Chemie, Medizini-

sche Fakultt Carl Gustav Carus, Technische Universitt Dresden, Fetscherstrasse

74, D-01307 Dresden, Germany. Fax: +49 351 458 6306.

E-mail address:Karina.Kettner@tu-dresden.de(K. Kettner).1 Equal contribution.2 Authors share senior authorship.

FEBS Letters 586 (2012) 455458

j o u r n a l h o m e p a g e : w w w . F E B S L e t t e r s . o r g

http://dx.doi.org/10.1016/j.febslet.2012.01.030mailto:Karina.Kettner@tu-dresden.dehttp://dx.doi.org/10.1016/j.febslet.2012.01.030http://www.febsletters.org/http://www.febsletters.org/http://dx.doi.org/10.1016/j.febslet.2012.01.030mailto:Karina.Kettner@tu-dresden.dehttp://dx.doi.org/10.1016/j.febslet.2012.01.030

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with Xpo1 [17]. Serine-15 phosphorylation may, therefore, con-

tribute to the control of the nucleo-cytoplasmic distribution of

Hxk2 depending on glucose availability. While dephosphorylation

of Hxk2, which is favoured in high glucose conditions, is mediated

by the phosphatase Glc7p and its regulatory subunit Reg1p [16],

the kinase phosphorylating Hxk2 at serine-15 is still unknown.

Although serine-15 is part of a protein kinase A consensus phos-

phorylation sequence, there is no evidence that cAMP-dependent

protein kinases participate in serine-15 phosphorylation in vivo

[18,19].

The current study presents a novel strategy to identify the Hxk2

kinase by a functional comparison of S. cerevisiae protein kinase

single-gene deletion mutant strains. The experimental approach

is based on the dissociation of the Hxk2 dimer in vitro [20]upon

serine-15 phosphorylation and the different electrophoretic migra-

tion of the monomeric and the dimeric form of the enzyme. Evi-

dence is provided that the S. cerevisiae YMR291W/TDA1 gene is

required for serine-15 phosphorylation of Hxk2 by encoding either

the actual Hxk2 kinase or a protein/enzyme being part of the Hxk2

phosphorylation network.

2. Materials and methods

2.1. Strains and media

TheEscherichia colistrain used in this work was DH5a (BRL).S.

cerevisiae wild type (WT) strain BY4741 (accession number

Y00000) and deletion strains Dpks1 (accession number Y00391),

Dsks1 (Y02802), Delm1 (Y04897), Dsak1 (Y06128), Dyak1

(Y07006), Dtos3 (Y04546), Dybr028c (Y03165), Dydl025c

(Y03721), Dykl171w (Y05021), Dymr291W (Y00878), Dypl141c

(Y02111), Dypl150w (Y02102), and Dykl168c (Y05018) were ob-

tained from Euroscarf. Mutant strain YKK1 (HXK2::hxk2S15A-

KlURA3) was generated in this work. E. coli was cultivated in LB

as described by Sambrook et al.[21]. Yeast cells were either grown

in YPD or in minimal medium (0.67% yeast nitrogen base w/o ami-

no acids, 2% glucose) supplemented with histidine, leucine, and

methionine[22].

2.2. Preparation of crude cell extracts

Yeast cells were harvested at anOD600of 2.0, washed with ice-

cold lysis buffer (50 mM imidazole/HCl, pH 7.0, 50 mM NaCl, 5 mM

e-aminocaproic acid, 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF), 1 proteinase inhibitor cocktail (EDTA-free;

Roche), phosphatase inhibitor cocktails I + II (1:100; SigmaAl-

drich)), and transferred into a tube containing 0.3 ml of the same

buffer and 0.5 g glass beads of 0.4 mm diameter (Roth). Crude cell

extracts were prepared by vigorously shaking five times for 1 min

at 25 Hz with 1-min intervals on ice using a Mixer Mill MM 200

(Retsch), followed by centrifugation at 12000g and 4 C for10 min. The supernatants were used for further analyses.

2.3. High resolution clear native electrophoresis (hrCNE) and Western

blot analysis

hrCNE was performed essentially as described previously [23]

using the cathode buffer variant 3 (50 mM tricine, 7.5 mM imidaz-

ole/HCl, pH 7.0, supplemented with 0.01% w/v dodecyl-b-D-malto-

side and 0.05% w/v deoxycholate before use). Proteins were

transferred onto a PVDF membrane (Millipore), probed with poly-

clonal antibodies directed against Hxk2 (BioGenes), and detected

with HRP-conjugated anti-rabbit IgG antibodies (Cell Signaling)

and ImmobilonTM Western Chemiluminescent HRP Substrate

(Millipore).

2.4. k-Phosphatase treatment

Crude cell extracts were prepared as described above using a

buffer containing 50 mM potassium phosphate, pH 7.4, supple-

mented with 1 mM AEBSF and 1 proteinase inhibitor cocktail

(EDTA-free, Roche) before use. Protein samples (20lg) wereincubated for 1 h at 30 C with 200 U k-phosphatase (New England

Biolabs) in the absence or presence of phosphatase inhibitors

(5mM EDTA, phosphatase inhibitor cocktails I and II

(SigmaAldrich)) in a final volume of 20 ll.

2.5. DNA manipulations

Standard DNA techniques were carried out as described by

Sambrook et al. [21]. New DNA constructs were confirmed by

sequencing. Restriction enzymes were obtained from New England

Biolabs. DNA Ligase was purchased from Promega.

2.6. Construction of plasmid p416YMR291W

The YMR291W open reading frame including 497 bp upstream

and 385 bp downstream sequences harbouring the putative tran-

scription initiation and termination sites, respectively, was ampli-

fied by PCR using genomic DNA of S. cerevisiae strain BY4741 as

the template. The PCR product was cleaved with SacI/KpnI and in-

serted into the SacI/KpnI sites of single-copy vector p416ADH

[24], thereby replacing the plasmid-born ADH1 promoter and

CYC1terminator.

2.7. Construction of the S15A mutant strain YKK1

TheHXK2 coding region was PCR-amplified by using genomic

DNA ofS. cerevisiae strain BY4741 as the template, employing a for-

ward primer with the S15A mutation. The URA3cassette compris-

ing the KlURA3 gene including its promoter and terminator

sequences was PCR-amplified using vector pBS1539 (Euroscarf)

as the template. Both PCR products were fused by overlap exten-sion PCR [25]. The final construct was transformed into BY4741

cells[26].

3. Results and discussion

3.1. Phosphorylated and non-phosphorylated Hxk2 is separated by

hrCNE

The experimental approach employed to identify the actual

Hxk2 kinase is based on the dissociation of the Hxk2 dimer as a re-

sult of serine-15 phosphorylation. Due to their different electro-

phoretic mobility, the monomeric and dimeric forms of Hxk2 are

separated in clear native gels. We used hrCNE known as a method

that preserves the oligomeric states of proteins [23], to identifyphosphorylated and non-phosphorylated Hxk2. In a wild type

(BY4741) extract, which served as a control, two well-separated

Hxk2 bands were detected with anti-Hxk2 antibodies (Fig. 1A, lane

1). Upon phosphatase treatment the faster migrating band disap-

peared (Fig. 1A, lane 2). In contrast, the protein pattern remained

unaffected, when phosphatase inhibitors were added (Fig. 1A, lane

3) or phosphatase was omitted from the sample (Fig. 1A, lane 4).

These findings confirm the existence of a phosphorylated mono-

meric form and a non-phosphorylated dimeric form of Hxk2[16]

and prove the suitability of hrCNE to discriminate between both

enzyme species.

In line with the finding that serine-15 represents the in vivo

phosphorylation site of Hxk2 [8], hrCNE analysis of a mutant

strain expressing a non-phosphorylatable form of Hxk2, in which

456 K. Kettner et al. / FEBS Letters 586 (2012) 455458

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serine-15 was replaced by alanine (Hxk2-S15A) revealed a single

band essentially corresponding to the dimeric form (Fig. 1B, lane

1). Phosphatase treatment had no influence on the migrationbehaviour of Hxk2-S15A (Fig. 1B, lane 2). The apparently slightly

lower molecular mass of Hxk2-S15A compared to the WT dimer

(Fig. 1B, lane 5) might reflect the reduced oligomeric stability of

the mutant enzyme[20].

3.2. Phosphorylation of Hxk2 depends on the YMR291W/TDA1 gene

product

TheS. cerevisiae genome encodes 120 nominal protein kinases,

of which 40 have clearly assigned substrates. Hxk2 is one of 23

known phosphoregulated metabolic enzymes; however, the

respective kinase has been identified for only seven of them [27].

In an attempt to identify the actual Hxk2 kinase, we screened by

hrCNES. cerevisiae mutant strains with single deletions of protein

kinase genes. The selection from 80 candidate genes considered

those that encode protein kinases with known functions in hexose

metabolism and kinases of unknown function, but with known

localisation in the cytoplasm and/or the nucleus (Table 1). The pro-

tein band corresponding to the Hxk2 phosphomonomer was not

detectable only when the YMR291W/TDA1 deletion mutant wasanalysed (Fig. 2). These data indicate that Ymr291W/Tda1 is re-

quired for Hxk2 phosphorylation at serine-15, either as the

searched-for Hxk2 kinase or as a protein/enzyme that is indirectly

involved in serine-15 phosphorylation. Both alternatives are sup-

ported by the complementation of the YMR291W/TDA1 deletion

mutant (Fig. 3, lane 2) with the YMR291W/TDA1 open reading

frame cloned into the single-copy vector p416. Like the WT strain,

the transformant exhibits two Hxk2 bands corresponding to the

phosphomonomer and dimer (Fig. 3,lanes 1 and 4). In contrast, a

transformant bearing the control plasmid displays only the dimeric

form of Hxk2 (Fig. 3, lane 3).

The present work identifies Ymr291W/Tda1 as a protein that is

indispensably required for the phosphorylation of Hxk2 at serine-

15. However, our data do not allow to discriminate between a di-rect effect of Ymr291W/Tda1 on Hxk2 and an indirect mode of ac-

tion, e.g., as an upstream protein kinase. The involvement of

Ymr291W/Tda1 in Hxk2 phosphorylation is somewhat surprising

146 kDa

UPP 66 kDa

-Phosphatase

Phosphatase inhibitors - -+ +

+

WT

- + +

WTHxk2-S15A

--

+

A

---

B

-+

-+

1 2 3 4 1 2 3 4 5

Fig. 1. hrCNEas a tool to separate phosphorylated andnon-phosphorylated forms of Hxk2. WTstrain BY4741 (A)and hexokinase mutant strainHxk2-S15A (B)were grownin

YPD. Crude cell extracts (20 lg protein) were incubated for 1 h at 30 C in the absence () or presence (+) ofk-phosphatase and phosphatase inhibitors. Proteins wereseparated by 520% hrCNE, blotted onto a PVDF membrane, probed with anti-Hxk2 antibodies and detected with HRP-conjugated anti-rabbit IgG using the Immobilon TM

Western Chemiluminescent HRP detection system. UP, unphosphorylated Hxk2; P, phosphorylated Hxk2.

Table 1

Localisation of protein kinases selected for hrCNE screening. Data on protein

localisation were obtained by fluorescence microscopy of yeast strains expressing

the respective proteins as GFP-fusions [29]. The kinase that is required for the

phosphorylation of Hxk2 at serine-15 is shown in bold.

Systematic name (protein name) Protein localisation

YAL017W(Pks1p) Cytoplasm

YPL026C(Sks1p) Cytoplasm, nucleusYKL048C(Elm1p) Cytoplasm

YER129W(Sak1p) Cytoplasm

YJL141C(Yak1p) Cytoplasm, nucleus

YGL179C(Tos3p) Cytoplasm

YBR028C Cytoplasm

YDL025C Unknown

YKL171W Cytoplasm

YMR291W(Tda1p) Cytoplasm, nucleus

YPL141C Cytoplasm

YPL150W Unknown

YKL168C Cytoplasm

ps

k1

66 kDa

146 kDaelm1

yb

r028

c

yak1

ym

r291

w

ykl171

w

ypl1

41c

yp

l150

w

ykl168

c

sak1

to

s3

ydl025

c

sks1

*

UP

P

Fig. 2. Identification ofYMR291W/TDA1 as a gene encoding a protein kinase required for Hxk2 in vivo phosphorylation at serine-15. The indicated single-gene deletion

mutant strains ofS. cerevisiae were grown in YPD. Crude cell extracts (40 lg protein) were analysed by hrCNE as described in the legend ofFig. 1. Asterisk, immunoreactiveprotein of unknown identity; UP, unphosphorylated Hxk2; P, phosphorylated Hxk2.

WT

ym

r291w

ym

r291w

+p416ADH

ym

r291w

+p416

YMR2

91w

66 kDa

146 kDa

UP

P

1 2 3 4

Fig. 3. The YMR291W/TDA1 gene product mediates Hxk2 phosphorylation. WT

strain BY4741 and deletion mutant Dymr291W without plasmid, with plasmid

p416ADH (negative control) and with plasmid p416YMR291W were grown in

minimal medium containing 2% glucose. Crude cell extracts (20 lg protein) wereanalysed by hrCNE as described in the legend ofFig. 1UP, unphosphorylated Hxk2;

P, phosphorylated Hxk2.

K. Kettner et al./ FEBS Letters 586 (2012) 455458 457

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because TDA1was identified in a high-throughput screen as a gene

affecting topoisomerase I-induced DNA damage[28]. The deletion

of YMR291W/TDA1 is apparently without major effects on growth

on various carbon sources (data not shown). Current experiments

employing tandem affinity purification and mass spectrometry

aim to identify proteins interacting with Ymr291W/Tda1 in order

to further characterise the role of this nominal protein kinase in

Hxk2 in vivo phosphorylation and beyond. Future studies will also

have to address the question of Ymr291W/Tda1 expression and

activity regulation.

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