Snf1 and the AMP-activated protein kinase

AMPK structure and function are conserved throughout eukaryotes.

AMPK is regarded as the central cellular energy regulator: it stimulates ATP-producing and inhibits ATP-consuming processes.

Activating AMPK is a potential target to treat metabolic life style diseases.

Together with the TOR (Target Of Rapamycin) kinases and PKA (cAMP-activated protein kinase), AMPK controls nutrient responses, energy homeostasis and stress responses.

The AMPK complexAMPK is a complex consisting of three subunits:

α-subunit, catalytic protein kinase, Snf1

β-subunit, scaffold, targeting, Sip1, Sip2, Gal83

γ-subunit, regulatory (ADP and AMP-binding), Snf4

The entire mammalian complex expressed in yeast lacking all own subunits is functional and regulated.

Activation of AMPK/Snf1 requires phosphorylation by (probably constitutive) upstream kinases (Sak1, Elm1, Tos3).

Deactivation (apparently the regulated step) requires phosphatases (Glc7-Reg1/Reg2).

ADP/AMP binding to γ-subunit stabilises the phosphorylated form of the α-subunit.

The AMPK ”network” in mammalian cells

The Snf1 network

Nielsen lab

Yeast carbon catabolite repressionSnf1 is best known for its role in carbon catabolite de/repression.In the presence of glucose unphosphorylated Mig1 represses expression of target genes.When glucose is limiting, Snf1 phosphorylates Mig1, which is then exported from the nucleus.Specific induction mechanisms (e.g. via Gal4 in the Gal regulon) then mediate upregulation of gene expression.The phosphorylation states of Snf1 and Mig1 can be monitored by Western blot.Snf1 activity can be measured in vitro.Target gene expression can be monitored.Real-time signalling can be observed via nuclear shuttling of Mig1-GFP.

Glucose responses at single cell level

Cell array in microfluidic device – rapidly changing medium

Moving cells repeatedly from glucose to no glucose and back

The role of the hexokinases in glucose repression

The hexokinases, esp Hxk2, are required for glucose repression.

Apparently, no further step in glycolysis is needed for glucose repression.

Hxk2 has been proposed to have a dual role as sensor and glycolytic enzyme.

Hxk seems to have regulatory functions in animals and plants.

The mechanism is unknown.

It has been proposed that Mig1, Hxk2 and Snf1 are in a complex on the DNA and Hxk2 affects access of Snf1 to Mig1. This model is not widely accepted.

We revisited some aspects of the role of Hxk2 by genetic analysis and monitoring Snf1 phosphorylation.

Role of hexokinase in Snf1 dephosphorylation upon glucose addition

Glucose addition to derepressed cells results in Snf1 dephosphorylation within a few seconds.

This also occurs in the Hxk2 mutant.

The problem of the hxk2 mutant is the inability to keep over a longer period Snf1 dephosphorylated.

Only the triple sugar kinase mutants lacks all glucose responses on Snf1 phosphorylation.

These data show that hexokinase affects the Snf1 phosphorylation state and most likely also glucose repression in this way (and not only by affecting Mig1 phosphorylation in the nucleus).

wild type

4%Glc 0.05% 4% Glucose pulse

20s 40s 60s 30 60 90min

hxk1Δ

4% 0.05% 4% Glucose pulse

20s 40s 60s 30 60 90min

hxk1Δ hxk2Δ

0.05% 4% Glucose pulse

20s 40s 60s 30 60 90min 4% 0.05% 4% Glucose pulse

20s 40s 60s 30 60 90min 4%

hxk2Δ

α-HA

Snf1-P

wild type hxk1Δ hxk2Δ glk1Δ

3%Gly+ 2%EtOH

4% glucose pulse20s 40s 60s 120s 5 30 60min

3%Gly+ 2%EtOH

4% glucose pulse20s 40s 60s 120s 5 30 60min

total-Snf1

Snf1-P

The role of the hexokinases in glucose repression

We revisited some aspects of the role of Hxk2 by genetic analysis and monitoring Snf1 phosphorylation.

Deletion of SAK1, TOS3, ELM1 plus HXK2 is lethal. We do not know why – but this suggests that they do not simply work in a linear pathway.

Deletion of REG1 and HXK2 also leads to effects that suggest that they work in parallel to affect Snf1 phosphorylation.

Snf1-P

reg1Δ reg2Δ

0.05% 4% Glucose pulse

20s 40s 60s 30 60 90min 4% 0.05% 4% Glucose pulse

20s 40s 60s 30 60 90min 4%

reg2Δ

α-HA

reg1Δ

0.05% 4% Glucose pulse

20s 40s 60s 30 60 90min 4%

Relation between the Reg1/2-Glc7 phosphatase and Hxk2

Deletion of both REG1 and REG2 completely abolishes glucose-induced dephosphorylation of Snf1.

Remarkably, deletion of REG1 plus HXK2 also completely abolishes dephosphorylation of Snf1 (and largely of Mig1 as well).

Hence Reg1 and Hxk2 act in parallel pathways.

As the reg2 hxk2 double mutant has the same phenotype as the hxk2 single mutant, Hxk2 may act via Reg2.

It may also act in a different way, perhaps by controlling Snf4 via the ADP/AMP levels.

wild type hxk2Δ

4%Glc 0.2% 4% Glucose pulse

20s 40s 60s 30 60 90min 4% 0.2% 4% Glucose pulse

20s 40s 60s 30 60 90min

hxk2Δ reg1Δ

0.2% 4% Glucose pulse

20s 40s 60s 30 60 90min 4% 0.2% 4% Glucose pulse

20s 40s 60s 30 60 90min 4%

reg1Δ

Mig1-HA

Mig1-P

Snf1-P

α-HA

wild type

4%Glc 0.05% 4% Glucose pulse

20s 40s 60s 30 60 90min

The Snf1 signalling pathway

Snf1 phosphorylation state is controlled by three upstream kinases (Sak1 is one of them) – probably constitutively.......and by a phosphatase – probably controlled by glucose.At low glucose the access of the phosphatase to the kinase appears to be impaired – Snf1 becomes phosphorylated.Snf1 phosphorylates Mig1 to relieve glucose repression.Glc7 performs a feed-forward loop by dephosphorylating both Snf1 and Mig1, probably to allow faster responses to glucose addition.

The signal is mediated via kinases (HOG) or via phosphatases (Snf1)

Phosphorylation of Snf1 seems to be constitutive.

It appears that glucose regulates access of the phosphatase to Snf1.

Dephosphorylation of Hog1 seems to be constitutive.

It appears that the phosphorylation rate of Hog1 by the upstream signalling system is controlled by stress.

Basal signalling in the absence of stimulus occurs in Snf1 and HOG

In both systems the upstream kinase seems to phosphorylate the effector kinase even in the absence of the physiological stimulus (deleting genes for phosphatases causes constitutive signalling).

The effector kinase is effectively dephosphorylated by the phosphatase under basal conditions.

In the Snf1 system the physiological signal then seems to diminish the dephosphorylation rate while in the Hog1 system the signal increases the phosphorylation rate.

These mechanisms may be important for rapid responses and adjusting thresholds and respond rapidly.

Snf1 is activated by salt stress

Addition of NaCl results in rapid and sustained phosphorylation and activation of Snf1.

Snf1 is required for normal acquisition of salt tolerance.

Snf1 phosphorylation and hence active Snf1 is needed for salt tolerance.

Snf1-P

α-HA

0 1 2 3 4 60 90 120 180 min at 0.8M NaCl

snf1Δ + pSNF1-HA

wild type + vector

snf1Δ + vector

snf1Δ + pSNF1

snf1Δ + pSNF1T210A

control 1M NaCl Raffinose

Snf1 is required for stimulation of ENA1expression under salt stress

Snf1 is required for normal upregulation of ENA1 expression (Na+

extrusion pump).

This effect is mediated by a different transcription factor, possibly the Nrg1 repressor.

ENA1-LacZ

0

100

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500

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700

LacZ

nmol

/min

/mg

prot

ein

control

0.8MNaCl

Snf1 activation by salt stress does not elicit glucose derepression

Snf1-activation by salt stress does not lead to major changes in Mig1 phosphorylation.

Mig1 also remains nuclear.

SUC2 expression is not stimulated under these conditions.

Hence Snf1 can be activated without mediating glucose derepression.

mig1Δ + pMIG1-HA

2% Glc + 0.8M NaCl 0.05% Glc 0.05% Glc + 0.8M NaCl 2% Glc 2% Glc2% Glc

0 5 10 30 60 0 5 10 30 60 0 5 10 30 60 min

Bright field GFP DAPI

2% glucose

0.05 % glucose

2% glucose+ 0.8M NaCl

Mig1-HAMig1-P

mig1Δ + pMIG1-GFP

Overexpression of Sak1 also activates Snf1....

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l)H L

Glucose Time after pulse

30’ 60’

pRS426 pSAK1-426

T210-P

α-HAH L

Glucose Time after pulse

30’ 60’

pRS426 pSAK1-4260

0.2

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Snf1

pho

spho

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1-P/

Snf1

-HA

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Sak1-TAP

...without causing glucose derepression...

Glucose (%)

H L

Time after Pulse (min)

1 10

Glucose (%)

H L

Time after Pulse (min)

1 10

Mig1-P

Mig1

Snf1-P

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(arb

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High glucose

Shift to low glucose

1 min after glucose pulse

10 min after glucose pulse

pRS426 pSAK1-426

pRS426 pSAK1-426 pSAK1-426pRS426High glucose Low glucose High glucose Low glucose

Simultaneous overexpression of Reg1 partly restores regulation

50-fold overexpression of Reg1 strongly affects growth, probably by recruiting Glc7 exclusively to Reg1.

This does not affect the Snf1 phosphorylation level, probably because access to Snf1 is strongly regulated by glucose.

However, it partly restores glucose regulation when Sak1 is overexpressed, suggesting that the balance between upstream kinase and phosphatase is important for controlling Snf1 phosphorylation.

Overexpression of Reg1 diminished expression of the Snf1 target SUC2 and this was not suppressed by overexpression of Sak1, consistent with the fact that Glc7-Reg1 controls both Snf1 and Mig1.

00.20.40.60.8

11.2

Snf1

pho

spho

ryla

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leve

l (S

nf1-

P/Sn

f1-H

A)

pCM185 pRS426

T210-Pα-HA

00.20.40.60.8

11.2

Snf1

pho

spho

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nf1-

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pREG1-TAPpRS426

T210-Pα-HA

00.20.40.60.8

11.2

Snf1

pho

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leve

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nf1-

P/Sn

f1-H

A)

pREG1-TAPpSAK1-TAP

T210-Pα-HA

Doxycycline: + -

H 30’ 60’Time after pulse

Glucose

L H 30’ 60’Time after pulse

Glucose

L

The Snf1 signalling pathway

How can we explain that in the presence of glucose Snf1 can be phoshorylated and activated in different ways (physiological and unphysiological), without causing any effect on Mig1 (no glucose derepression)?An obvious reason may be that the glucose regulated step is mediated by Glc7-Reg1, which targets both Snf1 and Mig1.Alternatively, one might invoke an additonal, presently unknown glucose-regulated step that allows Snf1 to target Mig1.

Snf1 networks considered

Models were generated using various types of data (time courses, protein levels, single cell data).

Simulations were compared to different types of data.

ConclusionsIt appears that the Glc7-Reg1 regulation of both Snf1 and Mig1 can not explain that Snf1 can be activated in the presence of glucose without causing Mig1 phosphorylation and glucose derepression.It further appears that glucose-control of the phosphatase is sufficient to explain the observed patterns of Snf1 (de)phosphorylation. Regulation of the upstream kinase is not required.But there needs to be an additional glucose-dependent regulation that allows Snf1 to phosphorylate Mig1.That Snf4 may bind ADP and activates Snf1 is probably not this mechanism because it appears to affect Snf1 phosphorylation.Hence there may be a different effect, mediated by Hxk2, that affects the Mig1 phosphorylation state.The Snf1 pathway is an interesting object to study network design and dynamic control as well as the link between signalling and metabolism.The exact role of Hxk2 in this system remains and interesting challenge.

Prof. Stefan HohmannDr. Maria Enge (project manager)Peter Dahl (lab manager)Takako Furukawa (technician)

MAPK PATHWAYS

(Dr. Bodil NordlanderDr. Marcus Krantz)Dr. Caroline Beck

(Dr. Carl Tiger)Dr. Kentaro Furukawa

(Elzbieta Petelenz)Doryaneh AhmadpourLars-Göran Ottosson

Jimmy KjellénRoja Babazadeh

ARSENITESIGNALLING

Prof. Markus Tamás(collaborating)Dr. Jenny Veide

Dr. Clara NavaretteTherese Johansson

NUTRIENT REGULATION

(Dr. Karin Elbing)Dr. Ye Tian

(Dr. Raul Salcedo)Loubna Bendrioua

AQUA(GLYCERO)PORINFUNCTION

Dr. Karin Lindkvist(collaborating)Cecilia Geijer

Madelene PalmgrenKarin Rodström

Hohmann lab

Funding:Swedish Research CouncilEuropean CommissionVinnovaFoundation for Strategic ResearchScience Faculty

Collaborators:Edda Klipp, BerlinFrancesc Posas, BarcelonaJens Nielsen, ChalmersMattias Goksör, Physics GUMorten Grøtli, Chemistry GU