Science - USA (2022-04-15)

diet [the fructose, palmitate, and cholesterol
(FPC) diet] (Fig. 1H). Phosphorylation of S6
was unchanged in mice fasted overnight and
then refed normal chow (Fig. 1G), and only
mildly decreased in mice fed an FPC diet

(Fig. 1H). Phosphorylation of 4E-BP1 was un-

changed (Fig. 1H). Phosphorylation of Lipin1,

an mTORC1 substrate reported to inhibit

SREBP-1c in the liver ( 8 ), was also unaffected

in LiFKO livers (Fig. 1I). Together, these data

indicate that hepatic FLCN activates mTORC1

selectively to regulate TFE3 localization, which

is known to be regulated by mTORC1-mediated

phosphorylation, with little impact on canon-

ical mTORC1 substrates (Fig. 1, A and B).

Gosiset al.,Science 376 , eabf8271 (2022) 15 April 2022 2 of 12

Fig. 1. Liver FLCN selec-
tively promotes mTORC1-
mediated cytoplasmic
sequestration of TFE3,
without affecting canoni-
cal mTORC1 signaling.
(AandB) Model of selective
mTORC1 regulation.
P, phosphorylated; PRAS40,
proline-rich AKT substrate
of 40 kDa; DEPTOR, DEP
domain-containing mTOR-
interacting protein; mLST8,
target of rapamycin
complex subunit LST8.
(C) Schematic of FLCN liver
deletion to yield control
and hepatocyte-specific
Flcn-null (LiFKO) mice.
(D) FLCN protein expression
in livers of control and
LiFKO mice fed normal
chow and euthanized after
overnight fast followed
by 4 hours of refeeding.
HSP90, heat shock protein

90. (E) TFE3 protein
    expression in the indicated
    subcellular fractions from
    livers of control, LiFKO, and
    Tfe3knockout mice fed
    normal chow and euthanized
    ad lib. S.E., short exposure;
    L.E., long exposure; H3, his-
    tone H3. (F) TFE3 protein
    expression in the nucleus
    from livers of control (Con.)
    andRaptorliver-KO mice,
    euthanized after overnight
    fasting and 4 hours of
    refeeding. See fig. S1A for
    whole cell and cytoplasmic
    fractions. p:total, fraction
    of phosphorylated protein
    to total protein. (Gand
    H) Phosphorylation of
    mTORC1 targets in control
    and LiFKO mice fed either
    (G) normal chow or
    (H) 7 or 8 days of FPC
    diet (TD190142) and sugar
    water, and euthanized after
    overnight fast and ~4 hours
    of refeeding. pS6 240/4, phospho-ribosomal protein S6 (Ser240/244); S6, ribosomal protein S6; p4E-BP1 S65, phospho-4E-BP1 (Ser^65 ); p4E-BP1 Th37, phospho-4E-BP1 (Thr^37 ).
    (I) Phosphorylation of Lipin1, assayed by immunoprecipitation (IP) with anti-Lipin1 followed by immunoblotting for phospho-Lipin1, in liver lysates of control and LiFKO
    mice fed 9 days of FPC diet (TD190142) and sugar water and euthanized at 10 p.m. ad lib. IgG, immunoglobulin G. (J) TFE3 protein expression in the indicated subcellular
    fractions from control and liverTsc1KO mice fed normal chow and euthanized ad lib. *P< 0.05, **P< 0.01; StudentÕs two-tailedttest. Data are depicted as mean ± SEM.

A

14-3-3

Cytoplasm

TFE3:14-3-3

Nuclear

TFE3:H3

Whole cell

TFE3:14-3-3

D

TFE3

(S.E.)

TFE3

(L.E.)

H3

14-3-3

Whole cell Cytoplasm Nucleus

lo

rt

no

C LiFKOTf

e3

KO

E

FLCN

TFE3

H3

Con.

Rap

KO

F

HSP90

Control LiFKO

Control LiFKO

pS6 240/4

S6

HSP90

G

Whole cell

p:total

4E-BP1 S65

p:total

4EBP Th37

p:total

S6

Arbitrary unit

Normal chow

Control

LiFKO

Control

LiFKO

J

TFE3

H3

HDAC2

Con.

Ts c 1

KO

Whole cell

Con.

Ts c 1

KO

Cytoplasm

Con.

Ts c 1

KO

Nucleus

FLCN

Arbitrary unit

Control

LiFKO

FLCN:HSP90

rt

no

C

lo

LiFKOT

fe3

KO

lo

rt

no

C LiFKOTf

e3

KO

Arbitrary unit

Cytoplasm

TFE3:

14-3-3

Nuclear

TFE3:

H3

Whole cell

TFE3:

14-3-3

Arbitrary unit

Nucleus

pS6 240/4

S6

p4E-BP1 Th37

p4E-BP1 S65

4E-BP1

Beta actin

Con. LiFKO

FPC diet Whole cell

p:total S6

Arbitrary unit

Control

Rap KO

Control

LiFKO

H

Arbitrary unit

Control

Tsc1 KO

Flcnlox/lox Flcn-/-

Control LiFKO

Flcnlox/lox

AAV8-TBG-GFP, or

AAV8-TBG-Cre

C

RagC

FLCN

S6K TFE3

Catabolism

(i.e., lipids)

Protein

synthesis

TSC1/2

mTORC1

Control

Flcn KO

Raptor KO

TSC KO

mTORC1

+

+

• ++

+

• 
  


• 
  


• 
  

Cytoplasm

TFE3:

14-3-3

Nuclear

TFE3:

HDAC2

Whole cell

TFE3:

14-3-3

Nuclear

TFE3:

H3

Whole cell

FLCN:

14-3-3

pLipin1 S106

Total Lipin1

FPC diet

I

IgG

ControlLiFKO ControlRap

KO

IP: Total Lipin1

ControlLiFKO

rol

Co

nt

Ra

p

KO

p:total

Lipin1 (IP)

Arbitrary unitControl

LiFKO

TSC1/2

mTORC1

Raptor mTOR

PRAS40

DEPTOR

mLST8

FLCN

RagC

P

S6K lysosome

4E-BP1

Lipin1

Selective regulation of mTORC1

B

TFE3

PAMPK

P

ACC ULK1

P

P

0

1

2

3

4

5

0.0

0.2

0.4

0.6

0.8

0

1

2

3

(^4) *
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5
0
2
4
(^6)
0.0
0.5
1.0
1.5
2.0
0.0
0.2
0.4
0.6
0.8
1.0
0
1
2
3
4
5 p=0.054
0.0
0.5
1.0
1.5
0
1
2
3
4
5 *
0.0
0.5
1.0
1.5
2.0
0
10
20
30
0
5
10
15
20
0.0
0.5
1.0
1.5
2.0
2.5
0
1
2
3
0
5
10
15
20 p=0.11
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