phosphorylation of human chromosome maintenance 1 mediates association with 14-3-3 proteins

Phosphorylation of human chromosome maintenance 1 mediates association with 14-3-3 proteins

Sang Sun Kang* and Sung Hwa Shin

Department of Biology Education, Chungbuk National University, 410 Seongbong Road, Heungdok-gu, Cheongju, Chungbuk361�763, Republic of Korea

(Received 7 March 2013; received in revised form 15 April 2013; accepted 25 April 2013)

Human chromosome maintenance 1 (CRM1) was originally cloned based on homology to a yeast gene. CRM1,which belongs to the family of importin b-related nuclear transport receptors, directly and specifically associates withnuclear export signals (NESs) and mediates nuclear export of proteins containing leucine-rich NESs. We presentevidence that CRM1 associates with a 22-kDa 14-3-3 scaffolding protein that is a principal structural and regulatorycomponent of Human embryonic kidney (HEK 293) cells. We found a potential 14-3-3-binding motif(1049KHKRQMSVPG1058) in the CRM1 C-terminal domain that depended on serine 1055 phosphorylation byProtein Kinase A (PKA). We demonstrated that CRM1 is a PKA substrate using an in vitro assay. Using a pull-downapproach and co-immunoprecipitation, we found that CRM1 interacted with the 14-3-3 motif in vivo and in vitro. Wealso detected colocalization of CRM1 and 14-3-3 proteins using confocal microscopy. Nuclear pore localization ofCRM1 was disrupted by treatment with a PKA activator or inhibitor or by a S1055D/S1055A mutation in theCRM1 14-3-3-binding motif. Transient transfection assays showed that the apoptosis rate of cells with the S1055Dconstruct was twice that of cells with wild type (WT) or S1055A construct. Our observations indicated thatphosphorylation on the serine 1055 residue of CRM1 by PKA promoted 14-3-3 binding and cytoplasmiclocalization, resulting in enhancement of cell apoptosis.

Keywords: 14-3-3; CRM1; PKA; phosphorylation; protein�protein interaction

Introduction

Yeast genetic data using temperature-sensitive mutants

showed that export of marker proteins containing

nuclear export signals (NESs) is disrupted in yeast

strains with conditional chromosome maintenance 1

(CRM1) mutations (Toda et al. 1992; Turi et al. 1994;

Shimanuki et al. 1995). In Xenopus oocytes, overexpres-

sion of human CRM1 increases the export of nuclear-

injected Rev protein (Fornerod et al. 1997a; Neville et

al. 1997). Moreover, the cytotoxin leptomycin B (LMB)

inhibited export of Rev protein in both mammalian

cells and in Xenopus oocytes (Kudo et al. 1999; Fasken

et al. 2000). These studies show that CRM1 is involved

in the nuclear export of NES-containing proteins

(Fornerod et al. 1997a; Yoneda et al. 1999).

The LMB effect is probably direct, because LMB

binds to in vitro-translated CRM1 and in Schizosa-

ccharomyces pombe, resistance to LMB maps to the

CRM1 gene (Hamamoto et al. 1985; Kudo et al. 1999;

Fasken et al. 2000). This places CRM1 in the family of

RanGTP-binding proteins that includes other known

and putative import and export receptors. Further

supporting CRM1 as an export receptor for NES-

containing proteins is the observation that the N-

terminal region of CRM1 is homologous to the

RanGTP-binding domain of importin b (Nishi et al.

1994; Fukuda et al. 1997). CRM1 has been identified

with at least two proteins associated with the human

nuclear pore complex, namely the nucleoporins CAN/

Nup214 and Nup88. Two-hybrid assays in Sacch-

aromyces cerevisiae show interactions between CRM1

and several nucleoporins, as well as Rev and Ran

(Fornerod et al. 1997b; Askjaer et al. 1999; Kehlenbach

et al. 1999).

The leucine-rich NES recognized by CRM1 was

first identified in protein A phosphorylation inhibitor

(PKI) and the viral HIV-1 Rev protein. Both sequences

contain four regularly spaced leucines (Fornerod et al.

1997a; Neville et al. 1997; Kehlenbach & Gerace 2000).

Numerous studies have contributed to the definition of

the leucine-rich NES consensus sequence as F-X2�3-F-

X2�3-F-X-F (F: L, I, F, V, M; X: any amino acid)

(Fukuda et al. 1997; Jensen et al. 2000). The presence

of leucine residues is not a prerequisite for NESs and

several NESs have been identified that diverge from

this postulated consensus sequence (Neumann et al.

2000; la Cour et al. 2003). Using the currently ill-

defined NES consensus sequence, most proteins are

predicted to harbor NES consensus sequences. This

hampers the annotation of valid export signals and

their characterization in vivo (Neumann et al. 2000; la

Cour et al. 2003).

Recent progress in structural characterization of

cyclic AMP-dependent protein kinase (Protein Kinase

A, PKA) has expanded our knowledge of kinase

signaling (Kuehn 1972; Makman & Klein 1972; Kleppe

et al. 2011; Chan et al. 2012). The PKA holoenzyme is

a heterotetramer of two catalytic (C) subunits held in

*Corresponding author. Email: [email protected]

MOLECULAR

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Animal Cells and Systems, 2013

Vol. 17, No. 3, 186�195, http://dx.doi.org/10.1080/19768354.2013.801366

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an inactive state by association with a regulatory (R)

subunit dimer. cAMP binds cooperatively to two sites

termed A and B on each R subunit (Li 2011; Luconi

et al. 2011). In the inactive holoenzyme, only the B site

is exposed and available for cAMP binding. When

occupied, this enhances the binding of cAMP to the A

site by an intramolecular steric change. Binding of four

cAMP molecules, two per R subunit, leads to a

conformational change and dissociation into an R

subunit dimer bound to four cAMP molecules, and twoC monomers (Schillace & Carr 2006; Li 2011; Luconi

et al. 2011). The C subunits become catalytically active

and phosphorylate nearby target substrates on serine

or threonine residues in the context of Arg-Arg-X-Ser/

Thr, Arg-Lys-X-Ser/Thr, Lys-Arg-X-Ser/Thr, or Lys-

Lys-X-Ser/Thr. Each R subunit of PKA contains an

N-terminal docking and dimerization (D/D) domain, a

PKA inhibitor site, and two tandem cAMP-binding

domains. The D/D domain is connected to the cAMP-

binding domain A by an extended, highly disordered

linker that contains an autoinhibitory sequence andseveral putative phosphorylation sites (Pidoux &

Tasken 2010; Pidoux et al. 2011).

The 14-3-3 proteins are intracellular, dimeric, phos-

phoserine-binding proteins that have been identified in

eukaryotic organisms and are found primarily in the

cytoplasm (Aitken et al. 1992; Yaffe et al. 1997). The

eight mammalian members of the 14-3-3 family are

encoded by b, g, o,h,s, u, t, and z genes. Mammalian 14-

3-3 proteins regulate tyrosine and tryptophan

hydroxylases in neurotransmitter synthetic pathways.The 14-3-3 proteins bind and inhibit PKC, PDK1, and

Ask1 (Shibuya 2003; Obsilova et al. 2008). Binding of

14-3-3 proteins to the apoptosis-promoting protein

BAD prevents its binding to Bcl-XL (Muslin & Xing

2000; Tzivion et al. 2001). Generally, the binding of 14-3-

3 proteins to partners depends on serine (Ser) or threonine

(Thr) phosphorylation in the specific binding motif

(RX1�2S/T*X2�3S/T or RX2�3S/T*XP, where * indicates

the phosphorylated residue) (Aitken et al. 1992; Yaffe

et al. 1997; Muslin & Xing 2000; Tzivion et al. 2001;Shibuya 2003; Chun et al. 2004; Obsilova et al. 2008).

Upon visual inspection of CRM1 amino acid

sequence with the 14-3-3 binding motif and PKA

target substrate information, we noticed the presence

of a potential 14-3-3 binding motifs (1049KHKR-

QMSVPG1058) in its C-terminal domain (Fornerod

et al. 1997b). Our results suggested that 14-3-3 proteins

interact with wild type (WT) CRM1 through phos-

phorylation of the 1055 Ser residue in the CRM1 C-

terminal domain by PKA. In addition, we present

evidence suggesting that interaction with 14-3-3 pro-teins mediates the subcellular localization of CRM1,

and leads to a decrease in cell survival. Thus, our

observations shed light on the molecular mechanism(s)

underlying CRM1 regulation, localization, and signal-

ing that involve binding to 14-3-3 proteins.

Materials and methods

Antibodies

Antibodies against green fluorescent protein (GFP),

glutathionine-S-transferase (GST), CRM1 and 14-3-3bwere from Santa Cruz Biotechnology Inc. (Santa Cruz,

CA, USA). Phosphor-Ser/Thr antibody was from Cell

Signaling Technology Inc. (Boston, MA, USA).

Cell culture

Human embryonic kidney (HEK 293) cells were from

ATCC (Manassas, VA, USA). Media and supplements

were from GIBCO (Grand Island, NY, USA). Cells

were maintained in Dulbecco’s Modified Essential

Medium containing 10% fetal bovine serum (FBS)heat-inactivated for 30 min at 568C, 100 U potassium

penicillin/ml, 100 mg streptomycin/ml, 2 mM gluta-

mine and 20 mM sodium bicarbonate. Incubation was

in 5% CO2, 95% humidity at 378C.

Cell treatment

Cells were treated with 60 mM forskolin (Cell Signaling)

or 10 mM H89 (Sigma Aldrich) for 24 h, starting 24 h

after medium replacement.

Double-immunofluorescence microscopy

HEK 293 cells were plated to about 30% confluence on

microscope cover glasses (Fisher, USA) in 4-well plates

(SPL, Korea), and transiently transfected with en-

hanced green fluorescent protein (EGFP)-CRM1 WT

or EGFP-CRM1 mutant plasmids using the lipofecta-mine (Life Technologies Corporation, USA) procedure.

Cells were serum-starved for 6 h and subsequently

treated with 10% FBS for 42 h. Cell confluence did not

exceed 70%. Cells were blocked in 5% BSA in

Phosphate Buffered Saline (PBS) for 1 h and incubated

with a 1:100 dilution of anti-14-3-3 or anti-CRM1

(Santa Cruz Biotechnology), for 2 h at room tempera-

ture. For indirect immunofluorescence microscopy,washed slides were incubated for 1 h at room tempera-

ture with a 1:200 dilution of goat anti-rabbit Alexa

Fluor 568 or goat anti-mouse Alexa Fluor 594 (Life

Technologies Corporation). Slides were washed and

mounted with Dako fluorescent mounting medium

(Dako Co., USA), and examined using an LSM710

confocal microscope (ZEISS, Germany) in the Core

Facility of Chungbuk National University (Chun et al.2004).

Animal Cells and Systems 187

Expression and purification of recombinant proteins

WT C-terminal fragment CRM1 (amino acids 960�1120) and CRM1 mutant (S1055A) tagged with GSTwere expressed in Escherichia coli BL21 and purified

with GST-agarose beads according to the manufac-

turer’s instruction (Amersham Biosciences Co.). Pur-

ified proteins were used as bait protein for pull-down

assays or as substrates in PKA assays.

Fluorescence-activated cell sorting (FACS)

EGFP-CRM1 WT and EGFP-CRM1 mutants were

transfected and the rate of apoptosis was measured by

FACS Calibur (BD Bioscience, USA). Cells were

trypsinized in 2-ml Petri plates with 70 ml of 1�trypsin. Transfected cells were washed twice in cold

PBS, and 2 ml of 70% cold EtOH was added while

vortexing gently. Cells were left in EtOH at �208Covernight for fixing. Cells were spun at 1500 rpm for 5

min, resuspended in 2 ml PBS and spun again at 1500

rpm for 5 min. After adding 500 ml FACS buffer (PBS

plus PI 4 mg/ml plus RNase 30 mg/ml), cells were

incubated at 48C for 1 h, and analyzed immediately toprevent clumping. The FACS Calibur was equipped

with a gated amplifier and upgraded for enhanced

system performance at The Core Facility of Chungbuk

National University (Shin et al. 2012).

Site-directed mutagenesis

In order to obtain the mutants, amino acid changes

were introduced using mutated oligonucleotides for

S1055A (up 5?-cat aaa cgt caa atg Gct gtc cct ggc atc-

3?, down 5?-aaa gat gcc agg gac agC cat ttg acg ttt-3?) orS1055D (up 5?-cat aaa cgt caa atg GAt gtc cct ggc atc-

3?, down 5?-aaa gat gcc agg gac aTC cat ttg acg ttt-3?),and WT CRM1 as a template. The CRM1 mutant

constructs were prepared using a QuickChange† Multi

Site-Directed agenesis Kit (Stratagene). The C-terminal

CRM1 960�1120 aa fragment was obtained using

oligonucleotides (up 5?-ggtt agg atc caa aca tca tta aat

cct gga aat cca-3?, down 5?-ggtt ctc gag tta atc aca cat ttcttc tgg aat-3ƒ), and wild type CRM1 as a template. The

polymerase chain reaction (PCR) product was cloned in

pGEX-1 vector BamH1 and Xho1 site. All CRM1

constructs were confirmed via DNA sequencing.

PKA assay

Assay kits and active PKA were from Promega. After

PKA reaction with CRM1 C-terminal recombinant

protein (960�1120 fragment), reactants were analyzed

by western blotting with anti-phospho-Ser/Thr (CellSignaling Technology Inc.).

Results

Interaction between CRM1 and 14-3-3 proteins

In the most well-characterized nuclear export mechan-

ism for proteins, the nuclear export receptor CRM1binds directly to leucine-rich NESs to translocate cargo

proteins through the nuclear pore from the nucleus to

the cytoplasm (Toda et al. 1992; Turi et al. 1994;

Shimanuki et al. 1995; Fornerod et al. 1997a; Neville

et al. 1997). CRM1 is the major nuclear export receptor

(Figure 1A). The CRIME domain (which stands for

CRM1, importin b, etc.) shares homology with impor-

tin b (Hutten & Kehlenbach 2007; Fox et al. 2011). In

the domain, 19 Huntingtin, elongation factor 3, protein

phosphatase 2A, and the yeast kinase TOR1 (HEAT)

repeat motifs have been have been defined by homology

modeling. The HEAT helices 11A and 12A form acargo-binding hydrophobic cleft. Leptomycin B (LMB)

modifies Cys528 in the NES-binding region (Nishi et al.

1994; Kudo et al. 1999; Fasken et al. 2000). The acidic

loop in the eighth HEAT repeat motifs is involved in

RanGTP binding (Askjaer et al. 1999; Yoneda et al.

1999; Hutten & Kehlenbach 2007; Fox et al. 2011).

We noticed consensus 14-3-3-binding motifs in the

C-terminus of CRM1 (1049KHKRQMSVPG1058) (Fig-

ure 1A), suggesting that CRM1 was a 14-3-3-binding

protein. To investigate formation of endogenous CRM1-

14-3-3 complexes in cells, CRM1 and 14-3-3 wereimmunoprecipitated with anti-14-3-3 or anti-CRM1,

using normal rabbit antibody as a negative control.

Reciprocal immunoprecipitation (IP) and immunoblot-

ting (IB) of HEK 293 cells with CRM1 or 14-3-3

antibody suggested that CRM1 formed a complex with

a 14-3-3 protein (Figure 1B and C). Confocal micro-

scopy was used to visualize complexes of endogenous 14-

3-3 proteins binding to CRM1. Merged images show

coincident distribution of WT CRM1 and 14-3-3

proteins (Figure 1D). These results demonstrated that

CRM1 interacted with 14-3-3 proteins in HEK 293 cells.

Formation of CRM1 and 14-3-3 protein-proteincomplexes requires Ser 1055 in the CRM1 C-terminaldomain

Since CRM1, which contained a conserved 14-3-3

binding motif, pulled down a 14-3-3 protein, we

investigated whether the 14-3-3 binding motif was

required for the association. To determine whether

CRM1 interacted with 14-3-3 through the motif, we

constructed the CRM1 S1055A point mutant, which

affects amino acids 960�1120 in the C-terminal region.

GST-CRM1 fusion proteins were purified and incu-

bated with HEK 293 cell lysates to determine binding to

14-3-3 proteins. The WT C-terminus of CRM1 precipi-tated large amounts of 14-3-3 proteins from HEK 293

188 S.S. Kang and S.H. Shin

cell lysates, while the CRM1 S1055A mutant protein did

not (Figure 2A). Co-IP was used to confirm the cellular

association between CRM1 and 14-3-3 proteins. As

shown in Figure 2B, EGFP-CRM1 WT immunopreci-

pitates contained 14-3-3 proteins. Antibodies against

EGFP also captured both CRM1 and 14-3-3 proteins

from the same lysates, supporting the hypothesis that the

two proteins were physically associated (Figure 2B).

However, antibodies against EGFP did not precipitate

14-3-3 proteins from lysates of cells with EGFP-CRM1

S1055A (Figure 2B). These results suggested that CRM1

interacted with 14-3-3 proteins through the 14-3-3

binding motif, and that the motif was required for the

interaction. Among the eight mammalian members of

the 14-3-3 family (b, g, o, h, s, u, t, and z), 14-3-3ushowed the strongest signal (data not shown).

Figure 1. Functional domains and mutants of chromosome maintenance1 (CRM1). (A) Schematic structure of CRM1. Boxes 1�19 represent the HEAT repeat motifs, as defined by homology modeling. The CRIME domain (CRM1, importin b, etc.), which

shares homology with importin b, and the acidic loop are involved in RanGTP binding. RanBP3-binding domain is indicated by

green color. Modification of Cys528 by LMB targets the region involved in NES binding (Hamamoto et al. 1985; Nishi et al.

1994; Kudo et al. 1999). The C-terminal fragment corresponding to residues 707�1034 (CTR) is indicated by blue color. The

consensus motif of Protein Kinase A (PKA) phpsphorylation site (Ser 1055) in the C-terminal domain is indicated above. The C-

terminal GST fusion protein fragment region (960�1120 aa) is shown below. (B and C) Reciprocal Immunoprecipitation (IP) and

Immunoblotting (IB) from HEK 293 cells. CRM1 and 14-3-3 immunoprecipitates were analyzed using anti-14-3-3 or anti-CRM1

antibody. Negative IP control was normal rabbit antibody. (D) Confocal microscopy. Endogenous CRM1 (red) or 14-3-3 (green)

in HEK 293 cells was visualized using appropriate primary antibodies, and Alexa Fluor 568-conjugated secondary antibodies.

Merged image (yellow) shows coincident distribution of wild type CRM1 and 14-3-3 proteins. Figures represent three or more

independent experiments.

Figure 2. Formation of CRM1 and 14-3-3 protein�protein

complexes. CRM1 and 14-3-3 immunoprecipitates were

analyzed using anti-14-3-3 and anti-CRM1. CRM1 precipi-

tated from HEK 293 cells (A). GST-tagged CRM1 mutants

were expressed in E. coli and prebound to GST-agarose beads

that were incubated with HEK 293 cell lysates and analyzed

using anti-14-3-3 (B).


Phosphorylation of Ser 1055 in CRM1

To verify the phosphorylation of CRM1, EGFP-

CRM1 WT and EGFP-CRM1 S1055A were trans-

fected and immunopurified CRM1 protein with EGFP

antibody. The western blot was performed with CRM1,

14-3-3, or an anti-phosphor Thr/Ser residue antibody.

In stark contrast to the results obtained with EGFP-

CRM1, EGFP-CRM1 S1055A utterly failed to interact

with 14-3-3 (Figure 3B), indicating that the serine

residue is the crucial factor with regard to the interac-

tion between CRM1 and 14-3-3. Furthermore, anti-

phosphor Thr/Ser residue antibody did not recognize

EGFP-CRM1 S1055A, suggesting that 1055 serine

residue is one of major phosphorylation sites in

CRM1 (Figure 3A).

In order to gain a better understanding of the

phosphorylation of 1055 serine residue, PKA was

performed in vitro with C-terminal of CRM1 WT and

S1055A fusion protein (960�1120 aa), which was

purified from E. coli. Similar to the results of Figure

3A, CRM1 S1055A fusion protein was not phosphory-

lated by the active PKA, while CRM1 WT was well

phosphorylated by it (Figure 3B). These results sug-

gested that PKA phosphorylated Ser 1055 of CRM1.

Colocalization of CRM1 WT with 14-3-3 proteins byconfocal microscopy

To verify phosphorylation of CRM1, EGFP-CRM1

WT and EGFP-CRM1 S1055A were transfected. In

HEK 293 cell, the transfected EGFP-CRM1 or -CRM1

S1055A or -CRM1 S1055D (green) was shown directly.

Endogenous 14-3-3 proteins (red) were visualized using

their appropriate primary antibodies, and Alexa Flour

568-conjugated secondary antibodies. Merged images

(yellow) show coincident distribution of WT CRM1 and

14-3-3 only (A). No coincident distribution with 14-3-3

proteins was seen on merged images with EGFP-CRM1

S1055A (B) or S1055D (C).

Thus, similar to the results in Figure 1D, EGFP-

CRM1 colocalized with endogenous 14-3-3 proteins in

the cytoplasm. These results also supported the

requirement for Ser 1055 for colocalization of CRM1

with 14-3-3 proteins.

Regulation of CRM1 subcellular localization depends onphosphorylation of Ser 1055

To define the role of CRM1 Ser 1055 phosphorylation,

we compared the subcellular localization of EGFP-

CRM1 WT, S1055A, or S1055D from plasmids trans-

fected into HEK 293 cells. The phosphorylated CRM1

analog S1055D was not localized at the nuclear

membrane but in the cytoplasm, whereas the unpho-

sphorylatable CRM1 S1055A was predominantly loca-

lized in the nuclear membrane (Figure 5A). Confocal

microscopy scanning determined relative protein

amounts. Scanning results are shown with micrographs.

Although EGFP-CRM1 WT was in the cytoplasm,

nucleus, and nuclear membrane, nuclear membrane

localization was clear (Figure 5A). These results

suggested that phosphorylation of CRM1 serine 1055

was crucial for CRM1 subcellular localization.

To further investigate the effect of CRM1 Ser 1055

phosphorylation, we determined the subcellular locali-

zation of EGFP-CRM1 WT in HEK 293 cells after

Figure 3. Phosphorylation of Ser 1055 in CRM1 by PKA was required for 14-3-3 binding. Plasmids with EGFP-CRM1 (WT)

and EGFP-CRM1 S1055A were transfected into HEK 293 cells and CRM1 protein was immunopurified with GFP antibody. (A)

Western blot with CRM1, 14-3-3, or phospho-Thr/Ser antibodies. CRM1 S1055A (lacking the PKA phosphorylation site) did not

form a 14-3-3 protein complex or react with anti-phospho-Thr/Ser. (B) PKA assay with GST-CRM1 WT C-terminal fragment

(amino acids 960�1120) or GST-CRM1 S1055A C-terminal fragment. GST-CRM1 WT, S1055A were expressed in E. coli and

prebound to agarose beads that were incubated with PKA in assay buffer and analyzed using anti-phospho-Ser/Thr. Figures

represent three independent experiments.


24 h of treatment with a PKA activator (60 mM

forskolin) or inhibitor (10 mM H89) (Chijiwa et al.

1990; Geilen et al. 1992). After transfection with

plasmids expressing EGFP-CRM1 WT, the subcellular

localization was determined after treatment with for-

skolin or H89. Similar to the subcellular localization of

CRM1 S1055D in Figure 5A, forskolin treatment

enhanced the cytoplasmic localization. Treatmentwith the PKA inhibitor H89 promoted nuclear locali-

zation (Figure 5B). These results suggested that phos-

phorylation of Ser 1055 of CRM1 regulated subcellular

localization. Furthermore, similar to the results in

Figure 3, Ser 1055 phosphorylation appeared to be

by PKA, because a specific PKA activator or inhibitor

modulated the CRM1 subcellular localization (similar

to Figure 5A).

Ser 1055 phosphorylation promoted cell apoptosis

To investigate the effect of the CRM1 phosphorylationon cell survival, we measured the apoptosis rate of cells

with CRM1 mutant constructs (Table 1). HEK 293

cells were split and transfected at day 3 with control

vector or CRM1 WT, S1055A, or S1055D constructs,

and harvested as indicated for FACS. The apoptosis

rate of cells with the S1055D construct was twice that

of cells with WT or S1055A constructs. Thus, the

disruption of nuclear pore integrity by overexpression

of CRM1 S1055D (Figure 5) might negatively affectcell survival. Therefore, 1055 Ser phosphorylation by

PKA (or another protein kinase) appeared to enhance

cell apoptosis (Table 1).

Interaction between CRM1 and 14-3-3 proteins throughSer1055 phosphorylation

The 14-3-3 proteins formed a complex with CRM1 in

the cytoplasm that depended on PKA phosphoryla-

tion. The C-terminal domain of CRM1 is involved in

the targeting of the export complex to the nuclear pore

complex (NPC), facilitating CRM1-dependent translo-cation of NES-containing proteins through the NPC.

Figure 4. Sublocalization of CRM1 WT, S1055A, and S1055D with confocal microscopy. HEK 293 cells were transfected with

plasmids for EGFP-CRM1, S1055A, or S1055D (green). Endogenous 14-3-3 proteins (red) were visualized using appropriate

primary antibodies, and Alexa Fluor 568-conjugated secondary antibodies. Merged image (yellow) shows coincident distribution

of wild type CRM1 and 14-3-3 proteins (A). No merged image (yellow) was seen with EGFP-CRM1 S1055A (B) or S1055D and

14-3-3 proteins (C). Figures represent three or more independent experiments.


CRM1 phosphorylated by PKA bound to 14-3-3

proteins as new partner proteins through the CRM1

C-terminal domain that contained a conserved 14-3-3

binding motif (1049KHKRQMSVPG1058). Binding with

14-3-3 proteins resulted in the cytoplasm localization of

CRM1. Unphosphorylated CRM1 was localized in the

nucleus, crossing the nuclear pore. Phosphorylation

of CRM1 on serine 1055 by PKA induced binding of

14-3-3 proteins to the 14-3-3 binding motif, and inhibited

CRM1 shuttle function by releasing CRM1 from the

nuclear pore. Thus, interaction with 14-3-3 proteins

depended on PKA phosphorylation, which regulated

CRM1 localization and function in vivo and in vitro.

Discussion

CRM1 appears to form a specific complex with the

NES of proteins that is necessary for NES-mediated

nuclear protein export (Fornerod et al. 1997a, 1997b;

Fukuda et al. 1997; Yoneda et al. 1999). By analogy

with nuclear import, Ran or a Ran-binding protein

might regulate the interaction of complexes of CRM1

and NES-containing proteins with the nuclear pore

complex before translocation out of the nucleus

(Askjaer et al. 1999; Kehlenbach et al. 1999; Yoneda

et al. 1999). We showed that CRM1 activity is cell

cycle-regulated, and were interested in determining

whether CRM1-14-3-3 interaction is critical for entry,

progression, or exit from mitosis.

The 14-3-3 binding motif (1049KHKRQMS-

VPG1058) in CRM1 seems to be overlapped with

PKA phosphorylation site (1055 serine residue), which

is not perfectly matched with the best PKA substrate

consensus sequence (R-R-X-S/T) (Pidoux & Tasken

2010; Li 2011). However, we demonstrated here that

CRM1 forms a protein complex with 14-3-3, and the

PKA phosphorylation on the 1055 serine residue

contributes both the protein complex formation and

CRM1 subcellular localization (Figures 3�5).

In this study, we found that the CRM1-14-3-3

interaction induced by PKA caused the cell apoptosis

(Table 1). As the expression of PKA induced mitotic

arrest and apoptosis, we wished to determine whether

Figure 5. Subcellular localization of CRM1 phosphorylated by PKA. (A) HEK 293 cells were transfected with plasmids for

EGFP-CRM1 WT, S1055A, or S1055D (green). CRM1 S1055D was detected in the cytoplasm as a large dot (right) and not on

the nuclear rim. Figures represent three independent experiments. Confocal microscopic pictures were scanned using profile in the

ZEN program. (B) Subcellular localization in HEK 293 cells of EGFP-CRM1 WT was examined after treatment with forskolin or

H89 for 24 h. EGFP-CRM1 WT (green) was observed by fluorescence microscopy.

Table 1. Serine 1055 phosphorylation of CRM1 promoted

cell apoptosis.

CRM1 construct Rate of apoptosis (%) by FACS

CRM1 WT 5.7193

CRM1 S1055A 6.5294

CRM1 S1055D 12.7194

pEGFP C2 (vector only) 6.9892

Mean value of five repeats

Note: Cells were split and transfected at day 3 and harvested post-transfection as indicated. HEK 293 cells transfected with controlvector or CRM1 constructs (WT, S1055A, or S1055D) wereharvested and subjected to FACS. CRM1 S1055D promotedsignificant cell apoptosis compared with control or CRM1 WT orS1055A.


there was the phosphorylated CRM1 protein analog

(S1055D) effect in cells. Apoptotic cell populations

were quantitated in parallel by annexin V-fluorescein

isothiocyanate staining and by propidium iodide stain-

ing (Table 1). The data clearly show that the phos-

phorylated CRM1 from the cells induces apoptosis.

Thus it seems to be that the PKA catalytic subunit

promotes cell death through the phosphorylation on

1055 Ser residue of CRM1. Alternatively, because the

activity of PKA is dependent on the cell cycle, PKA

phosphorylates on Ser 1055 residue of CRM1 and

prompt the binding with 14-3-3, and the disassembly of

nuclear pore or membrane. And then, during the M

phase, the unphosphorylated CRM1 which is disasso-

ciated with 14-3-3, again reforms the nuclear pore or

membrane (Figure 6). The functional role of the

phosphorylation on Ser 1055 residue of CRM1 remains

to be characterized.

Although our data suggests that the interaction of

CRM1 and 14-3-3 controls its subcellular localization,

our findings also raise several questions regarding the

interaction of CRM1 and 14-3-3. It remains for

researchers to characterize the fashion and mechanisms

by which the interactions between CRM1 and 14-3-3

are controlled, depending on physiological conditions.

In addition, it remains to be determined whether

CRM1 mutation (1049KHKRQMSVPG1058) itself af-

fects the shuttle activity, regardless of protein-protein

interactions with 14-3-3. It is also necessary to ascer-

tain whether the phosphorylation of 14-3-3 is required

for the activation and/or regulation of CRM1, or for

the interaction between CRM1 and 14-3-3, because the

bacterial expressed CRM1 C-terminal fragment, which

is an unphosphorylated CRM1 form, also slightly pull-

down 14-3-3 (Figure 2A). Therefore, it seems that this

difference reflects the affinity between the CRM1 and

14-3-3, and the cytoplasm localization of CRM1 and

14-3-3. Thus, it seems that 14-3-3 preferentially associ-

ates with the inactive conformations of the signaling

molecules. Nevertheless, the high affinity of 14-3-3 for

the inactive conformation of CRM1 interacting pro-

teins is not reflected in CRM1, since the constitutively

active CRM1 mutant binds readily to 14-3-3 (data not

shown). Importantly, the phosphorylation and activity

of CRM1 were neither necessary nor required for its

functional interaction with 14-3-3. The 1055 Ser

residue in motifs (1049KHKRQMSVPG1058) of CRM1

seems be one of the regulation points for the cell cycle.

During the M period in cell mitotic division, the

disassembly of nuclear membrane is triggered by the

phosphorylation on 1055 Ser residue of CRM1.

The leucine-rich NES recognized by CRM1 was

also identified in 14-3-3 proteins (Aitken et al. 1992;

Yaffe et al. 1997; Obsilova et al. 2008). However, we do

not know how the 14-3-3 NES motif contributes to

binding with CRM1. Although association of 14-3-3

proteins with CRM1 contributes to CRM1 function by

mediating its subcellular localization, whether differ-

ences in CRM1 subcellular localization are due to

differences in cell lines or differences in CRM1 function

remains to be seen. The subcellular localization of

CRM1 was unaffected by treatment with growth

factors and chemokines (28�30).

Figure 6. Schematic diagram of interaction between CRM1 and 14-3-3 proteins, with PKA. The nuclear pore export complex

facilitates CRM1-dependent translocation of NES-containing proteins. Phosphorylation of CRM1 by PKA results in binding to

14-3-3 proteins as a new partner protein through the CRM1 C-terminal domain, which contains a conserved 14-3-3-binding motif

(1049KHKRQMSVPG1058). Interaction regulates CRM1 nuclear pore localization and NES function in vivo and in vitro.

Phosphorylated CRM1 seems to be dephosphorylated by a phosphoprotein phosphatase (PPA).


Molecular details of the CRM1 interaction with

RanGTP have been solved (Hutten & Kehlenbach

2007; Fox et al. 2011). The model suggests that the

HEAT helices 11A and 12A form a cargo-binding

hydrophobic cleft (Hutten & Kehlenbach 2007; Fox et

al. 2011). RanGTP contact areas are on the CRM1 C-

terminus. Further, CRM1 is hypothesized to switch

between a relaxed cytoplasmic and a strained nuclear

conformation, depending on RanGTP binding (Figure 1).

In the hypothetical cytoplasmic conformation, the contact

sites for RanGTP inside the CRM1 toroid are toofar apart to bind Ran with high affinity (Hutten &

Kehlenbach 2007; Fox et al. 2011). Also, the hydrophobic

cleft on the outer side of the toroid is closed. Rigid body

movements allow transition to the nuclear conformation.

Accordingly, in the model, the conformational change

also alters the curvature of the toroid near the cargo-

binding site, opens the hydrophobic cleft, and allows the

export cargo to dock. Thus, during CRM1 shuttling

between the cytoplasm and nuclear across the pore,

phosphorylation on Ser 1055 in the CRM1 C-terminal

domain by PKA contributes to the conformationalchange to alter the curvature of the toroid near the

cargo-binding site, closing the hydrophobic cleft to release

the export cargo. Phosphorylation on Ser 1055 by PKA

seems to inhibit or block RanGTP inside the CRM1

toroid (Figure 6). We are determining whether the binding

affinity of RanGTP to CRM1 is changed by phosphor-

ylation.

During the consensus motif database search, we

also noticed that RSK (90 kDa ribosomal S6 kinase)

might also phosphorylate CRM1 Ser 1055 (Roux et al.

2003; Romeo et al. 2012). RSK is characterized as ahub kinase that regulates diverse cellular processes

including cell growth, proliferation, survival and motility

(Roux et al. 2003; Romeo et al. 2012). Phosphorylation

by RSK might induce CRM1 to bind 14-3-3 proteins,

similar to PKA (Figure 6).

Phosphorylation on serine 1055 of CRM1 appeared

to contribute to cell apoptosis (Table 1). We are

pursuing whether RSK also phosphorylates CRM1.

Regardless of the kinases that phosphorylate Ser 1055,

the residue appears to be an important regulation sitefor CRM1 function.

In conclusion, this study identified 14-3-3 proteins

as new binding partners for CRM1 through the motif1049KHKRQMSVPG1058 in the CRM1 C-terminal

domain. We demonstrated that CRM1 is a PKA

substrate. Although the functional significance of this

interaction remains poorly understood, the regulation

of CRM1 nuclear pore localization by 14-3-3 proteins

could be a relevant consequence of different signaling

pathways involving CRM1. Results on both CRM1

localization and PKA activity revealed that these arechanged by 14-3-3 protein binding. The 14-3-3 proteins

appeared to be antagonistic to the CRM1 nuclear pore

localization. Our results suggested the interaction

of CRM1 with 14-3-3 proteins and 14-3-3 proteins

function as negative regulators of CRM1 signaling.

However, the precise control mechanisms underlying

the subcellular localization of CRM1 by 14-3-3 pro-

teins requires further characterization to determine the

overall function of 14-3-3 proteins in CRM1 signal

transduction pathways.

Future studies are needed to probe the molecular

mechanisms modulating the association of CRM1 with

14-3-3 proteins, the dephosphorylation of CRM1, and

the effect of PKA activity on CRM1 in and out of the

nuclear pore.

Acknowledgments

This work was supported by Chungbuk National UniversityResearch Grant (2011) to S S Kang. We also appreciated TheCore Facility of Chungbuk National University for theirexcellent skills.

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