Brain-selective Kinase 2 (BRSK2) Phosphorylation on
PCTAIRE1 Negatively Regulates Glucose-stimulated Insulin
Secretion in Pancreatic ?-Cells*□
Xin-Ya Chen‡1, Xiu-Ting Gu‡1, Hexige Saiyin‡, Bo Wan‡, Yu-Jing Zhang§, Jing Li§, Ying-Li Wang‡, Rui Gao‡,
Yu-Fan Wang¶, Wei-Ping Dong¶, Sonia M. Najjar?, Chen-Yu Zhang§, Han-Fei Ding**2, Jun O. Liu‡‡, and Long Yu‡3
Background: BRSK2 has never reported to be functional in pancreatic islets.
Results: BRSK2 interacts with PCTAIRE1 and phosphorylates it at Ser-12. Knockdown of BRSK2 augmented low glucose-
stimulated insulin secretion.
Conclusion: BRSK2 negatively regulates insulin secretion in ?-cells via a PCTAIRE1-dependent mechanism.
Significance: This study reveals a novel function of BRSK2 in insulin secretion and uncovers its related regulation mechanism.
Brain-selective kinase 2 (BRSK2) has been shown to play an
essential role in neuronal polarization. In the present study, we
show that BRSK2 is also abundantly expressed in pancreatic
islets and MIN6 ?-cell line. Yeast two-hybrid screening, GST
fusion protein pull-down, and co-immunoprecipitation assays
reveal that BRSK2 interacts with CDK-related protein kinase
PCTAIRE1, a kinase involved in neurite outgrowth and neu-
rotransmitter release. In MIN6 cells, BRSK2 co-localizes with
PCTAIRE1 in the cytoplasm and phosphorylates one of its ser-
ine residues, Ser-12. Phosphorylation of PCTAIRE1 by BRSK2
reduces glucose-stimulated insulin secretion (GSIS) in MIN6
cells. Conversely, knockdown of BRSK2 by siRNA increases
serum insulin levels in mice. Our results reveal a novel function
of BRSK2 in the regulation of GSIS in ?-cells via a PCTAIRE1-
dependent mechanism and suggest that BRSK2 is an attractive
target for developing novel diabetic drugs.
Brain-selective kinase 2 (BRSK2,4also known as SAD-A) is a
ily of serine/threonine kinases (1). BRSK2 and closely-related
BRSK1 (also named SAD-B) are orthologs of SAD-1 in
tion in neuronal polarization (2). Neurons of SAD-AB?/?-null
mutant mice have extended axons, and neurons from hip-
pocampus- and cortex-specific mutant mice also failed to form
distinct axons and dendrites in culture (2). Subsequently,
BRSK1 was identified to be a novel SV (synaptic vesicle), and
active zone cytomatrix-associated protein kinase that is
involved in the regulation of neurotransmitter release; it most
vesicle priming factor RIM1, among other potential targets in
SVs and/or active zones (3).
AMPK has been shown to be a potential therapeutic target
for type 2 diabetes (4), largely due to its regulatory function in
glucose and lipid metabolism. AMPK undergoes activation at
of insulin-containing secretory vesicles and hence, insulin
secretion (5, 6). Activation of AMPK has been reported to
pancreatic ?-cells and islets (7–9). AMPK is activated by high
AMP (and low ATP) concentrations through multiple mecha-
nisms, through modulation of both the intrinsic kinase activity
and its phosphorylation and activation by an upstream kinase,
tumor suppressor kinase implicated in the pathogenesis of
Peutz-Jeghers Syndrome (13–15). LKB1 has been reported to
phosphorylate and activate 13 AMPK family members, includ-
ing BRSK2 (1). Mutation of residue Thr-174 within the T-loop
to the AMPK family, it has not been shown to play a role in
regulating insulin secretion and/or energy metabolism.
identified as a Cdc2-like kinase (16, 17). As an uncharacterized
* This work was supported in whole or in part by grants from the National
Institutes of Health (R01DK054254 and R01DK083850 (to S. M. N.), and
CA124982(toH. F. D.),bytheNational973ProgramofChina,the863Proj-
ects of China, and the National Natural Science Foundation of China
SThis article contains supplemental Figs. S1–S3.
1Both authors made equal contributions to this work.
2A Georgia Cancer Coalition Distinguished Scholar.
3To whom correspondence should be addressed: State Key Laboratory of
Genetic Engineering, School of Life Science, Fudan University, Handan
Road 220, Shanghai 200433, China. Tel.: 86-21-65643954; Fax: 86-21-
65643404; E-mail: Longyu@fudan.edu.cn.
4The abbreviations used are: BRSK2, brain selective kinase 2; AMPK, AMP-
activated protein kinase; CDC25C, cell division cycle 25 homolog c; CDK,
mTOR, mammalian target of rapamycin; NSF, N-ethylmaleimide-sensitive
fusion protein; PCTAIRE1, CDK-related protein kinase; SV, synaptic vesicle;
KD, kinase dead.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 36, pp. 30368–30375, August 31, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
by guest on October 28, 2015
branch of the cyclin-dependent kinase (CDK) family,
PCTAIRE1 has two isoforms in higher organisms, PCTAIRE2
and PCTAIRE3 (17), both of which contain a large N-terminal
domain. PCTAIRE kinases are ubiquitously expressed, and it
has been found to be predominantly expressed in terminally
cysteine mutation in their conserved cyclin-binding consensus
to be essential for PCTAIRE1 activity targeting presynaptic
components to axons (20). PCTAIRE1 modulates secretory
cargo transport by interacting with the COPII complex (21),
and regulates secretion of growth hormone from PC12 cells
through phosphorylation of residue Ser-569 of the N-ethylma-
leimide-sensitive fusion protein (NSF) (22).
In this study, we uncovered new functions of BRSK2 and
PCTAIRE1 in pancreatic ?-cells. We demonstrated that both
kinases are highly expressed in human pancreatic islets and
MIN6 murine ?-cell line. Importantly, we found that
PCTAIRE1 is a substrate of BRSK2 and the phosphorylation of
PCTAIRE1 by BRSK2 plays a crucial role in regulating insulin
secretion in response to glucose.
Plasmids and Antibodies—cDNA encoding full-length
human BRSK2 or PCTAIRE1 was subcloned into a BD
(pGBKT7)/AD (pGADT7) vector (ClonTech) and a PCMV-
sion vector. Full-length BRSK2 cDNA was subcloned into a
and T174E) or PCTAIRE1 (S12A, S12E, S153A, and K194M)
mutants were conducted using a Quick Change mutation kit
(Stratagene). The antibodies used were as follows: Rabbit anti-
PCTAIRE1, PCTAIRE2, PCTAIRE3, or mouse anti-CDC25C
(Santa Cruz Biotechnology); Rabbit anti-phospho-CDC25C
Ser216 (Cell Signaling); mouse anti-HA, Myc, ?-actin/tubulin
lin, glucagon (Abcam); rabbit anti-E-cadherin (PTG).
Cell Culture and Transfection—Murine MIN6 pancreatic
?-cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) containing 25 mM glucose, supplemented with 15%
heat-inactivated fetal calf serum (FCS), 4 mM L-glutamine, and
100 ?M ?-mercaptoethanol, at 37 °C with 5% CO2unless spec-
ified otherwise. Panc-1, HEK 293T, COS-7, and Hela cell lines
were cultured in DMEM with 25 mM glucose, 10% FCS. All
transfections were using Lipofectamine 2000TMaccording to
the manufacturer’s instructions (Invitrogen).
Northern Blot Analysis—Northern blot was performed as
previously described (2) using a full-length human BRSK2
cDNA hybridization probe. Human multiple tissue Northern
ized for ?-actin expression, were purchased from Clontech.
Immunohistochemistry—Human or mice pancreas tissues
were fixed with 4% paraformaldehyde and sectioned at 10 ?m.
Immunostaining was performed with antibodies specific for
BRSK2, insulin, glucagon, E-cadherin, or PCTAIRE1, followed
dine tetrahydrochloride (DAB/H2O2) or fluorescein isothio-
cyanate-conjugated goat anti-rabbit/mouse antibody (Alexa
Fluor 488 or 555 from Invitrogen). DAPI (Sigma) was also
stained for nucleus. Sections were then washed and mounted
for confocal microscopy (Leica).
Tissue Source—Human pancreatic tissues were obtained
from the pancreatic tumor patients, who had undergone
resection at General Surgery Unit of Zhongshan Hospital at
Fudan University. We carried out immunostaining in pan-
creatic tumor tissues that contain a large region of normal
pancreatic exocrine and endocrine parts. And these patients
were all informed and approved before using their tissues.
All tissues were used in accordance with applicable laws and
with the Declaration of Helsinki for research involving
Mice tissues were isolated from adult BALB/c mice (male,
6-week-old). Mice experiments followed the principles of lab-
oratory animal care and were approved by Fudan University
Life Science Ethic Committee.
siRNA Duplexes—Small interfering RNA (siRNA) duplexes
were designed and synthesized by GeneChem or GenePharma
Biotech. siRNA sequences were as follows: BRSK2: 5?-GCUA-
CUGAGGACAUCtt-3?; nonsilence: 5?-UUCUCCGAACGU-
Intravenous siRNA Delivery, ELISA Assay, and Glucose Tol-
erance Tests—6-week-old BALB/c mice (20 g body weight)
received tail-vein injections of saline, control siRNA, or siRNA
against BRSK2 for one to three consecutive days. Mice were
a 12:12 h light/dark cycle. Everyday mice were injected at 4:00
pm and starved at 9:00 pm for 12 hours (water allowed). The
Blood sample collections and islet isolations were performed
18 h after the last injection.
Following an overnight fast, mice were intraperitoneal (IP)
were assessed using a freestyle glucometer (Abbott). Serum
insulin levels were assayed by a 96-well plate ELISA assay
(Linco Research). Serum glucagon levels were assessed by a
96-well plate ELISA assay (ALPCO Diagnostics). Tissues were
harvested, snap-frozen, and stored at ?80 °C.
Isolated Pancreatic Islet and ?-Cell Size Studies—Pancreatic
28 min, and then purified by single layer Histopaque (Sigma).
Isolated islets were cultured in RPMI 1640 medium containing
11 mM glucose, 7.5% FCS, and 10 mM Hepes (Sigma). Islets of
BRSK2-RNAi mice and control mice were isolated, spread and
photographed using microscopy, followed by analysis using
Image J software.
?-Cells were marked by E-cadherin/insulin co-immuno-
staining. To calculate single ? cell size, the area of 50–100 sin-
measured using Image J software.
Measurements of Insulin Secretion—MIN6 cells were seeded
ing Myc-BRSK2, PCTAIRE1 or their mutant forms. 48 h after
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cose-free Krebs-Ringer bicarbonate (KRB) medium (125 mM
NaCl, 4.74 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM
at 37 °C for 30 min. Cells were then incubated in KRB contain-
ing 3 mM or 25 mM glucose at 37 °C for 30 min. The amount of
insulin released into the incubation medium was assayed using
a radioimmunoassay (Linco Research) following the manufac-
Yeast Two-hybrid Assay—A yeast two-hybrid assay was per-
formed following the Matchmaker III system protocol (Clon-
as bait, and identified by DNA sequencing. The interaction of
ified by re-transformation in yeast.
Western Blot Analysis and Immunoprecipitation—For im-
munoprecipitation, cells were lysed in chilled lysis buffer (Cell
Signaling lysis buffer with complete protease inhibitors).
Lysates (200 ?g) were collected and incubated with 40 ?l pro-
tein G-Sepharose beads (Amersham Biosciences) and 1–2 ?g
corresponding antibody for 4–5 h at 4 °C with gentle rotation.
The samples were washed with cold lysis buffer, and subjected
to Western analysis or to an in vitro phosphorylation assay (see
For Western blot analysis, cell and tissue extracts were pre-
kit (Bio-Rad). Samples were equally loaded onto a 4% to 10% or
12% gradient SDS-PAGE gel and transferred onto a nitrocellu-
lose membrane using standard techniques.
In Vitro Phosphorylation Assay—HA-BRSK2 overexpressed
in 293T cells and immunoprecipitated with HA antibody were
assayed for kinase activity (see immunoprecipitation assay) by
incubating with recombinant GST-PCTAIRE1 (full length,
deletion mutants and site-directed mutants) as substrates in
kinase buffer (20 mM MOPS, PH 7.4, 15 mM MgCl2, 100 ?M
ATP) containing 1 ?Ci of [?-32P]ATP at 30 °C for 30 min.
Samples were separated on SDS-PAGE and visualized by
Fusion Protein and Pull-down Assay—GST-PCTAIRE1, its
fragments and/or mutant proteins were expressed in the BL21
sciences). GST-tagged fusion proteins or GST proteins were
incubated with 40 ?l beads and 200 ?g lysates from 293T cells
expressing HA-BRSK2 for 4 h at 4 °C. Proteins were then sub-
jected to SDS-PAGE and immunoblotted using anti-HA anti-
body. The fusion proteins were also detected by Western blot
using an anti-GST antibody.
Immunofluorescence—MIN6 cells were transfected with
PCMV-Myc-BRSK2 and/or the EGFPN1-PCTAIRE1 for
48 h, fixed with 4% paraformaldehyde and permeabilized
with Triton X-100. After washes with TBS, cells were stained
with DAPI (Sigma) and stained with an anti-Myc antibody
mouse antibody. Images were acquired with a Leica confocal
Expression and Activity of BRSK2 in Pancreatic Islets and
MIN6 ?-Cells—Using Northern blot analysis, we determined
were surprised to find that BRSK2 mRNA was expressed at an
similar expression pattern was seen at the protein level by
BRSK2 (Fig. 1B and supplemental Fig. S1, A and B). Immuno-
histochemical analysis of human pancreatic tissue with BRSK2
antibody also revealed abundant staining in pancreatic islets
and ducts (Fig. 2B and supplemental Fig. S1C). Moreover,
BRSK2 was found to be specifically co-localized with insulin,
but not glucagon (Fig. 1C). In agreement with those observa-
a murine ?-cell line (Fig. 1D), and in homogenates of isolated
mouse islets (Fig. 4A). Importantly, when MIN6 cells were
treated with varying concentrations of glucose for 5 h, the
expression of BRSK2 was down-regulated in a dose-dependent
manner, which was accompanied by decreases in the phos-
phorylation of CDC25C (Fig. 1E and supplemental Fig. S1, D
and E). These preliminary observations hinted at a potential
role of BRSK2 in pancreas, raising the possibility that it might
be involved in the regulation of insulin secretion.
Identification of PCTAIRE1 as a Novel BRSK2-interacting
Protein—To gain insights into the function of BRSK2, we
tem and identified PCTAIRE1 as a novel BRSK2-interacting
protein. The full-length PCTAIRE1 cDNA was cloned by PCR,
firmed by the two-hybrid assay (Fig. 2A). Similar to BRSK2 (2,
3), PCTAIRE1 was previously known to regulate neurite out-
growth and secretion of growth hormones (18, 22). Unlike
BRSK2, PCTAIRE1 is ubiquitously expressed with the highest
abundance in terminally differentiated cells and transformed
cell lines (18, 19). Using immunohistochemical staining and
Western blot analysis, we found that PCTAIRE1 was highly
expressed in both pancreatic islets and MIN6 cells (Fig. 2B and
supplemental Fig. S2A). In contrast to PCTAIRE1, the other
isoforms, PCTAIRE2 and PCTAIRE3, were not detected in
MIN6 cells (supplemental Fig. S2A).
We next determined the specificity of the interaction
Thus, recombinant HA-BRSK2 was purified and incubated
with immobilized GST-PCTAIRE1 or GST as a control. HA-
BRSK2 was pulled down by GST-PCTAIRE1, but not by GST
(Fig. 2D). The BRSK2-PCTAIRE1 interaction was further veri-
body in MIN6 cells (Fig. 2E). HA-BRSK2 and Myc-PCTAIRE1
proteins were found to be localized in the cytoplasm in MIN6
cells (Fig. 2F). We then employed the GST pull-down assay to
determine the domain in PCTAIRE1 that mediates its interac-
tion with BRSK2. We generated three PCTAIRE1 deletion
mutants, GST-PCTAIRE1-Fa, Fb, and Fc, corresponding to
shown in Fig. 2D, BRSK2 was specifically captured by immobi-
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lized GST-PCTAIRE1, GST-PCTAIRE1 Fb (aa129–289) and
Fc (aa290–496), but not by Fa (aa1–128), indicating that
C-terminal domains, located in the aa129–496 region.
substrate for the serine/threonine kinase CAMK2 (Ca2?/cal-
modulin-dependent kinase 2). Because BRSK2 is a serine/thre-
onine kinase belonging to the AMPK subfamily of the CAMK
family (23), we investigated whether BRSK2 is capable of phos-
BRSK2 has been reported to preferentially phosphorylate
serine/threonine residues within the (L/M/I/F/V)X(R/K/
H)XX(S/T) consensus sequence (1). Interestingly, PCTAIRE1
contains 9 BRSK2 phosphorylation consensus sequences, fur-
ther suggesting that PCTAIRE1 may be a substrate of BRSK2
(Fig. 2C). Using an in vitro kinase assay, we found that BRSK2
phosphorylated PCTAIRE1 while the catalytically inactive
mutants of PCTAIRE1, each of which contained three consen-
sus BRSK2 phosphorylation sequences (Fig. 2C). Of the three
deletion mutant proteins, GST-PCTAIRE1 Fa (N-terminal
domain, aa1–128), but not GST-PCTAIRE1 Fb or Fc (kinase
domain and C-terminal domain, aa129–496) was phosphory-
lated by wild type, but not catalytically inactive BRSK2 (Fig. 3).
To further narrow down the site(s) of BRSK2 phosphorylation
in PCTAIRE1 Fa domain, we mutated the putative serine resi-
dues into alanines and subjected each mutant to the in vitro
kinase assay. While BRSK2 phosphorylated S78A and S119A
mutants, it failed to phosphorylate the S12A mutant (Fig. 3),
suggesting that serine 12 constitutes the main phosphorylation
site by BRSK2.
BRSK2 Negatively Regulates Insulin Secretion in MIN6 Cells—
The selective expression of BRSK2 in pancreatic tissue and
MIN6 cell line prompted us to investigate its potential role in
regulating insulin secretion. We used glucose-induced insulin
secretion in MIN6 cells as the model system (24). MIN6 cells
showed an ?3-fold increase in insulin secretion when the glu-
cose concentration in the medium was increased from 3–25
mM (Fig. 4A).
We transiently expressed Myc-tagged wild-type BRSK2, a
constitutively active mutant T174E or a kinase-dead mutant
K48M of BRSK2 in MIN6 cells and measured insulin secretion
upon incubation with or without glucose for 36 h. Each of the
mutant proteins was found to be expressed at a comparable
level by Western blot analysis (Fig. 4A). Overexpression of
BRSK2 and the constitutively active T174E mutant had no sig-
nificant effect on insulin secretion when MIN6 cells were
treated with 3 mM glucose, but caused a marked reduction of
insulin secretion when glucose concentration was increased to
25 mM glucose (Fig. 4A). In contrast, the inactive K48M form
had no effect on insulin secretion in response to 3–25 mM glu-
cose (Fig. 4A).
Reciprocally, down-regulation of BRSK2 by siRNA, as con-
firmed by Western blot analysis (Fig. 4A), caused a marked
increase in insulin secretion from MIN6 cells at 3 mM glucose,
but not 25 mM glucose (Fig. 4A). Together, these results
FIGURE 1. Expression and activity of BRSK2 in pancreatic islets and MIN6 ?-cells. A, Northern blot analysis using full length BRSK2 probe showed BRSK2
expression in human brain and pancreatic tissues. B, Western blot of BRSK2 with high level in mouse pancreas besides in brain. C, immunostaining of human
pancreatic tissues with anti-BRSK2 antibody or/and anti-insulin antibody or anti-glucagon antibody followed by fluorescein isothiocyanate-conjugated goat
anti-rabbit/mouse antibody. Scale bar, 25 ?m. D, expression of BRSK2 in MIN6 ?-cells compared with other cell lines by Western blot with BRSK2 antibody.
activity detection according to our unpublished data (supplemental Fig. S1E). Loading of each lane was controlled by immunolabeling of ?-actin.
AUGUST31,2012•VOLUME287•NUMBER36 JOURNALOFBIOLOGICALCHEMISTRY 30371
by guest on October 28, 2015
in glucose-stimulated insulin secretion (GSIS) in MIN6 cells.
BRSK2 Regulates Insulin Secretion in MIN6 Cells via
PCTAIRE1 Depending on Its Kinase Activity—Given that
BRSK2 interacted with and phosphorylated PCTAIRE1, we
asked whether PCTAIRE1 and its kinase activity are critical in
mediating the effect of BRSK2 on insulin secretion in MIN6
cells. Myc-tagged wild-type PCTAIRE1, and the phosphoryla-
tion-defective PCTAIRE1-S12A and phosphorylation-mimetic
constitutively active (S153A) and the kinase-dead (K194M)
and its active forms S153A mutants or BRSK2 phosphoryla-
tion-mimetic S12E mutants markedly inhibited insulin secre-
tion at 25 mM, but not at 3 mM glucose in MIN6 cells (Fig. 4B).
In contrast, overexpression of the phosphorylation-defective
PCTAIRE1 S12A or the kinase-dead (K194M) mutant did not
affect insulin secretion (Fig. 4B). Similar to BRSK2, siRNA
down-regulation of PCTAIRE1 (Fig. 4B) significantly stimu-
at 25 mM glucose (Fig. 4B). These results suggested that BRSK2
phosphorylation of PCTAIRE1 at Ser-12 is required for its reg-
ulation of insulin secretion from MIN6 cells.
To verify that BRSK2 lies upstream of PCTAIRE1 in the reg-
expressed in MIN6 cells in which BRSK2 had been knocked
down by RNAi and PCTAIRE1 expression was found to par-
tially reverse the effect of BRSK2 knockdown on insulin secre-
tion (Fig. 4B). In contrast, down-regulation of PCTAIRE1 in
MIN6 cells overexpressing BRSK2 failed to inhibit insulin
secretion at 25 mM glucose (Fig. 4B). These results suggested
that PCTAIRE1 mediated the regulatory effect of BRSK2 on
insulin secretion in MIN6 cells. Together, these observations
indicated that BRSK2 regulated insulin secretion in MIN6 cells
through phosphorylating PCTAIRE1 kinase.
Attenuation of BRSK2 in Mice Islets Increases Serum Insulin
Levels—Finally, we attempted to verify the role of BRSK2 in
regulating insulin secretion in vivo. It has been shown that
intravascular delivery of fluorophoro-labeled siRNA could
a similar approach (tail vein injection) to delivering BRSK2
siRNA into mice (BRSK2-RNAi mice), which led to ?1.8-fold
reduction in BRSK2 protein levels in mouse pancreatic islets
(Fig. 5A). The down-regulation of BRSK2 resulted in a signifi-
cant increase (1.5–1.8-fold) in serum insulin levels relative to
the control mice (Fig. 5B), but no significant changes in mouse
serum glucagon levels (supplemental Fig. S3A). Importantly,
FIGURE 2. Identification of BRSK2-interacted protein PCTAIRE1. A, interaction of BRSK2 and PCTAIRE1 verified by re-transformation in Yeast. The dots
represent yeast colonies. Positive clone showed the blue color. pCL1 was used as positive control, and BD (pGBKT7)-Lam (Lamin) as negative control.
of PCTAIRE1: Fa (aa1–128), Fb (aa129–289), and Fc (aa290–496). All potential phosphorylation sites modified by BRSK2 were pointed out. D, interaction of
lysates were immunoprecipitated (IP) with PCTAIRE1 antibody and immunoblotted with BRSK2 antibody (top panel) or PCTAIRE1 antibody (bottom panel).
cell lysates. F, immunostaining of PCTAIRE1 (green, GFP) and BRSK2 (red, anti-Myc) in MIN6 cells counterstained with DAPI (blue). Magnification, 600?.
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BRSK2-RNAi mice displayed improved glucose tolerance (Fig.
5C) and possibly showed a tendency toward enlarged islets and
increased ?-cell size (supplemental Fig. S3, B and C). These
findings suggest a physiological function of BRSK2 in the
control of insulin secretion and possibly pancreatic islet
FIGURE 3. In vitro phosphorylation performance of BRSK2 (wide-type WT or kinase-dead mutant KD) phosphorylation on PCTAIRE1. HA-BRSK2 was
expressed in 293T for kinase activity assay. PCTAIRE1 deletion mutants (Fa, Fb, and Fc fragments described in Fig. 2C) and site-directed mutants of Fa (S12A,
S78A, and S119A) were used as BRSK2 substrates. The arrowheads in32P-reaction visualized by autoradiography (top panel) indicated the bands of BRSK2
auto-phosphorylation, PCTAIRE1 fragments phosphorylation and positive control CDC25C F phosphorylation by BRSK2. Corresponding bands using CBB
(Coomassie Brilliant Blue) staining (bottom panel) were also arrow-pointed. GST protein showing no BRSK2 phosphorylation signal was used as negative
FIGURE 4. BRSK2 inhibits glucose-stimulated insulin secretion in MIN6 cells. A, insulin secretion in BRSK2 overexpression or BRSK2-RNAi depletion MIN6
cells incubated in 3 mM glucose or 25 mM glucose, respectively. ***, p ? 0.001, n ? 6/condition. B, insulin secretion in PCTAIRE1 overexpression or PCTAIRE1-
RNAi deletion MIN6 cells incubated in 3 mM glucose or 25 mM glucose, respectively. Overexpressing PCTAIRE1 could partly reverse the BRSK2-depletion
a nonsilence RNAi construct are used as a control for overexpression or RNAi, respectively.
AUGUST31,2012•VOLUME287•NUMBER36 JOURNALOFBIOLOGICALCHEMISTRY 30373
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and PCTAIRE1 are selectively expressed in high abundance in
pancreatic tissues and MIN6 ?-cells. At high, but not low con-
centrations, glucose reduced BRSK2 activity in MIN6 ?-cells.
Upon activation, BRSK2 interacted with and phosphorylated
secretion in MIN6 cells. Conversely, knockdown of BRSK2
resulted in a significant increase in serum insulin levels and a
tendency toward enlarged islets and increased ?-cell size in
mice. These findings identify a novel signaling pathway
involved in the regulation of GSIS in pancreatic ?-cells.
It has been previously shown that BRSK2 is activated by the
kinase LKB1 (1). LKB1 is a tumor suppressor implicated in
Peutz-Jeghers Syndrome (15) and has been implicated in a
number of distinct biological processes. Loss of LKB1 in adult
?-cells has been recently shown to increase ?-cell mass and
through the AMPK-mTOR pathway (26, 27). The existence of
in vivo was also suggested. Given that BRSK2 is a downstream
phosphorylation target of LKB1 (1), LKB1 may also activate
BRSK2 to down-regulate insulin secretion in ?-cells.
BRSK2 knockdown by siRNA in mice was found to increase
serum insulin levels, improve GTT and show a tendency of
increased islet/?-cell size, further supporting a physiological
atic homeostasis. This is consistent with the findings by Hezel
et al. that Lkb1 deficiency in pancreas reduced expression of
BRSK1 and BRSK2 in pancreatic tissues (28), though another
group failed to detect BRSK1/2 expression in islets isolated
from 12-week-old male C57B1/6 mice (29). While both adult
ited improved GTT and p-Lkb1 mice at PD1 (postnatal day 1)
have smaller islets (28), the latter showed increased plasma
insulin levels and increased ?-cell mass with impaired GSIS
(29). Whether the increased insulin secretion is due to the aug-
mented islet/?-cell size remains to be verified.
dant. The positive band of BRSK2 mRNA showed distribution
from 1.5–10 kB, suggesting that it might have several multi-
BRSK2 contains about 11 variants which are likely to code pro-
teins. This explains the observations that while we detected
protein in islets (26). It also raised the interesting question of
whether different BRSK2 variants have distinct functions.
PCTAIRE kinases are structurally related to CDKs, but have
proteins (16–18). Hence, their role in cell cycle regulation has
remained unclear. More recent studies, however, have demon-
strated that PCTAIRE1 modulates the secretory cargo trans-
port (21), phosphorylates NSF (N-ethylmaleimide-sensitive
factor) and inhibits growth hormone release from PC12 cells
(22). It has been suggested that PCTAIRE1 is a component of
the NSF-SNAP-SNARE complex and plays a regulatory role in
Ca2?-dependent exocytosis through NSF phosphorylation
of presynaptic membranes (30, 31), which acts as a molecular
SNARE complex (32). Complex formation between NSF,
SNAP, and SNARE membrane receptors plays an important
role in membrane fusion and constitutes an essential mediator
of many transport reactions (33). Similar to neurotransmitter
release (34), the mechanism underlying insulin secretion in
?-cells is also regulated by the SNARE complex (35, 36). The
current study demonstrates that PCTAIRE1 functions down-
kinase activity. We speculate that the ability of PCTAIRE1 to
phosphorylate and inhibit NSF activity is also a major mecha-
nism by which it regulates insulin secretion.
In summary, we propose a novel signaling pathway regulat-
ing insulin secretion in pancreatic ?-cells (Fig. 5D). In our
model, low levels of glucose activates the kinase activity of
BRSK2 possibly by its upstream kinase LKB1. BRSK2, in turn,
interacts with and phosphorylates PCTAIRE1 at Ser-12, lead-
ing to the inhibition of insulin secretion through phosphoryla-
tion of NSF and inactivation of the NSF-SNARE complex,
which is required for insulin secretion from pancreatic ?-cells.
That knockdown of BRSK2 alone caused a significant increase
in insulin secretion at low glucose concentration implied that
BRSK2 dysfunction might be related to pancreatic ?-cell dys-
function. Together with the highly specific expression of
BRSK2 in pancreas and the brain, our study also suggests that
BRSK2 may serve as a novel target for developing drugs for the
treatment of diabetes.
indicated siRNA duplexes by tail vein injections. B, serum insulin levels in
BRSK2-RNAi mice and control mice were measured. *, p ? 0.05, n ? 3/condi-
tion. C, BRSK2-RNAi mice demonstrate enhanced glucose tolerance (GTT)
signaling pathway for insulin secretion regulation in pancreatic ?-cells.
by guest on October 28, 2015
Acknowledgments—We thank Dr. Dang Yong-Jun for critically read-
ing the manuscript. We thank Dr. Li Xiaoying at the Rui Jin Hospital
for kindly providing the MIN6 cell line.
J., Hawley, S. A., Udd, L., Mäkelä, T. P., Hardie, D. G., and Alessi, D. R.
(2004) LKB1 is a master kinase that activates 13 kinases of the AMPK
subfamily, including MARK/PAR-1. EMBO J. 23, 833–843
kinase are required for neuronal polarization. Science 307, 929–932
Rikitsu, E., Inoue, M., Yao, I., Takeuchi, K., Kitajima, I., Setou, M., Oht-
suka, T., and Takai, Y. (2006) SAD: a presynaptic kinase associated with
mitter release. Neuron 50, 261–275
Rev. Pharmaco. Toxicol. 47, 185–210
5. Rajan, A. S., Aguilar-Bryan, L., Nelson, D. A., Yaney, G. C., Hsu, W. H.,
Kunze, D. L., and Boyd, A. E., 3rd. (1990) Ion channels and insulin secre-
tion. Diabetes Care 13, 340–363
6. Tsuboi, T., da Silva Xavier, G., Leclerc, I., and Rutter, G. A. (2003) 5?-
AMP-activated protein kinase controls insulin-containing secretory vesi-
cle dynamics. J. Biol. Chem. 278, 52042–52051
7. da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and
Rutter, G. A. (2003) Role for AMP-activated protein kinase in glucose-
stimulated insulin secretion and preproinsulin gene expression. Biochem.
J. 371, 761–774
Metab. 286, E1023-E1031
9. Richards, S. K., Parton, L. E., Leclerc, I., Rutter, G. A., and Smith, R. M.
(2005) Overexpression of AMP-activated protein kinase impairs pancre-
atic ?-cell function in vivo. J. Endocrinol. 187, 225–235
10. Carling, D., Clarke, P. R., Zammit, V. A., and Hardie, D. G. (1989) Purifi-
cation and characterization of the AMP-activated protein kinase. Copu-
rification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglu-
taryl-CoA reductase kinase activities. Eur. J. Biochem. 186, 129–136
and Hardie, D. G. (1995) 5?-AMP activates the AMP-activated protein
kinase cascade, and Ca2?/calmodulin activates the calmodulin-depen-
dent protein kinase I cascade, via three independent mechanisms. J. Biol.
Chem. 270, 27186–27191
12. Davies, S. P., Helps, N. R., Cohen, P. T., and Hardie, D. G. (1995) 5?-AMP
inhibits dephosphorylation, as well as promoting phosphorylation, of the
protein phosphatase-2C alpha and native bovine protein phosphatase-
2AC. FEBS Lett. 377, 421–425
13. Shaw, R. J., Kosmatka, M., and Bardeesy, N. (2004) The tumor suppressor
LKB1 kinase directly activates AMP-activated kinase and regulates apo-
ptosis in response to energy stress. Proc. Natl. Acad. Sci. U.S.A. 101,
14. Jenne, D. E., Reimann, H., and Nezu, J. (1998) Peutz-Jeghers syndrome is
caused by mutations in a novel serine threonine kinase. Nat. Genet. 18,
15. Hemminki, A., Markie, D., and Tomlinson, I. (1998) A serine/threonine
kinase gene defective in Peutz–Jeghers syndrome. Nature 391, 184–187
16. Meyerson, M., Enders, G. H., and Wu, C. L. (1992) A family of human
cdc2-related protein kinases. EMBO J. 11, 2909–2917
17. Okuda, T., Cleveland, J. L., and Downing, J. R. (1992) PCTAIRE-1 and
gene family. Oncogene 7, 2249–2258
18. Graeser, R., Gannon, J., Poon, R. Y., Dubois, T., Aitken, A., and Hunt, T.
(2002) Regulation of the CDK-related protein kinase PCTAIRE-1 and its
possible role in neurite outgrowth in Neuro-2A cells. J. Cell Sci. 115,
and kinase activity of the Cdk family member Pctaire1 in the adult mouse
20. Ou, C. Y., Poon, V. Y., and Maeder, C. I. (2010) Two cyclin-dependent
ponents. Cell 141, 846–858
21. Palmer, K. J., Konkel, J. E., and Stephens, D. J. (2005) PCTAIRE protein
kinases interact directly with the COPII complex and modulate secretory
cargo transport. J. Cell Sci. 118, 3839–3847
22. Liu, Y., Cheng, K., Gong, K., Fu, A. K., and Ip, N. Y. (2006) Pctaire1 Phos-
phorylates N-ethylmaleimide-sensitive fusion protein: implications in the
regulation of its hexamerization and exocytosis. J. Biol. Chem. 281,
23. Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S.
(2002) The protein kinase complement of the human genome. Science
and Meda, P. (2003) Connexin 36 controls synchronization of Ca2?oscil-
lations and insulin secrection in MIN6 cells. Diabetes 52, 417–424
25. Bradley, S. P., Rastellini, C., and da Costa, M. A. (2005) Gene silencing in
the endocrine pancreas mediated by short-interfering RNA. Pancreas 31,
? cell size, polarity, and function. Cell Metabolism 10, 296–308
27. Fu, A., Ng, A. C., and Depatie, C. (2009) Loss of Lkb1 in Adult ? Cells
Increases ? Cell Mass and Enhances Glucose Tolerance in Mice. Cell
Metabolism 10, 285–295
28. Hezel, A. F., Gurumurthy, S., and Granot, Z. (2008) Pancreatic Lkb1 dele-
tion leads to acinar polarity defects and cystic neoplasms. Mol. Cell. Biol.
29. Sun, G., Tarasov, A. I., and McGinty, J. A. (2010) LKB1 deletion with the
RIP2. Cre transgene modifies pancreatic ? cell morphology and enhances
insulin secretion in vivo. Am. J. Physiol. Endocrinol. Metab. 298,
30. Block, M. R., Glick, B. S., Wilcox, C. A., Wieland, F. T., and Rothman, J. E.
(1988) Purification of an N-ethylmaleimide-sensitive protein catalyzing
vesicular transport. Proc. Natl. Acad. Sci. U.S.A. 85, 7852–7856
31. Beckers, C. J., Block, M. R., Glick, B. S., Rothman, J. E., and Balch, W. E.
(1989) Vesicular transport between the endoplasmic reticulum and the
Golgi stack requires the NEM-sensitive fusion protein. Nature 339,
32. Morgan, A., and Burgoyne, R. D. (2004) Membrane traffic: controlling
membrane fusion by modifying NSF. Curr. Biol. 14, 968–970
33. Woodman, P. G. (1997) The roles of NSF, SNAPs and SNAREs during
membrane fusion. Biochim. Biophys. Acta 1357, 155–172
34. Wheeler, M. B., Sheu, L., and Ghai, M. (1996) Characterization of SNARE
35. Barg, S., Ma, X., and Eliasson, L. (2001) Fast exocytosis with few Ca2?
channels in insulin-secreting mouse pancreatic B cells. Biophys. J. 81,
36. Barg, S., Eliasson, L., Renstrom, E., and Rorsman, P. A. (2002) A subset of
for first-phase insulin secretion in mouse ?-cells. Diabetes 51, S74–S82
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Han-Fei Ding, Jun O. Liu and Long Yu Download full-text
2012, 287:30368-30375.J. Biol. Chem.
Dong, Sonia M. Najjar, Chen-Yu Zhang,
Wang, Rui Gao, Yu-Fan Wang, Wei-Ping
Bo Wan, Yu-Jing Zhang, Jing Li, Ying-Li
Xin-Ya Chen, Xiu-Ting Gu, Hexige Saiyin,
Secretion in Pancreatic
Regulates Glucose-stimulated Insulin
Phosphorylation on PCTAIRE1 Negatively
Brain-selective Kinase 2 (BRSK2)
doi: 10.1074/jbc.M112.375618 originally published online July 13, 2012
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