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Molecular Biology and Genetics |
Upstate Medical University, Pathology Department, Syracuse, New York 13210
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSResults and DiscussionREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSResults and DiscussionREFERENCES |
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSResults and DiscussionREFERENCES |
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Isolation of mirk cDNA Clone.
Mirk was purified from diolein-treated (5 min) HP1 colon
carcinoma cells by sequential fractionation of S100 cytosols by
DEAE-cellulose and phenyl Sepharose fast protein liquid chromatography,
followed by affinity purification on immobilized pan-MAPK polyclonal
antibody directed to the conserved kinase subdomain 11 sequence
DRLTAEEALSHPYMSIYSFPTDE. Eluted protein was fractionated by SDS-PAGE
and electroblotted onto nitrocellulose, and the Ponceau S-stained
Mr 57,000 band was excised and
processed for internal amino acid analysis. HPLC peak fractions were
subjected to chemical sequencing by an Applied Biosystems 477A
sequenator optimized for femtomol level analysis. One peak corresponded
to amino acids 379–390 of mirk and was used to identify its cDNA
clone. Degenerate oligonucleotide primers from conserved protein kinase
subdomains II and VIb were used to amplify a cDNA fragment made using
HD3 colon carcinoma cell mRNA with the Access RT-PCR system (Promega).
Sixteen different cDNA bands were amplified, purified on agarose gel,
inserted into the pGEM-T Easy vector (Promega), and then transfected
into Escherichia coli JM109. Of 180 colonies isolated, 36
were sequenced, yielding three erk2s, one erk1, one MEKK, and one novel
440-bp fragment that was used as a probe to screen a human SW480
colorectal adenocarcinoma cDNA (Clontech) library. More than
1,000,000 plaques were screened. Seventeen positive clones were
obtained, subcloned into the pGEM vector, and analyzed by sequencing
for the presence of amino acids 379–390 in purified mirk. pG9R
contained a 2541-bp long mirk cDNA with a start codon,
poly(A) sequence, and coded for the amino acid sequence found in
purified mirk. The DNA sequence was deposited in GenBank under
accession number AF205861.
Preparation of Expression Plasmids.
After EcoRI digestion, mirk cDNA was ligated into
pcDNA3.1/HisA (Invitrogen), yielding the hexahistidine fusion plasmid
pHX9. Hexahistidine fusion mirk cDNA was isolated from pHX9
by HindIII, XhoI double digestion and blunt
ended. After StuI digestion, the blunted mirk
cDNA was ligated into the pBacPAK8 (Clontech) plasmid, yielding
recombinant baculovirus pBH. After mirk was excised from
pG9R by StuI digestion, it was ligated into pGEX-4T-1
(Pharmacia), yielding the GST fusion plasmid pGD. After mirk
was excised from pG9R by EcoRI digestion, it was ligated
into pLXSN (Clontech), yielding pLXSN-D2. pLXSN-D2 was transfected into
the PT67 retrovirus packaging cells, and recombinant retrovirus was
harvested from the supernatant at
105–106 virions/ml. Stable
mirk transfectants were isolated in G418 after retroviral
infection of HD3 colon carcinoma cells.
Mutagenesis/Coupled Transcription Translation/in
Vitro Kinase Assays.
Site-directed mutagenesis was performed with mutagenic oligonucleotides
using the pGEM-11Zf(+) vector (Promega) to transform PMH 71–18 mutS
competent cells, and the products were sequenced to verify the
mutation. Hexahistidine fusion mirk was produced by a coupled in
vitro transcription and translation system (Promega) using 1 µg
of pHX plasmid/reaction. Produced protein was immunoprecipitated
overnight by using 6x(His) mAb (Clontech) and 10 µl of 50% Protein
A-Sephadex (Sigma). The immunoprecipitate was washed three times with
RIPA buffer and three times with kinase buffer [10
mM Tris (pH 7.4), 150 mM
NaCl, 10 mM MgCl2, and 0.5
mM DTT]. The immunocomplex was incubated for 15
min at 37°C with 40 µl of the kinase buffer containing 25
µM ATP, 2.5 µCi
[32P
]ATP, and 1.0 mg/ml MBP as substrate and
then analyzed by electrophoresis and autoradiography.
Phosphatase Treatment of Phosphorylated MBP.
The kinase reaction mixture described above was incubated for 30 min at
30°C with either protein phosphatase-1 [0.5 unit in 20
mM 4-morpholinepropanesulfonic acid (pH 7.5), 60
mM 2-mercaptoethanol, 1 mM
MgCl2, and 0.1 mg/ml serum albumin], protein
tyrosine phosphatase-ß [2 units in 25 mM HEPES (pH 7.2),
50 mM NaCl, 5 mM DTT, and 2.5 mM
EDTA], or PP-1 (0.5 unit) with okadaic acid (100 nM) and
then analyzed by electrophoresis and autoradiography.
Western Blotting.
Samples were prepared according to Deng et al.
(6)
. All mirk blots used affinity-purified polyclonal
antibody directed to the mirk unique C' terminus unless noted, except
blots of the human tissue samples, which used monoclonal antibody.
Western blotting for activated and total MAPKs was performed exactly
according to Yan et al. (7)
. Band density in
autoradiograms was measured using a Lacie Silverscanner and
silverscanner III software and analyzed by the IP LabGel program.
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Results and Discussion |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSResults and DiscussionREFERENCES |
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. BLAST search confirmed that this gene was a homologue of a
Drosophila gene called minibrain (Mnb;
Ref. 8
) or dyrk (9
, 10)
;
therefore, it was called mirk. A initial report of this work
has been presented (11)
. Mutant Mnb flies are
characterized by a marked reduction in size of the optic lobes and
central brain hemispheres (8)
. Dyrk1/minibrain
is a strong candidate for the gene duplicated in Down Syndrome
(12)
, but no function has been established for this gene
aside from its kinase activity. More recently, several other members of
the Mnb/dyrk family have been cloned, but no function for
any of these genes has been elucidated (13)
. One
Mnb/dyrk gene, dyrk1B, has three splice variants,
encoding putative proteins of 629, 601, and 589 amino acids
(14)
, the longest being identical to mirk. A
mirk splice variant of 601 amino acids had been isolated in
our initial screen but was not studied further because it did not
encode the peptide found in mirk protein purified from colon carcinoma
cells (see "Methods and Materials"). This splice variant was
generated by alternative splicing within exon 9 of mirk
(Fig. 1B)
, which encodes amino acids found in the conserved
kinase domain between subdomains X and XI.
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. The amino acid sequence of mirk is 56% identical
within the conserved kinase domain to the human forms of dyrk1
(minibrain), dyrk2, and dyrk3, with much less identity in the N'
terminus and C' terminus. All dyrk family members are serine/threonine
kinases that also can autophosphorylate on tyrosine; therefore, they
are dual function kinases (8
, 9 , 10
, 13)
. Their activation
sequence is YQY or YTY, with both tyrosines phosphorylated. This YxY
sequence aligns with the TxY activation sequence in MAPKs. The cDNA
sequence of mirk was compared by BLAST search to sequences
deposited in GenBank from the Human Genome Project. The genome sequence
of the mirk gene was found in chromosome 19q13.1, and
intron/exon junctures were determined by comparison of the two
sequences. mirk has 11 exons and 10 introns encompassing 8.8
kb of genomic DNA (Fig. 1B)
. mirk was not mutated
in the SW480 colon carcinoma cells from which it was cloned, because
its sequence did not differ from the genomic DNA sequence.
Recombinant mirk expressed in sf21 insect cells was purified and then
either assayed for protein kinase activity on immobilized MBP in an
in gel kinase assay or Western blotted with
anti-phosphotyrosine antibody. Recombinant mirk had MBP kinase activity
at Mr 70,000, the molecular weight of
mirk deduced from its amino acid sequence, and was phosphorylated on
tyrosine (Fig. 1C)
. Phosphotyrosine was also detected in
mirk expressed in E. coli, indicating that mirk has tyrosine
autophosphorylation capacity (data not shown). Mirk activity is
dependent on phosphorylation of tyrosines 271 and 273 (Fig. 1C)
located between subdomains VII and VIII in the sequence
YQY, which aligns with the erk1 activation sequence TEY
(15)
. A mirk mutant at the ATP-binding site (K140R) and a
double mutant at the putative activation sequence YQY, to FQF, were
generated. Both mutations greatly decreased the kinase activity of mirk
on MBP, as well as the capacity of mirk for autophosphorylation (Fig. 1C)
and the autophosphorylation of mirk on tyrosine (data
not shown). Western blotting confirmed that equal levels of the
wild-type and mutant proteins were present in each reaction (Fig. 1C)
. Analysis of the phosphorylated amino acids in MBP by
TLC and of labeled MBP for phosphatase sensitivity (Fig. 1C)
demonstrated that mirk phosphorylates only serine and threonine
residues in vitro. Mirk-phosphorylated MBP was a substrate
for the serine-threonine phosphatase PP1 but not for the
phosphotyrosine-specific phosphatase, PTP-ß (Fig. 1C)
. In
addition, mirk had no detectable in vitro activity on two
synthetic phosphotyrosine substrates while phosphorylating certain
substrates of the Ser/Thr kinase MAPKAP kinase-2 (Ref. 16
;
data not shown). Therefore, although mirk kinase is activated by
autophosphorylation on tyrosine at the Y271/Y273 site, mirk itself is a
serine-threonine kinase on exogenous substrates.
mirk exhibits restricted mRNA expression in normal tissue
(Fig. 2, A and B)
, with highest expression in skeletal
muscle, testes, heart, and brain, and with little expression in normal
colon (Fig. 2A
, Lane c) and normal lung (Fig. 2B
, Lane
lu). mirk is highly expressed in SW480 colon carcinoma
cells and A549 lung carcinoma cells (Fig. 2C)
and other
tumor cell lines including HeLa ovarian carcinoma cells, K562 chronic
myelogenous leukemia cells, molt4 lymphoblastic leukemia cells, and
G361 melanoma cells. mirk is not detectably expressed in
HL60 promyelocytic leukemia cells or Raji lymphoma cells (Fig. 2C)
. Thus, mirk is expressed in few normal
tissues but in many types of cancer.
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, upper panel), with N'-terminal antibody (Fig. 3C
,
lower panel, upper arrow), or with monoclonal antibody 11 (Fig. 3B)
, mirk was detected at the expected translational size of
Mr 70,000. A
Mr 67,000 mirk form was detected in
several lines by both N'-terminal and C'-terminal antibodies.
Therefore, the Mr 67,000 mirk must be
caused by an internal deletion. A splice variant caused by internal
deletion within exon 9 was cloned in our initial screen (see
"Materials and Methods"). This splice variant encoded a
Mr 67,000 mirk protein after in
vitro coupled transcription/translation (data not shown) and
therefore is very likely to be the source of the smaller mirk form. In
addition to the splice variants, a mirk breakdown form of
Mr 57,000 was detected by N'-terminal
antibody and monoclonal antibody but was not detected by
C'-terminal-directed antibody, indicating that it was generated by
C'-terminal deletion.
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; also data not shown). Cells were cultured in
serum-containing growth media. Fractionation of cells into nuclear
(N) and cytoplasmic (C) fractions
demonstrated that a Mr 57,000 mirk breakdown
product was found solely in the nucleus of each cell type, whereas
higher levels of full-length, Mr 70,000 mirk
are found in each mirk transfectant. The Mr
57,000 mirk breakdown product could not be detected with
C'-terminal-directed antibody (not shown), indicating that the C'
terminus of mirk was deleted in the nuclear species. \.
antibody raised to recombinant mirk was used to detect mirk in the
stable mirk transfectant T24 and a vector control transfectant V1. Mirk
was more abundant in the T24 overexpressing line than the control
cells, and a Mr 57,000 degradation
product of mirk was also detected in the overexpressing line (Fig. 3B)
. Monoclonal antibody activity was removed by
preabsorption with recombinant mirk protein, and preimmune mouse serum
did not detect mirk in parallel blots (Fig. 3B)
. The blots
were stripped and reblotted for ß-actin, demonstrating equal loading
and transfer (Fig. 3B
, lower panels). When eight cases of
colon cancer tissue with paired normal colon were examined by Western
blotting with this monoclonal antibody, mirk was found to be much more
abundant in the cancer tissue in three of eight cases (nos. 1, 3, and
6) when normalized to actin levels (Fig. 3A
, upper panel).
Lack of degradation of protein within the clinical samples was
demonstrated by reblotting for ß-actin protein (Fig. 3A
, lower
panel). Mirk was localized predominately within the cytoplasm of
the colon carcinoma cells within sectioned colon cancer tissue from
three additional cases, not within the stroma (data not shown), as
shown by immunocytochemistry with monoclonal antibody to mirk.
Therefore, the greater abundance of mirk protein by Western blotting in
a subset of colon cancer tissues was attributable to increased mirk
expression within the colon cancer cells themselves, not another cell
type.
Recombinant mirk is phosphorylated by all three classes of MAPKs, the
erks, the SAP/c-Jun NH2-terminal kinases and p38
(Fig. 4A)
. Western blotting then showed that mirk protein levels are
down-regulated by activated erks in vivo. Inhibition of erk
activity by removal of serum mitogens and by addition of a MEK
inhibitor increased endogenous mirk protein levels 18–20-fold. When U9
colon carcinoma cells were placed in serum-free medium for 1, 2, and 3
days, levels of endogenous mirk were increased 5-, 6-, and 7-fold,
respectively (Fig. 4B)
. These data suggested that serum
mitogens activated some signaling path that either down-regulated mirk
mRNA, mirk protein levels, or both. U9 cells exhibit autocrine
regulation by epidermal growth factor, fibroblast growth factor, and
transforming growth factor ß family members (17, 18, 19)
;
therefore, U9 cells exhibit some activation of erks in the absence of
serum mitogens (Fig. 4C)
. Treatment of U9 cells for 24 h in serum-free medium with the MEK inhibitor PD98059 induced a 4-fold,
dose-dependent increase in mirk levels, above that caused by serum-free
culture alone (Fig. 4B
, Lanes 1–4), probably because the
MEK inhibitor blocked the activation of erks by autocrine growth
factors. This total 20-fold increase in mirk above background levels
was reversed by concurrent treatment of cells with the serum mitogen
IGF-I (Fig. 4B
, Lanes 5–8), which activated erk1 and erk2
for at least 3 h (data not shown). Therefore, blocking erk
activation by removal of serum mitogens and blocking any residual erk
activation from autocrine growth factors by the MEK inhibitor elevated
mirk levels 20-fold, whereas activation of erks with IGF-I reduced mirk
levels to those seen in cells cultured in serum-containing medium.
Similar data were seen with SW480 and HD3 colon carcinoma cell lines
(data not shown).
|
. The
mirk double band is better resolved than in A or
B because the gel fractionation was extended for a longer
period. No variation in total erk1/erk2 abundance was seen
(lowest panel). U9 colon carcinoma cells were placed in
serum-free media with or without 10 ng/ml IGF-I. Parallel immunoblots
were probed for mirk protein (upper blot), for
phosphorylated, activated erk1 and erk2 using monoclonal antibody
directed to the erk1 and 2 phosphorylated activation sequence TEY
(middle blot), and for total erk1/erk2 using an antibody
which did not distinguish between activated and inactivated erk forms
(lower blot). Mirk levels began to increase by 3 h of
serum-free culture and continued to rise for 24 h, coincident with
the decrease in activated erk forms. The slow decrease in activated
erks may be attributable to autocrine growth factors. However, when
IGF-I was added to maintain activation of erk1 and erk2 (Fig. 4C
,
Lanes 7–12), no increase in mirk levels was observed.
Serum-free culture increased the level of transfected mirk (Fig. 4D)
in the T3 and T7 stable, clonal mirk transfectants of U9
colon carcinoma cells, as well as endogenous mirk in the vector
controls, 9V1 and 9V2. Addition of IGF-I to maintain activated erks
caused a decrease in both transfected and endogenous mirk levels (Fig. 4E)
. The stable mirk transfectant T3 expressed about 4-fold
the amount of mirk as vector control V1 cells after growth in
serum-free medium (Fig. 4E)
.
mirk mRNA levels were analyzed by RT-PCR in 9V2 vector
control and T7 mirk transfectant cells cultured in control
serum-containing medium or in serum-free media for 24 h with or
without 10 µM PD98059 (MEK inhibitor) or 10
ng/ml IGF-I. Primers for glyceraldehyde-3-phosphate dehydrogenase were
included in every reaction mixture as an internal control. Added
PD98059 had little effect on endogenous mirk mRNA levels in 9V2 vector
control transfectants (Fig. 4F)
, while dramatically
up-regulating mirk protein levels (Fig. 4B)
. IGF-I had no
discernible effect on the combination of exogenous and endogenous
mirk mRNA levels in the T7 transfectant (Fig. 4F)
but slightly decreased mRNA levels of endogenous mirk (seen
in three experiments). Thus, mirk protein levels are primarily
modulated at the posttranscriptional level by the MAPKs erk1 and erk2.
MAPKs dimerize after their activation, and dimers translocate to the
nucleus, where they modify transcription factors (20)
.
Phosphorylation of proteins by MAPKs has been shown to be a trigger for
their degradation (21
, 22)
. Western blotting with a mirk
N'-terminal antibody demonstrated that a mirk
Mr 57,000 breakdown product was found
solely in nuclear fractions of both mirk HD3 transfectants (T19, T24)
and vector control HD3 transfectants (V1, V2; Fig. 4G
, upper
panel), placing the mirk breakdown product in the nucleus with
activated MAPKs. Similar cell fractionation data were observed with U9
cells (Fig. 4G
, lower panel). We hypothesize that after
activation by serum factors and translocation to the nucleus, erks
signal the rapid turnover of mirks by phosphorylation of mirk at the
canonical MAPK S557 site in its nonconserved C' terminus and possibly
at a second site within the PEST
region.4
Mirk protein levels were generally higher in resected colon cancers
than in established cell lines by Western blotting (data not shown),
which is not surprising because carcinoma cell lines are established in
serum-containing medium that keeps mirk levels low. Cell lines may
up-regulate expression of autocrine growth factors to adapt to passage,
and in this way also contribute to the down-regulation of mirk. We
hypothesized that because mirk abundance is rather low in cell lines,
we would have to overexpress mirk in established cell lines to study
its function. Clonal stable transfectants expressing elevated
endogenous levels of mirk were isolated using two human colon carcinoma
cell lines, HD3 and U9 (23)
. Mirk levels in these
transfectants remained elevated 3–4-fold over vector controls in
serum-free conditions (Fig. 4, E and G)
. Each of
the three mirk HD3 cell transfectants and both of the mirk U9 cell
transfectants proliferated when switched to serum-free media, whereas
none of the five vector control transfectants was able to sustain
growth under these conditions (Fig. 5)
. Mutation of mirk at the putative ATP binding site (KR) and at the YQY
activation site (FQF) completely inhibited the kinase activity of mirk
on MBP (Fig. 1)
. Stable transfectants of these mirk mutants lacked the
ability to proliferate when switched to serum-free conditions (Fig. 5)
.
Similar data were obtained by direct cell counting and by MTT assay.
Overexpression of mirk by stable transfection increased cell numbers
3–5-fold in HD3 cells and 5–9-fold in U9 cells after 5 days of
serum-free growth, whereas mutations that blocked the kinase activity
of mirk also blocked this biological activity (Fig. 5, A, B,
E, and F
). After 7 days in serum-free medium, mirk
transfectants remained viable and readily resumed rapid growth when
serum was added to the cultures (Fig. 5H)
, whereas vector
control transfectant cultures did not re-establish rapid growth,
indicating that some cells had lost viability.
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and in a subset of colon carcinomas (Fig. 3)
,
indicating that selection for mirk-expressing cells is maintained after
cancers evolve from premalignant stages. Mirk may mediate tumor cell
survival, especially in solid tumors, during periods when carcinoma
cells temporarily outgrow their nutrient support.
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FOOTNOTES |
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1 Supported by National Cancer Institute RO1
CA67405 (to E. F.). 
2 To whom requests for reprints should be
addressed, at Pathology Department, Upstate Medical University, State
University of New York, 2305 Weiskotten Hall, 750 East Adams Street,
Syracuse, NY 13210. Phone: (315) 464-7148; Fax: (315) 464-8419;
E-mail: friedmae{at}mail.upstate.edu
3 The abbreviations used are: MAPK,
mitogen-activated protein kinase; erk, extracellular signal-regulated
kinase; IGF, insulin-like growth factor; MBP, myelin basic protein;
mirk, minibrain-related kinase; MEK, MAPK kinase; RT-PCR, reverse
transcription-PCR.
4 X. Deng and E. Friedman, manuscript in
preparation.
Received 11/10/99. Accepted 4/27/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSResults and DiscussionREFERENCES |
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as they differentiate. Exp. Cell Res., 219: 8-14, 1995.[Medline]
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) with
cell number assayed by MTT analysis (A, C, D, E, and
G; bars, SE) or by direct cell counting
(B and F). Bars are not
shown in the line graphs if the SE measurement is <5% of the mean
value, which is the default level for the graphing program. However,
all data are the mean of triplicate measurements. Similar experiments
using [3H]thymidine incorporation as a proliferation
assay had been performed with similar outcomes with each point a mean
of triplicate measurements (not shown). In H, U9 mirk
transfectants were cultured 7 days serum free and then switched to
serum-containing media and showed rapid resumption of growth, whereas
two vector control transfectants showed no sustained growth.